CA2035674A1 - Non-instrumented cholesterol assay - Google Patents
Non-instrumented cholesterol assayInfo
- Publication number
- CA2035674A1 CA2035674A1 CA002035674A CA2035674A CA2035674A1 CA 2035674 A1 CA2035674 A1 CA 2035674A1 CA 002035674 A CA002035674 A CA 002035674A CA 2035674 A CA2035674 A CA 2035674A CA 2035674 A1 CA2035674 A1 CA 2035674A1
- Authority
- CA
- Canada
- Prior art keywords
- region
- sample
- cholesterol
- transport
- measurement
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
- G01N33/521—Single-layer analytical elements
- G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Abstract
NON--INSTRUMENTED CHOLESTEROL ASSAY
ABSTRACT
An assay for determining the cholesterol level in a sample involving a bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, a sample receiving region in fluid communication with said transfer region, and a measurement region in fluid communication with said sample receiving region, and a detectable signal reagent system comprising unbound conversion reagent and bound reagent wherein said conversion reagent reacts with cholesterol to form an intermediate product and wherein said bound reagent reacts with said intermediate product to produce a detectable border, in which the conversion reagent is placed in the transfer region of the strip or in a region of the strip between the sample receiving and measurement regions and the signal reagent is non-diffusively bound to the strip in the measurement region, and which upon contact with the sample and the transport medium results in the production of a detectable border in the measurement region which is related to the level of cholesterol in the sample.
012790 33.
ABSTRACT
An assay for determining the cholesterol level in a sample involving a bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, a sample receiving region in fluid communication with said transfer region, and a measurement region in fluid communication with said sample receiving region, and a detectable signal reagent system comprising unbound conversion reagent and bound reagent wherein said conversion reagent reacts with cholesterol to form an intermediate product and wherein said bound reagent reacts with said intermediate product to produce a detectable border, in which the conversion reagent is placed in the transfer region of the strip or in a region of the strip between the sample receiving and measurement regions and the signal reagent is non-diffusively bound to the strip in the measurement region, and which upon contact with the sample and the transport medium results in the production of a detectable border in the measurement region which is related to the level of cholesterol in the sample.
012790 33.
Description
2~3~7~
S NON-INSTRU~NTED C~O~ST13 ROI- ~SSAlr -The field of this invention is non-in~trumen-ted diagno~tic device~ and sssays for determining chole~terol level~ in samples.
lS Chole~terol is a hydrophobic molecule found in all animal3 as an essential component of cell membranes and hormones and $8 known to be involved in other vital functions a~ well. Since cholesterol i~ present in all animal tissue, cholesterol i8 consumed every time food of animal origin i~ eaten. Although cholesterol is essential for body processes, it is not necessary for this compound to be ingeste~, since the liver can produce ~11 that i3 needed.
Cholesterol i8 found and stored in blood as a fstty acid ~ster which is complex~d with serum proteins.
The~e cholesterol ester-protein complexe~ found in blood are termed llpoproteins. In th$~ way "nature" hs3 found a method to allow water insoluble chole~terol to become soluble in whole blood. There are two types of l$poprotein~ which have been identified in blood, and these two t~pes are low density lipoprotein~ (~DL) and high dQnsity llpoproteins (HDL). LDL cholesterol is known to contain a high proportion of cholesterol and has been indicted a~ being the agent responsible for the deposition of cholesterol in artery walls. HDL
cholesterol, on the other hsnd, is believed to transport cholesterol to the liver for removal from the blood.
Thus, LDL cholesterol has been ch~racterized a~ "bad"
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2~35~7~1 cholesterol, while HDL cholesterol has been charac-terized as "good" cholesterol.
A relationship has been established between total blood cholesterol (which is primarily the LDL
S fraction) and coronary artery disease. Guidelines have been established for adults over 20 years of age to identify risk groups associated with blood cholesterol level. These levels are as follows: <200 mg/dl is desirable blood cholesterol; 200 to 239 mg/dl i~ border-line high blood cholesterol; >240 mg/dl is considered high blood cholesterol.
Cholesterol levels can be controlled by both diet and cholesterol-lowering drugs. The key to such efforts to control cholesterol levels is to identify those individuals at risk. There has been an effort in the past several years to identify individuals with elevated cholesterol levels and initiate treatment.
This effort is expected to lower mortality from coronary heart disea~e. A procedure whereby cholesterol levels could be ea~ily and conveniently determined at home, therefore, would be particularly useful in the effort to lower mort~l~ty from coron~ry heart disease.
The followLn~ mQthodology include~ ~ssay ~trip design and perfonmance characteristics for ~ non-instrumented whole blood cholesterol as~ay which is well-suited for home u~e. This type of hom~ te~ting will improve the method for identification of those at risk and further reduce death from coronary heart disease.
Relevant Literature Demacker et al., Clin. Chem. (1983) 29tl916-1922 reports the evaluation of cholesterol assay kits.
Studies associated with enzyme assays include Gochman and Schmitz, Clin. Chem. (1971) 17:12; Paul, The EnzYmes (1963) 8s227-274; Current Status of ~lood Cholesterol Mea~urement in Clinical L~boratorles in the United State~s A RePort from the L boratorv Standardization 012790 2.
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Panel of the National Cholesterol Education Program (1988) 34(1):193-201, and V.S. Patent Nos. 4,391,904, 4,366,241, 4,168,146, 4,435,504, 4,533,629, 4,504,659, and the references cited therein. See also, Zuk et al., Clin.
Chem. (1985~ 31:114~-1150.
German Patent No. 22 951 describes a filter assembly containing chemical reagents for removing cells from blood and measuring CPK.
The present invention involves methods and apparatus for use in determining cholesterol levels in samples by using a continuous flow path assay in which the cholesterol is reacted with a conversion reagent to provide an intermediate product which in turn reacts with a bound reagent on the path to produce a detectable signal having a detectable border whose distance from a predetermined site is related to the amount of cholesterol in the sample. Improved accuracy and sensitivity are obtained in the assay by placing the conversion reagent, and/or additional reagents needed to produce the detectable signal from the intermediate product and bound reagent, on the path upstream or downstream of the path region initially contacted with the sample, such that the reagents are transported by a transport medium through the bound reagent region.
The invention is illustrated in the drawings in which:
Figure 1 :i~ a diagrammatic ~plan view oE a 30 measuring strip according -to this invention;
Figure 2 is a diagrammatic plan view of the base plate and slide of an alternate embodiment according to this invention;
Figures 3a and 3b are diagrammatic plan views 35 of an intermediate plate which covers the base plate, and of the plate inverted to show the underside, respectively;
S NON-INSTRU~NTED C~O~ST13 ROI- ~SSAlr -The field of this invention is non-in~trumen-ted diagno~tic device~ and sssays for determining chole~terol level~ in samples.
lS Chole~terol is a hydrophobic molecule found in all animal3 as an essential component of cell membranes and hormones and $8 known to be involved in other vital functions a~ well. Since cholesterol i~ present in all animal tissue, cholesterol i8 consumed every time food of animal origin i~ eaten. Although cholesterol is essential for body processes, it is not necessary for this compound to be ingeste~, since the liver can produce ~11 that i3 needed.
Cholesterol i8 found and stored in blood as a fstty acid ~ster which is complex~d with serum proteins.
The~e cholesterol ester-protein complexe~ found in blood are termed llpoproteins. In th$~ way "nature" hs3 found a method to allow water insoluble chole~terol to become soluble in whole blood. There are two types of l$poprotein~ which have been identified in blood, and these two t~pes are low density lipoprotein~ (~DL) and high dQnsity llpoproteins (HDL). LDL cholesterol is known to contain a high proportion of cholesterol and has been indicted a~ being the agent responsible for the deposition of cholesterol in artery walls. HDL
cholesterol, on the other hsnd, is believed to transport cholesterol to the liver for removal from the blood.
Thus, LDL cholesterol has been ch~racterized a~ "bad"
012790 1.
2~35~7~1 cholesterol, while HDL cholesterol has been charac-terized as "good" cholesterol.
A relationship has been established between total blood cholesterol (which is primarily the LDL
S fraction) and coronary artery disease. Guidelines have been established for adults over 20 years of age to identify risk groups associated with blood cholesterol level. These levels are as follows: <200 mg/dl is desirable blood cholesterol; 200 to 239 mg/dl i~ border-line high blood cholesterol; >240 mg/dl is considered high blood cholesterol.
Cholesterol levels can be controlled by both diet and cholesterol-lowering drugs. The key to such efforts to control cholesterol levels is to identify those individuals at risk. There has been an effort in the past several years to identify individuals with elevated cholesterol levels and initiate treatment.
This effort is expected to lower mortality from coronary heart disea~e. A procedure whereby cholesterol levels could be ea~ily and conveniently determined at home, therefore, would be particularly useful in the effort to lower mort~l~ty from coron~ry heart disease.
The followLn~ mQthodology include~ ~ssay ~trip design and perfonmance characteristics for ~ non-instrumented whole blood cholesterol as~ay which is well-suited for home u~e. This type of hom~ te~ting will improve the method for identification of those at risk and further reduce death from coronary heart disease.
Relevant Literature Demacker et al., Clin. Chem. (1983) 29tl916-1922 reports the evaluation of cholesterol assay kits.
Studies associated with enzyme assays include Gochman and Schmitz, Clin. Chem. (1971) 17:12; Paul, The EnzYmes (1963) 8s227-274; Current Status of ~lood Cholesterol Mea~urement in Clinical L~boratorles in the United State~s A RePort from the L boratorv Standardization 012790 2.
2~3~7~
Panel of the National Cholesterol Education Program (1988) 34(1):193-201, and V.S. Patent Nos. 4,391,904, 4,366,241, 4,168,146, 4,435,504, 4,533,629, 4,504,659, and the references cited therein. See also, Zuk et al., Clin.
Chem. (1985~ 31:114~-1150.
German Patent No. 22 951 describes a filter assembly containing chemical reagents for removing cells from blood and measuring CPK.
The present invention involves methods and apparatus for use in determining cholesterol levels in samples by using a continuous flow path assay in which the cholesterol is reacted with a conversion reagent to provide an intermediate product which in turn reacts with a bound reagent on the path to produce a detectable signal having a detectable border whose distance from a predetermined site is related to the amount of cholesterol in the sample. Improved accuracy and sensitivity are obtained in the assay by placing the conversion reagent, and/or additional reagents needed to produce the detectable signal from the intermediate product and bound reagent, on the path upstream or downstream of the path region initially contacted with the sample, such that the reagents are transported by a transport medium through the bound reagent region.
The invention is illustrated in the drawings in which:
Figure 1 :i~ a diagrammatic ~plan view oE a 30 measuring strip according -to this invention;
Figure 2 is a diagrammatic plan view of the base plate and slide of an alternate embodiment according to this invention;
Figures 3a and 3b are diagrammatic plan views 35 of an intermediate plate which covers the base plate, and of the plate inverted to show the underside, respectively;
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Figure 4 i8 a planar view of an assembled device; and Figures 5-8 are diagrammatic plan views of various embodiments of a laminated measuring strip according to the invention.
Method~ and apparatus are provided for the measurement of cholesterol in a sample employing a continuous flow path, which has a transfer region, sample receiving region, an optional detectable signal reagent capturing region, and a measurement region. The presence of cholesterol and conversion reagent of the detectable signal reagent system results in production of an intermediate product, which directly or indirectly reacts with another member of the detectable signal reagent system which i~ bound to the surface of the measurement region. The result is the production of a detectable signal, generally a colored zone, in the measurement region. The distance of the border of the detectable signal from a predetermined site can be related to the amount of cholesterol in a ~ample.
Various technique~ may be employed for organizing the flow path and providing for flow of a r~agent eolution through the ~mple reglon, the reagent capturing region, and the measurement region. In addition, varioue ch~mistriee may be employed, using different type~ of reagent systeme to produce the de~ired eignal. The assay may be eemiquantltative or quantltative. For quantitative aeeaye, the dletance of the border of the detectable elgnal, l.e., colored border, from ~ predetermlned elte lndicatee the amount of choleeterol ln the sample.
The general design of euch aseaye is set forth ln copendlng U.S. Patent Appllcatlone Serial No. 433,538 filed November 8, 1989, which application i~ a continua-tion-in-part of Serial No. 357,045, filed May 29, 1989, which application i8 a continuation-in-part of Serial 012~90 4.
2 ~ 4 No. 324,407, :Eiled March L6, 1989, which is a continua-tion-in-par~ of Serial No. 195,881l fil.ed May 19, 1988, and Serial No. 064,883, filed June 22, 1987.
The flow path comprises several regions, where certain regions may be overlapping, in whole or in part:
(a) transfer region, ~b~ sample receiving region, (c) optional capture region, and (d) measurement region. The device embodying the flow path may be of any suitable configuration, preferably a strip which has the various regions aligned in the direction of transport medium flow.
The -transfer region receives the transport medium and initiates the flow of the transport medium into the flow path. For the most part, particularly with extended strips, it will be a bibulous short element which serves by capillary action to wick or transport the transport medium to the next region, normally the sample receiving region. For the most part, the -transfer region will be a bibulous strip which absorbs a hydrophilic liquid and allows for transport of any reagents contained in the ~0 transport medium with or without chromatographing the components of the transport medium. Where the device is a circular disc, the transfer region will be at the centre of the disc and allow for transport of the medium radially away from the centre through the other regions.
The sample receiving region serves to receive the sample and to act as a bridge .For t:ransferr.i.ng the transport solutic)n to tl)e nexl reyi.on. :rn c~ne emt)odi.me~nl, prior to the time that the transfer region serves to transport the medi.um to the sample rece:iving element, adjacent regions to the sample recei.ving region will not be in fluid :rece:iving relationshi.p with -the sample receiving region. In that embodiment, after receiving the sample, the sample receiv:ing element is 2~3~7~
then permitted to be a bridging elemsnt which allows for the flow of the transport ~olution through the sample receiving region and into the next region.
The sample receiving region may take a number of forms but may be simply a site on the bibulous strip where the sample i~ placed. Alternatively, it may be a pad in fluid transferring relationship with an underly-ing strip with or without enzyme~s) to enzymetically convert cholesterol to produce hydrogen peroxide.
Variou~ techniques may be employed for providing a measured amount of sample to the sample receiving region. The sample may be measured by any convenient means, such as a micropipet, capillary or the like, touching the measuring device to the sample receiving region. Alternatively, an automatic mechanism may be provided as part of the device, which receives the sample on a pad having a predetermined liquid absorbing cspacity, where the pad is moved from a site where the sample is received past a wiping mechanism to the site where the pad serves as a bridge between two regions in the flow path.
The capture region i~ optional but pr~ferred and oss~ntLally prev~nts a predetermined amount of cholesterol from affecting the measurement region, thereby allowLng for a shorter measurem~nt region and reduced wicking time while still expanding the distance traversed for an incremental value of cholestQrol. Th~
capture region will have a reagent which will react with the chole~terol or intermediate product wh$ch re~ult~
from the reactlon of the chole~terol and conversion reaqent and which is present in an amount related to the amount af chole~terol present in the sample. The capture region may completely or partially overlap the sample receiving region, extend upstream from the sample receiving region, or be upstream and separate from the qample receiving region.
The capture region may employ the same reagent as the measurement region, but at a higher density.
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2~3~g74 Generally, the density will be at least 20% greater, usually at least 1.5 fold greater and may be 5 fold or more greater. Usually the capture region will be less than half the area of the measurement region, usually les~ than about one-quarter. The capture region ~hould be large enough to ensure substantially complete capture of the amount of reagent desired.
By providing for a capture region, which is involved with reacting with a component involved with the production of the detectable signal, between the sample receiving element and the mea~uring region, the dynamic range of the assay may be modified. Specifi-cally, because a proportion of the cholesterol or inter-mediate product becomes bound or is reacted chemically to produce a non-interacting compound in the capture region, the amount of reagent non-diffusively bound in the measurement region may be reduced and spread over a greater area giving a lower density per unit area of the reagent in the measurement region. This results in having greater separation per unit of cholesterol, thus allowing for a more sensitive assay. It is found that greater separa~ions can be obtained while stilL obtain-ing a clearly d~tectable borde~. Distribution of th~
reagent such ag a leuco dye on the mea~urement region allow~ the signal generating material, ~uch as H202, to move further, thereby providing additional signal height for a fixed amount of the sample. This provides higher sensitivity and precision in the assay. The capture region can b~ provided with a secondary reagent which reacts with the detectable signal reagent system product, 80 that a predetermined threshold value becomes the zero or low value observed in the measuring region.
The reaction in the capture region may involve specific binding pair member complex formation, chemical reactions involving transformation of reactants into a product, or the like. For example, one may provide for antibodies to the cholesterol, which prevent the 012790 7.
2~3~;74 cholesterol from reacting with the conversion reagents.
Thu~, one can withdraw a predetermined amount of cholesterol from the transport medium or sample prior to reaction, ~o that in order to have a signal in the measurement region, the amount of chole~terol mu~t be greater than the amount that reacts with the capturing region. Alternatively, one can provide for a relatively high den~ity of a component of the detectable signal reagent sy~tem in a relatively ~mall area, 80 that there will be a ~trong band of signal produced in this narrow area, which may be disregarded. Rather than using a member of the detectable signal reagent system, one may u~e a different compound which react~ with a detectable ~ignal reagent component which is Pre~ent in a llmited amount. For example, in the case of hydrogen peroxide as a detectable signal reagent component, various reactants may be present which will react with hydrogen peroxide to produce unreactlve products. Illu~trative reagents include metal ions or ions such as lodide.
The measuring region wlll usually be an extended member, which allows for the flow of the tran~port solution through the measuring element by means of capillary action. The measuring region will have one or more members of the detectable signal rQagent syst~m non-diffu~ively bound to the meaJuring region. The bound reagent reacts, either directly or indirectly, with the product of the cholesterol and conversion reagent to produce a detectable signal, e.g., a colored region with a discernible border. The height or distance of the observable border a8 a result of a detectable slgnal of the detectable 8ignal reagent ~ystem, e.g., dl~tance from the sample receivlng element to the signal front, will be related to the amount of cholesterol ln the sample. Reagents on the measuring region may be uniformly dl~tributed or spread in the form of alternating equal or unequal helght band~ to extend the signal helght for a given sample concentra-tion. Alternatively, appearance of a signal at a 012790 8.
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predetermined area can indicate the presence or ab~ence of cholesterol.
The detectable signal reagent system comprisec a conversion reagent which reacts with the cholesterol to produce a stoichiometric amount of an intermediate product which in turn reacts, directly or indirectly, with another member of the detectable signal reagent system bound to the continuous flow path or strip to produce a detectable, e.g., colored, signal on the path or strip. The detectable signal reagent system may also comprise additional reagents, such as other intermediate components or catalysts needed to produce a detectable signal. The conversion reagent preferably includes reagents which react with chole~terol esters and cholesterol to form hydrogen peroxide. Such reagents are most preferably cholesterol esterase (EC:3.1.1.13) and cholesterol oxidase (EC:1.1.3.6). The serum cholesterol ester is hydrolyzed by the cholesterol esterase, and subsequent oxidation of the cholesterol is accomplished by the cholesterol oxidase to produce a stoichiometric amount of hydrogen peroxide. The hydrogen peroxide can in turn react with a pQroxidas~
substrate on the cont~nuous flow path in the prQsence of horseradish peroxidase to form a colored reqion on the path, which reqion relates to th~ level of c~olesterol in the sample. The cholesterol esterase can be immo-bilized ~t 5 to 50 unit~/ml most prefe!rably at 18 units/ml. The cholesterol oxidase can be immobilized at 10 to 100 unitsfml, most preferably at 50 unit~/ml.
The horseradish peroxidase can be immobilized along the flow path of the as~ay strip at immobilization concen-tr~tions 0.05 to 2.5 mg/ml, usually at 0.5 mg/ml.
Peroxidase may also be included in the transport medium at 0.0005 to 0.050 mg/ml.
By appropriate choice of members of the detectable signal reagent system, visually observable OlZ790 9.
2~3~
color fronts, fluorescent signals, or the like may be obtained for a quantitative assay.
The conversion reagent of the detectable signal reagent system is placed in the continuous flow path in the transport region, the sample receiving pad or the region between the sample receiving and measure-ment regions, preferably in the region between the sample receiving and measurement regions. Upon movement of the transport medium through the path, the conversion reagent is transported into contact with the sample or vice versa depending upon whether the conversion reagent is placed upstream or downstream of the sample receiving region. Any additional reagents of the detectable signal reagent system, other than the bound reagent in lS the measurement region, may be a part of the transport medium or placed in the continuous flow path in the transport region or the region between the sample receiving and measurement regions, preferably in the region between the sample receiving and measurement regions. The transport medium transports these additional reagents throuqh the path.
Other reagents may al~o be present in the assay. For ex~mple, detergent~ find particul~r use in the present a~say to minimize or eliminats the binding of chole~terol to other proteins in the samplQ. Thus, detergents such a8 non-ionic, anionic, or cationic detergenta may be employed. Of particular interest are polyoxyalkylenes, etho~ylated alkylphenols, octyI-pheno~ypolyethoxy-ethanol, octylphenol-ethylene oxide condensates and polyoxyethylene lauryl ether~, or anionic deterqent~, such ag bile acids, e.g., sodium cholate and sodium taurochol~te. In addition, various sticking agents or adhesives may be employed, such as gum arabic. Also of interest will be proteins which are substantially non-interfering, which may include gelatin, caseln, serum albumin, or gamma-globulin~. In addition, the reagent containing regions may include preservatives, such as sucrose, polyvinyl alcohol, poly-012790 10.
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vinyl pyrrolidone, dextran, or sodium azide. Finally, a buffered ~olution will normally be employed for impreg-nating the reagent containing regions. Any convenient buffer may be employed, preferably a substantially dilute buffer, which may include phosphate, TRIS, MOPSO, borate, carbonate, or the like. Usually, the buffered solution will be at a pH in the range of about 4 to 9.
The buffer concentration will generally be from about 10 to 500 mM.
The conversion medium applied to the chip typically will contain about 0.1 to 5 weight percent detergent. In the case of detergent mixtures, the weight of non-ionic detergents may be about 10 to 90 weight percent, usually about 25 to 75 weiqht percent, of the total detergent mixture. The binding agents or adhesives will generally be in the range of about 0.2 to 10 weight percent, more usually about 1 to 5 weight percent, of the medium. A preservative or hydrogen bonding agent may be present in about 1 to 20 weight percent, more usually about 2 to 10 weight percent. The remaining additiues will generally be present in total amount of Iess than about 10 weight percent, more u~ually o le~s than about 5 w~ight percent. The remaining compo~ition may be water, non-reactive ingredien~, excipient~, extender~, and the like.
The as~ay i8 carried out by impregnating a sample receiving regian with the aample. In a preferred embodiment, the ~ample receiving reg~on i8 a pad, which serve~ as a bridge between the other flow path element~
positioned in tandem ~uxtaposition along thelr long axes. Thus the elements define one long flow path, usually comprised of differently sized bibulous strip~
or areas, conveniently with a separation between strips, where the sample receiving element may act as a bridge to allow for fluid flow between strip~.
Blood will be the sample typically assayed for cholesterol. The sample receiving reg$on, therefore, is preferably positioned under a red blood cell removing 012790 11.
2~3~74 filtering device. The blood ~ample will normally be one or a series of small drops, qenerally having a total volume under about 100 ~ m''l, more u~ually about 10-50 ~ m ~l. The layers through which the sample flow~
will usually include a mesh layer, a first membrane, and a second membrane cooperating with the first membrane to ensure the substantially complete removal of any inter-fering cells from the blood sample. The first cellular separation member is used to reduce the concentration of red and white blood cells received by the second filtration member. By lowering the red blood cell content about 10 to 90%, usually about 30 to 90%, of the original red blood cell content with the first - membrane member, the second membrane member is able to efficiently and accurately remove at least substantially all of the red blood cells from the blood sample. Since the first membrane acts as a coarse separation means, the first membrane may take any of a wide variety of forms.
Various packings or sieving depths of filters may be employed, such as glass fibers, cellulose filters treated with red blood cell capture reagents, glass fiber filt~rs, or ~ynthetic fiber filters. Glass fiber filters are available from such manufacturers as Whatman, Schleicher and Schuell, NSI, and~ Pall. The glas~ fiber filters are further characterized by a gla~a fi~er diam~ter in the range of about 0.5-~ ~nmn~, and a density of about 50 to 150 g/m2. The glass fiber filters may be illustrated by S&S Glass 30, Whatm~n GFD, and S&S 3362.
Other coar~e ~eparation membranes may include cellulo~ic membranes, e.g., filter paper, to which red blood cell binding proteins or agglutination agents are immobilized. Such proteins may include lectins, anti-bodies specific for RBC surface membrane proteins,thrombin, ion exchange agent~, etc. The preparation of such filters by con~ugating proteins or other agents to 012790 12.
2 ~ 7 4 cellulose is well known. Cellulose may be activated in a wide variety of ways employing carbodiimide, carbonyl diimidazole, cyanogen bromide, chloroacetic acid, where the acid may then be activated with carbodiimide, or S the like. The literature i8 replete with examples of binding of protein~ to cellulosic membranes for a variety of reasons, which technigues may be employed here. Alternatively, multiple layers of coar~e ~eparation membranes may be employed.
When two membranes are u~ed, the second membrane will preferably be immediately beneath the fir~t membrane and will be in fluid recelving relation-~hip with the fir~t membrane, either in contact with the first membrane or in close proximity thereto.
Generally, the spacing between the first and second membranes will not exceed a distance which inhibits flu$d flow, 80 that fluid readily flows from the first to the second membrane. The non-a~ymmetric membranes which are employed will be those in the medium porosity range, having an average porosity in the range of about 0.65 '~m~' to 7 '~m~', preferably about 1 to 5 ~m~, where the pores may or may not be of substantially uniform diameter through the membrane. By contrast, where an asymmetric membrane (i.e., one wherein the diameter of the pore~ vary from one surfaco to the other) i8 employed, desirably the membrane ~ill ha~e a min~mum porosity not less than about 0.2 ~m~', preferably not le~ than about 0.45 ~m~', and the max~mum porosity will generally not exceed about 40 '~m~', more usually not exceeding about 20 '~m~'.
Illustratlve mlcroporou~ membrane~ which m~y find use include Filterlte pol~ulfone asymmetric, 20 '~m~' -.45 '~m~'; Sartorious cellulose acetate, 1.2 '~m~'; and Nucleopore l.O'~m~'.
The choice of the second membrane 18 lmpor-tant, since the amount of red blood cell ly~is i8 dependent on ~ number of factors. Depending on the size of the pores, the amount of lys~s will greatly vary.
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* Trade mark 2~3~7~.
Since lysis results in release of colored cell compo-nents, which interfere with detection of the border in the measuring strip and may act to decompose detectable signal reagent system components, particu-larly hydrogen peroxide, merely removing cells is insufficient. A further consideration is the pressure differential across the membranes. Again, the appropriate choice of membranes will affect the pressure drop and forces acting on the cell~, which in turn can affect the stability of the cell~.
Thus, the two membranes serve to act together to efficiently and accurately remove red blood cell~
from the blood sample with little, if any, hemolysi~, so as to provide a plasma or serum sample which may be accurately analyzed without interference from hemolytic products, such as heme.
The sample receiving region will be immediately beneath the red blood cell removing membrane~ and in fluid receiving relationship with the membranes. The sample receiving region will normally be a bibulous member able to absorb the fluid. Various bi~ulous material~ may be use~, such a~ cellulosic materials, e.~., p~per, or the like. The sample receiv-ing region will usually be of a size in the range of 5 to 100 mm2 surface area, usually not more than 50 mm2 surface area, and a thickness in the range of about .1 to 2 mm, having a volume capacity of from~about 1 to 75 ~m~'l. When the sample receiving region is a pad, the pad may be round, square, rectangular, quadrila-teral, or polygonal, depending on the manner in which it i8 to be used to act as a bridge for the other members of the flow path. For further charscterization, see copending U.S. patent application Serial No. 195,881, filed May 19, 1988.
The applied sample will be absorbed in the sample receiving region and may extend outside the region, both upstream and downstream, depending upon the size of the region, the nature of the assay, and the 012790 14.
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nature of the upstream and downstream regions. In one embodiment, the sample is prevented from interacting with the ad~acent bibulous members when sample is transferred to the sample receiving region.
various techniques may be employed to prevent transfer of the sample from the sample receiving region (e.g.r a pad) to the other regions prior to flow of the transport medium. Of particular interest is the use of a slide which can be moved from a first position, where the sample receiving region receives the sample, to a second position where the sample receiving region serves as a bridge between the two other regions of the flow path. The slide therefore prevents sample from spread-ing to the other regions of the flow path, before it is lS time to carry out the assay. The path of the sample receiving region, in moving from the site at which the sample is received to the site where it is in the flow path, may provide for means for removing excess sample from the sample receiving region. Such means provides for a quantitative measure of the amount of sample received by the sample receiving region. Thus, by having a region in the path of the slide which is~
narrowed, so as to remOVQ unabsorbed sample me~ium, without significantly squeezing the sample receLving --25~ region, th~ amount of sample ~b~or~ed by the s~mple receiving region can be relatively accurately reproduced. Th~ n~rrowing may be a~ a reault of a convexity, ~uch as a rod in relief, a ro~ler, or any conv~nient scraping means. Ths narrowing of the path should provide a space about equal to or slightly le~
than the wet thickness of the sample receivlng region.
The slide, therefare, not only ~erves to move the sample receiving region, but also to meter the amount of fluid absorbed by the s~mple receiving region.
Besides the slide mechanism, other flow inhibition means may be employed, usually comprising an inert non-porous film, which blocks transfer from the sample receiving element to the bibulous members of the 012790 15.
2 ~ 7 '~
flow path. The amount of ~ample accepted by the sample receiving element and involved in the assay medium may be controlled by providing for transfer of fluid beyond the amount saturating the sample receiving element through a non-wetting screen into an absorbent layer.
After addition of the sample to the ~ample receiving element, and an incubation of up to about 30 minutes, the porous non-wetting material and absorbent layer are removed, leavinq the sample receiving element as the sole repository of sample for the assay. Where a wiping film i~ employed it will be removed upon ~aturation of the sample receiving element. (See U.S. patent application Serial No. 324,407, filed March 16, 1989.) The entire flow path may have a length of about 25 to 200 mm, more usually from about 50 to 150 mm, preferably about 100 mm. About 25 to 90% of the length of the flow path will be the mea~urement region comprising the quantitation zone, optionally a mixing region and/or a threshold value region. The mixing and/or thre~hold value regions will generally be about 5 to 35% of the flow path. The strips which provide for flow of fluid to ~nd from the ~ample receiving ~lement may be o the ~amQ or different length and each will generally bo from about 5 to 25 mm, more usually about 10 to 20~, of the length of the~flow path. The upstream strips may be part of the moasurement region strip, or independent entities. Alternatively, this atrip may be used to control the thre~hold value. The ~ample receiving region will generally be from about 1 to 10%, more usually from about 2 to 8~ of the length of the flow path; the longer the flow path, the larger the sample rec~iving rogion m~y normally be. The width of the strip~ may be varied, u~ually being at least about 2 mm and not more than about 10 mm, preferably about 3 to 7 mm.
Any convenient material may be used for the various bibulous part~ of the a~say ~trips forming the flow path. Usually, the thickness of the bibulou~
012790 16.
~3~7L~:
components will be in the range of about 0.05 to 2.0 mm, more usually 0.15 to 0.75 mm. A wide variety of bibulous supports may be employed, particularly cellulosic qupports, ~uch as chromatography paper, silica on a support, alumina on a support, and polymeric membranes such as nitrocellulose and nylon. The characteristics of the bibulous material employed for the measurement region include the need in many instances to covalently or irreversibly bind an indicator molecule to the support, to develop a clear and sharp color, and to ensure that the fluid is capable of flowing at a convenient rate through the bibulous members.
Of particular interest is an assay device which is self-contained and only requires the sample for carrying out the assay. The device may ~erve as a one-step diagnostic test device using a disposable cassette format. The device may be fabricated of three individual in~ection molded parts into which various components of the assay system are associated. These include the filtration medium designed to separate plasma from whole blood, means for metering a precise sample voLume, a glide to transfer the sample receiving element to the transfer and measurement elements and to --25 release a transport and reaqent solution. The transport solution initiatea capillary migration through the flow path rQsulting in the de~elopment of a detectable boundary related to the amount of analyte in the sample.
Where a reduced amount of the product result-ing from reaction of the cholesterol with an enzyme is present in the measuring region due to capture of such product in the capture region, it w$11 frequently be desirable to have a lower concentration of members of the detectable signal reagent system than would be used in the absence of the capturing region. For example, where hydrogen peroxide is the product in a cholesterol assay, the optimum amount of non-diffusively bound measurement reagent such a~ a substitute for horseradish 012790 17.
~3~
peroxida~e i8 in the range of about 0.25 to 0.50 mg/ml in the dip immobilization solution, while in the present embodiment using a capturinq region the dip concentra-tion of peroxidase substrate may be lowered by 10 to 75%
to a range of 0.05 mg/ml to 0.45 mg/ml.
The sub~ect invention i8 now considered in light of the drawings which depict several preferred embodiments of the pre~ent invention. In a preferred embodiment in Figure 1, a strip 10 is provided which has a ~cored area 12 which includes pad 14 as the sample receiving element for receiving the sample. Pad 14 may be removed from the scored area 12 and dipped in the sample to provide for a semiquantitative measurement of the amount of sample absorbed by the sample receiving -- 15 element 14. After dipping the sample receiving element into the sample, it is then returned to the scored area 12 and firmly fitted into the scored area 80 as to be part of device 10. The region below, i.e., upstream, from the sample receiving region i~ the transport region which is used to wick up the transport medium.
Downstream from the sample receiving element 14 is conversion reagent region 16. In this region, for example, one would have cholesterol e~terase and ~ chole~terol oxidase for reacting with the cholestQrol to -25 produce hydrogen peroxide.
Downstream from the conver~ion reagent region is the capture region, a narrow zone which serves to capture a predetermlned amount of a component of the detectable signal reagent system which is pre~ent in limited amount as a result of being present in an amount related to the amount of cholesterol in the sample. In this sltu~tion, again uJing hydrogen peroxide production as illustrative, one could use a leuco dye which reactR
with hydrogen peroxide in the pre~ence of peroxidase.
Thus, in region 18, one would produce a deep color if cholesterol iB present in excess of a threshold value determined by the amount of dye in region 18. This dye could be spotted and dried at the base of region 20 thus 012790 18.
2a3~7~
eliminating the need for a discrete region and producing a similar step-gradient of reactive dye along the flow path of the as~ay strip. Reqion 20 would have a much lower density of dye throughout the region so as to allow for production of a border, where one can detect the end of the reaction between the hydrogen peroxide and the dye. Desirably a rocket as depicted by broken line 22 is obtained, where one can clearly delineate the top of the rocket from the region immediately upstream from line 22. Desirably, the side edges 24 are perforated, since this appears to provide for a sharper delineation of the border. See U.S. Patent No.
Figure 4 i8 a planar view of an assembled device; and Figures 5-8 are diagrammatic plan views of various embodiments of a laminated measuring strip according to the invention.
Method~ and apparatus are provided for the measurement of cholesterol in a sample employing a continuous flow path, which has a transfer region, sample receiving region, an optional detectable signal reagent capturing region, and a measurement region. The presence of cholesterol and conversion reagent of the detectable signal reagent system results in production of an intermediate product, which directly or indirectly reacts with another member of the detectable signal reagent system which i~ bound to the surface of the measurement region. The result is the production of a detectable signal, generally a colored zone, in the measurement region. The distance of the border of the detectable signal from a predetermined site can be related to the amount of cholesterol in a ~ample.
Various technique~ may be employed for organizing the flow path and providing for flow of a r~agent eolution through the ~mple reglon, the reagent capturing region, and the measurement region. In addition, varioue ch~mistriee may be employed, using different type~ of reagent systeme to produce the de~ired eignal. The assay may be eemiquantltative or quantltative. For quantitative aeeaye, the dletance of the border of the detectable elgnal, l.e., colored border, from ~ predetermlned elte lndicatee the amount of choleeterol ln the sample.
The general design of euch aseaye is set forth ln copendlng U.S. Patent Appllcatlone Serial No. 433,538 filed November 8, 1989, which application i~ a continua-tion-in-part of Serial No. 357,045, filed May 29, 1989, which application i8 a continuation-in-part of Serial 012~90 4.
2 ~ 4 No. 324,407, :Eiled March L6, 1989, which is a continua-tion-in-par~ of Serial No. 195,881l fil.ed May 19, 1988, and Serial No. 064,883, filed June 22, 1987.
The flow path comprises several regions, where certain regions may be overlapping, in whole or in part:
(a) transfer region, ~b~ sample receiving region, (c) optional capture region, and (d) measurement region. The device embodying the flow path may be of any suitable configuration, preferably a strip which has the various regions aligned in the direction of transport medium flow.
The -transfer region receives the transport medium and initiates the flow of the transport medium into the flow path. For the most part, particularly with extended strips, it will be a bibulous short element which serves by capillary action to wick or transport the transport medium to the next region, normally the sample receiving region. For the most part, the -transfer region will be a bibulous strip which absorbs a hydrophilic liquid and allows for transport of any reagents contained in the ~0 transport medium with or without chromatographing the components of the transport medium. Where the device is a circular disc, the transfer region will be at the centre of the disc and allow for transport of the medium radially away from the centre through the other regions.
The sample receiving region serves to receive the sample and to act as a bridge .For t:ransferr.i.ng the transport solutic)n to tl)e nexl reyi.on. :rn c~ne emt)odi.me~nl, prior to the time that the transfer region serves to transport the medi.um to the sample rece:iving element, adjacent regions to the sample recei.ving region will not be in fluid :rece:iving relationshi.p with -the sample receiving region. In that embodiment, after receiving the sample, the sample receiv:ing element is 2~3~7~
then permitted to be a bridging elemsnt which allows for the flow of the transport ~olution through the sample receiving region and into the next region.
The sample receiving region may take a number of forms but may be simply a site on the bibulous strip where the sample i~ placed. Alternatively, it may be a pad in fluid transferring relationship with an underly-ing strip with or without enzyme~s) to enzymetically convert cholesterol to produce hydrogen peroxide.
Variou~ techniques may be employed for providing a measured amount of sample to the sample receiving region. The sample may be measured by any convenient means, such as a micropipet, capillary or the like, touching the measuring device to the sample receiving region. Alternatively, an automatic mechanism may be provided as part of the device, which receives the sample on a pad having a predetermined liquid absorbing cspacity, where the pad is moved from a site where the sample is received past a wiping mechanism to the site where the pad serves as a bridge between two regions in the flow path.
The capture region i~ optional but pr~ferred and oss~ntLally prev~nts a predetermined amount of cholesterol from affecting the measurement region, thereby allowLng for a shorter measurem~nt region and reduced wicking time while still expanding the distance traversed for an incremental value of cholestQrol. Th~
capture region will have a reagent which will react with the chole~terol or intermediate product wh$ch re~ult~
from the reactlon of the chole~terol and conversion reaqent and which is present in an amount related to the amount af chole~terol present in the sample. The capture region may completely or partially overlap the sample receiving region, extend upstream from the sample receiving region, or be upstream and separate from the qample receiving region.
The capture region may employ the same reagent as the measurement region, but at a higher density.
012790 6.
2~3~g74 Generally, the density will be at least 20% greater, usually at least 1.5 fold greater and may be 5 fold or more greater. Usually the capture region will be less than half the area of the measurement region, usually les~ than about one-quarter. The capture region ~hould be large enough to ensure substantially complete capture of the amount of reagent desired.
By providing for a capture region, which is involved with reacting with a component involved with the production of the detectable signal, between the sample receiving element and the mea~uring region, the dynamic range of the assay may be modified. Specifi-cally, because a proportion of the cholesterol or inter-mediate product becomes bound or is reacted chemically to produce a non-interacting compound in the capture region, the amount of reagent non-diffusively bound in the measurement region may be reduced and spread over a greater area giving a lower density per unit area of the reagent in the measurement region. This results in having greater separation per unit of cholesterol, thus allowing for a more sensitive assay. It is found that greater separa~ions can be obtained while stilL obtain-ing a clearly d~tectable borde~. Distribution of th~
reagent such ag a leuco dye on the mea~urement region allow~ the signal generating material, ~uch as H202, to move further, thereby providing additional signal height for a fixed amount of the sample. This provides higher sensitivity and precision in the assay. The capture region can b~ provided with a secondary reagent which reacts with the detectable signal reagent system product, 80 that a predetermined threshold value becomes the zero or low value observed in the measuring region.
The reaction in the capture region may involve specific binding pair member complex formation, chemical reactions involving transformation of reactants into a product, or the like. For example, one may provide for antibodies to the cholesterol, which prevent the 012790 7.
2~3~;74 cholesterol from reacting with the conversion reagents.
Thu~, one can withdraw a predetermined amount of cholesterol from the transport medium or sample prior to reaction, ~o that in order to have a signal in the measurement region, the amount of chole~terol mu~t be greater than the amount that reacts with the capturing region. Alternatively, one can provide for a relatively high den~ity of a component of the detectable signal reagent sy~tem in a relatively ~mall area, 80 that there will be a ~trong band of signal produced in this narrow area, which may be disregarded. Rather than using a member of the detectable signal reagent system, one may u~e a different compound which react~ with a detectable ~ignal reagent component which is Pre~ent in a llmited amount. For example, in the case of hydrogen peroxide as a detectable signal reagent component, various reactants may be present which will react with hydrogen peroxide to produce unreactlve products. Illu~trative reagents include metal ions or ions such as lodide.
The measuring region wlll usually be an extended member, which allows for the flow of the tran~port solution through the measuring element by means of capillary action. The measuring region will have one or more members of the detectable signal rQagent syst~m non-diffu~ively bound to the meaJuring region. The bound reagent reacts, either directly or indirectly, with the product of the cholesterol and conversion reagent to produce a detectable signal, e.g., a colored region with a discernible border. The height or distance of the observable border a8 a result of a detectable slgnal of the detectable 8ignal reagent ~ystem, e.g., dl~tance from the sample receivlng element to the signal front, will be related to the amount of cholesterol ln the sample. Reagents on the measuring region may be uniformly dl~tributed or spread in the form of alternating equal or unequal helght band~ to extend the signal helght for a given sample concentra-tion. Alternatively, appearance of a signal at a 012790 8.
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predetermined area can indicate the presence or ab~ence of cholesterol.
The detectable signal reagent system comprisec a conversion reagent which reacts with the cholesterol to produce a stoichiometric amount of an intermediate product which in turn reacts, directly or indirectly, with another member of the detectable signal reagent system bound to the continuous flow path or strip to produce a detectable, e.g., colored, signal on the path or strip. The detectable signal reagent system may also comprise additional reagents, such as other intermediate components or catalysts needed to produce a detectable signal. The conversion reagent preferably includes reagents which react with chole~terol esters and cholesterol to form hydrogen peroxide. Such reagents are most preferably cholesterol esterase (EC:3.1.1.13) and cholesterol oxidase (EC:1.1.3.6). The serum cholesterol ester is hydrolyzed by the cholesterol esterase, and subsequent oxidation of the cholesterol is accomplished by the cholesterol oxidase to produce a stoichiometric amount of hydrogen peroxide. The hydrogen peroxide can in turn react with a pQroxidas~
substrate on the cont~nuous flow path in the prQsence of horseradish peroxidase to form a colored reqion on the path, which reqion relates to th~ level of c~olesterol in the sample. The cholesterol esterase can be immo-bilized ~t 5 to 50 unit~/ml most prefe!rably at 18 units/ml. The cholesterol oxidase can be immobilized at 10 to 100 unitsfml, most preferably at 50 unit~/ml.
The horseradish peroxidase can be immobilized along the flow path of the as~ay strip at immobilization concen-tr~tions 0.05 to 2.5 mg/ml, usually at 0.5 mg/ml.
Peroxidase may also be included in the transport medium at 0.0005 to 0.050 mg/ml.
By appropriate choice of members of the detectable signal reagent system, visually observable OlZ790 9.
2~3~
color fronts, fluorescent signals, or the like may be obtained for a quantitative assay.
The conversion reagent of the detectable signal reagent system is placed in the continuous flow path in the transport region, the sample receiving pad or the region between the sample receiving and measure-ment regions, preferably in the region between the sample receiving and measurement regions. Upon movement of the transport medium through the path, the conversion reagent is transported into contact with the sample or vice versa depending upon whether the conversion reagent is placed upstream or downstream of the sample receiving region. Any additional reagents of the detectable signal reagent system, other than the bound reagent in lS the measurement region, may be a part of the transport medium or placed in the continuous flow path in the transport region or the region between the sample receiving and measurement regions, preferably in the region between the sample receiving and measurement regions. The transport medium transports these additional reagents throuqh the path.
Other reagents may al~o be present in the assay. For ex~mple, detergent~ find particul~r use in the present a~say to minimize or eliminats the binding of chole~terol to other proteins in the samplQ. Thus, detergents such a8 non-ionic, anionic, or cationic detergenta may be employed. Of particular interest are polyoxyalkylenes, etho~ylated alkylphenols, octyI-pheno~ypolyethoxy-ethanol, octylphenol-ethylene oxide condensates and polyoxyethylene lauryl ether~, or anionic deterqent~, such ag bile acids, e.g., sodium cholate and sodium taurochol~te. In addition, various sticking agents or adhesives may be employed, such as gum arabic. Also of interest will be proteins which are substantially non-interfering, which may include gelatin, caseln, serum albumin, or gamma-globulin~. In addition, the reagent containing regions may include preservatives, such as sucrose, polyvinyl alcohol, poly-012790 10.
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vinyl pyrrolidone, dextran, or sodium azide. Finally, a buffered ~olution will normally be employed for impreg-nating the reagent containing regions. Any convenient buffer may be employed, preferably a substantially dilute buffer, which may include phosphate, TRIS, MOPSO, borate, carbonate, or the like. Usually, the buffered solution will be at a pH in the range of about 4 to 9.
The buffer concentration will generally be from about 10 to 500 mM.
The conversion medium applied to the chip typically will contain about 0.1 to 5 weight percent detergent. In the case of detergent mixtures, the weight of non-ionic detergents may be about 10 to 90 weight percent, usually about 25 to 75 weiqht percent, of the total detergent mixture. The binding agents or adhesives will generally be in the range of about 0.2 to 10 weight percent, more usually about 1 to 5 weight percent, of the medium. A preservative or hydrogen bonding agent may be present in about 1 to 20 weight percent, more usually about 2 to 10 weight percent. The remaining additiues will generally be present in total amount of Iess than about 10 weight percent, more u~ually o le~s than about 5 w~ight percent. The remaining compo~ition may be water, non-reactive ingredien~, excipient~, extender~, and the like.
The as~ay i8 carried out by impregnating a sample receiving regian with the aample. In a preferred embodiment, the ~ample receiving reg~on i8 a pad, which serve~ as a bridge between the other flow path element~
positioned in tandem ~uxtaposition along thelr long axes. Thus the elements define one long flow path, usually comprised of differently sized bibulous strip~
or areas, conveniently with a separation between strips, where the sample receiving element may act as a bridge to allow for fluid flow between strip~.
Blood will be the sample typically assayed for cholesterol. The sample receiving reg$on, therefore, is preferably positioned under a red blood cell removing 012790 11.
2~3~74 filtering device. The blood ~ample will normally be one or a series of small drops, qenerally having a total volume under about 100 ~ m''l, more u~ually about 10-50 ~ m ~l. The layers through which the sample flow~
will usually include a mesh layer, a first membrane, and a second membrane cooperating with the first membrane to ensure the substantially complete removal of any inter-fering cells from the blood sample. The first cellular separation member is used to reduce the concentration of red and white blood cells received by the second filtration member. By lowering the red blood cell content about 10 to 90%, usually about 30 to 90%, of the original red blood cell content with the first - membrane member, the second membrane member is able to efficiently and accurately remove at least substantially all of the red blood cells from the blood sample. Since the first membrane acts as a coarse separation means, the first membrane may take any of a wide variety of forms.
Various packings or sieving depths of filters may be employed, such as glass fibers, cellulose filters treated with red blood cell capture reagents, glass fiber filt~rs, or ~ynthetic fiber filters. Glass fiber filters are available from such manufacturers as Whatman, Schleicher and Schuell, NSI, and~ Pall. The glas~ fiber filters are further characterized by a gla~a fi~er diam~ter in the range of about 0.5-~ ~nmn~, and a density of about 50 to 150 g/m2. The glass fiber filters may be illustrated by S&S Glass 30, Whatm~n GFD, and S&S 3362.
Other coar~e ~eparation membranes may include cellulo~ic membranes, e.g., filter paper, to which red blood cell binding proteins or agglutination agents are immobilized. Such proteins may include lectins, anti-bodies specific for RBC surface membrane proteins,thrombin, ion exchange agent~, etc. The preparation of such filters by con~ugating proteins or other agents to 012790 12.
2 ~ 7 4 cellulose is well known. Cellulose may be activated in a wide variety of ways employing carbodiimide, carbonyl diimidazole, cyanogen bromide, chloroacetic acid, where the acid may then be activated with carbodiimide, or S the like. The literature i8 replete with examples of binding of protein~ to cellulosic membranes for a variety of reasons, which technigues may be employed here. Alternatively, multiple layers of coar~e ~eparation membranes may be employed.
When two membranes are u~ed, the second membrane will preferably be immediately beneath the fir~t membrane and will be in fluid recelving relation-~hip with the fir~t membrane, either in contact with the first membrane or in close proximity thereto.
Generally, the spacing between the first and second membranes will not exceed a distance which inhibits flu$d flow, 80 that fluid readily flows from the first to the second membrane. The non-a~ymmetric membranes which are employed will be those in the medium porosity range, having an average porosity in the range of about 0.65 '~m~' to 7 '~m~', preferably about 1 to 5 ~m~, where the pores may or may not be of substantially uniform diameter through the membrane. By contrast, where an asymmetric membrane (i.e., one wherein the diameter of the pore~ vary from one surfaco to the other) i8 employed, desirably the membrane ~ill ha~e a min~mum porosity not less than about 0.2 ~m~', preferably not le~ than about 0.45 ~m~', and the max~mum porosity will generally not exceed about 40 '~m~', more usually not exceeding about 20 '~m~'.
Illustratlve mlcroporou~ membrane~ which m~y find use include Filterlte pol~ulfone asymmetric, 20 '~m~' -.45 '~m~'; Sartorious cellulose acetate, 1.2 '~m~'; and Nucleopore l.O'~m~'.
The choice of the second membrane 18 lmpor-tant, since the amount of red blood cell ly~is i8 dependent on ~ number of factors. Depending on the size of the pores, the amount of lys~s will greatly vary.
012790 13.
* Trade mark 2~3~7~.
Since lysis results in release of colored cell compo-nents, which interfere with detection of the border in the measuring strip and may act to decompose detectable signal reagent system components, particu-larly hydrogen peroxide, merely removing cells is insufficient. A further consideration is the pressure differential across the membranes. Again, the appropriate choice of membranes will affect the pressure drop and forces acting on the cell~, which in turn can affect the stability of the cell~.
Thus, the two membranes serve to act together to efficiently and accurately remove red blood cell~
from the blood sample with little, if any, hemolysi~, so as to provide a plasma or serum sample which may be accurately analyzed without interference from hemolytic products, such as heme.
The sample receiving region will be immediately beneath the red blood cell removing membrane~ and in fluid receiving relationship with the membranes. The sample receiving region will normally be a bibulous member able to absorb the fluid. Various bi~ulous material~ may be use~, such a~ cellulosic materials, e.~., p~per, or the like. The sample receiv-ing region will usually be of a size in the range of 5 to 100 mm2 surface area, usually not more than 50 mm2 surface area, and a thickness in the range of about .1 to 2 mm, having a volume capacity of from~about 1 to 75 ~m~'l. When the sample receiving region is a pad, the pad may be round, square, rectangular, quadrila-teral, or polygonal, depending on the manner in which it i8 to be used to act as a bridge for the other members of the flow path. For further charscterization, see copending U.S. patent application Serial No. 195,881, filed May 19, 1988.
The applied sample will be absorbed in the sample receiving region and may extend outside the region, both upstream and downstream, depending upon the size of the region, the nature of the assay, and the 012790 14.
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nature of the upstream and downstream regions. In one embodiment, the sample is prevented from interacting with the ad~acent bibulous members when sample is transferred to the sample receiving region.
various techniques may be employed to prevent transfer of the sample from the sample receiving region (e.g.r a pad) to the other regions prior to flow of the transport medium. Of particular interest is the use of a slide which can be moved from a first position, where the sample receiving region receives the sample, to a second position where the sample receiving region serves as a bridge between the two other regions of the flow path. The slide therefore prevents sample from spread-ing to the other regions of the flow path, before it is lS time to carry out the assay. The path of the sample receiving region, in moving from the site at which the sample is received to the site where it is in the flow path, may provide for means for removing excess sample from the sample receiving region. Such means provides for a quantitative measure of the amount of sample received by the sample receiving region. Thus, by having a region in the path of the slide which is~
narrowed, so as to remOVQ unabsorbed sample me~ium, without significantly squeezing the sample receLving --25~ region, th~ amount of sample ~b~or~ed by the s~mple receiving region can be relatively accurately reproduced. Th~ n~rrowing may be a~ a reault of a convexity, ~uch as a rod in relief, a ro~ler, or any conv~nient scraping means. Ths narrowing of the path should provide a space about equal to or slightly le~
than the wet thickness of the sample receivlng region.
The slide, therefare, not only ~erves to move the sample receiving region, but also to meter the amount of fluid absorbed by the s~mple receiving region.
Besides the slide mechanism, other flow inhibition means may be employed, usually comprising an inert non-porous film, which blocks transfer from the sample receiving element to the bibulous members of the 012790 15.
2 ~ 7 '~
flow path. The amount of ~ample accepted by the sample receiving element and involved in the assay medium may be controlled by providing for transfer of fluid beyond the amount saturating the sample receiving element through a non-wetting screen into an absorbent layer.
After addition of the sample to the ~ample receiving element, and an incubation of up to about 30 minutes, the porous non-wetting material and absorbent layer are removed, leavinq the sample receiving element as the sole repository of sample for the assay. Where a wiping film i~ employed it will be removed upon ~aturation of the sample receiving element. (See U.S. patent application Serial No. 324,407, filed March 16, 1989.) The entire flow path may have a length of about 25 to 200 mm, more usually from about 50 to 150 mm, preferably about 100 mm. About 25 to 90% of the length of the flow path will be the mea~urement region comprising the quantitation zone, optionally a mixing region and/or a threshold value region. The mixing and/or thre~hold value regions will generally be about 5 to 35% of the flow path. The strips which provide for flow of fluid to ~nd from the ~ample receiving ~lement may be o the ~amQ or different length and each will generally bo from about 5 to 25 mm, more usually about 10 to 20~, of the length of the~flow path. The upstream strips may be part of the moasurement region strip, or independent entities. Alternatively, this atrip may be used to control the thre~hold value. The ~ample receiving region will generally be from about 1 to 10%, more usually from about 2 to 8~ of the length of the flow path; the longer the flow path, the larger the sample rec~iving rogion m~y normally be. The width of the strip~ may be varied, u~ually being at least about 2 mm and not more than about 10 mm, preferably about 3 to 7 mm.
Any convenient material may be used for the various bibulous part~ of the a~say ~trips forming the flow path. Usually, the thickness of the bibulou~
012790 16.
~3~7L~:
components will be in the range of about 0.05 to 2.0 mm, more usually 0.15 to 0.75 mm. A wide variety of bibulous supports may be employed, particularly cellulosic qupports, ~uch as chromatography paper, silica on a support, alumina on a support, and polymeric membranes such as nitrocellulose and nylon. The characteristics of the bibulous material employed for the measurement region include the need in many instances to covalently or irreversibly bind an indicator molecule to the support, to develop a clear and sharp color, and to ensure that the fluid is capable of flowing at a convenient rate through the bibulous members.
Of particular interest is an assay device which is self-contained and only requires the sample for carrying out the assay. The device may ~erve as a one-step diagnostic test device using a disposable cassette format. The device may be fabricated of three individual in~ection molded parts into which various components of the assay system are associated. These include the filtration medium designed to separate plasma from whole blood, means for metering a precise sample voLume, a glide to transfer the sample receiving element to the transfer and measurement elements and to --25 release a transport and reaqent solution. The transport solution initiatea capillary migration through the flow path rQsulting in the de~elopment of a detectable boundary related to the amount of analyte in the sample.
Where a reduced amount of the product result-ing from reaction of the cholesterol with an enzyme is present in the measuring region due to capture of such product in the capture region, it w$11 frequently be desirable to have a lower concentration of members of the detectable signal reagent system than would be used in the absence of the capturing region. For example, where hydrogen peroxide is the product in a cholesterol assay, the optimum amount of non-diffusively bound measurement reagent such a~ a substitute for horseradish 012790 17.
~3~
peroxida~e i8 in the range of about 0.25 to 0.50 mg/ml in the dip immobilization solution, while in the present embodiment using a capturinq region the dip concentra-tion of peroxidase substrate may be lowered by 10 to 75%
to a range of 0.05 mg/ml to 0.45 mg/ml.
The sub~ect invention i8 now considered in light of the drawings which depict several preferred embodiments of the pre~ent invention. In a preferred embodiment in Figure 1, a strip 10 is provided which has a ~cored area 12 which includes pad 14 as the sample receiving element for receiving the sample. Pad 14 may be removed from the scored area 12 and dipped in the sample to provide for a semiquantitative measurement of the amount of sample absorbed by the sample receiving -- 15 element 14. After dipping the sample receiving element into the sample, it is then returned to the scored area 12 and firmly fitted into the scored area 80 as to be part of device 10. The region below, i.e., upstream, from the sample receiving region i~ the transport region which is used to wick up the transport medium.
Downstream from the sample receiving element 14 is conversion reagent region 16. In this region, for example, one would have cholesterol e~terase and ~ chole~terol oxidase for reacting with the cholestQrol to -25 produce hydrogen peroxide.
Downstream from the conver~ion reagent region is the capture region, a narrow zone which serves to capture a predetermlned amount of a component of the detectable signal reagent system which is pre~ent in limited amount as a result of being present in an amount related to the amount of cholesterol in the sample. In this sltu~tion, again uJing hydrogen peroxide production as illustrative, one could use a leuco dye which reactR
with hydrogen peroxide in the pre~ence of peroxidase.
Thus, in region 18, one would produce a deep color if cholesterol iB present in excess of a threshold value determined by the amount of dye in region 18. This dye could be spotted and dried at the base of region 20 thus 012790 18.
2a3~7~
eliminating the need for a discrete region and producing a similar step-gradient of reactive dye along the flow path of the as~ay strip. Reqion 20 would have a much lower density of dye throughout the region so as to allow for production of a border, where one can detect the end of the reaction between the hydrogen peroxide and the dye. Desirably a rocket as depicted by broken line 22 is obtained, where one can clearly delineate the top of the rocket from the region immediately upstream from line 22. Desirably, the side edges 24 are perforated, since this appears to provide for a sharper delineation of the border. See U.S. Patent No.
4,757,004.
` A more sophisticated device i8 shown in Figures 2 to 4. The invention may be fabricated from three in~ection molded parts or by any other convenient process. The parts comprise a base plate 40, a slide 42 and a cover plate 44, as shown in Pig. 2 and 3. The base plate 40 consists of a cutout to accept the glide 42, a slot 46 with locating pins 48 into which the quantitation strip 50 and bibulous strip 52 are precisely positioned, maintaining about a 2 mm gap 54 between them, and a well 5* de~igned to capture the J released tran~port ~olution, e.g., wicking buffer.
Th~ slide 42 consist~ of a vented receptor site 58 into which the sample receiving pad 59 is inserted, an ~nm 60 with she~ring action designed~to facilitate the relqase of the transport soLution from a pouch which i~ housed in well 62 of cover plate 44, and a snap 61 to lock the slide in place, once pulled. The sample receiving pad may have antibodie~ which react with the chole~terol to prevent the chole~terol from reacting with detectable signal reagent system compo-nents. Thus, for example, a predetermined amount of cholesterol can be inhibited from reacting with enzyme to produce hydrogen peroxide.
The cover plate 44 consi~ts of a well 62, which houses a sealed foil pouch (not shown) containing 012790 19.
2 ~ 7 4 the transport ~olution or the well 62 may be filled with transport ~olution and covered with a peelable foil seal. The cover plate has an orifice 64 for the introduction of the sample. Underneath orifice 64 are filters 66, for separating cells from blood sample~. The filtration system may comprise dual glass fiber disks and a final filtration membrane in order to deliver cell free plasma to the ~ample receiving element. The cover plate also comprises the squeegee metering bar 68, which serves to control the volume of ~ample absorbed by the sample receiving element, as well as a viewing slot 70.
At the top of the viewing slot 70 is an indicator hole 72, which changes color when the test is complete to ! inform the user that a reading may be taken.
- 15 The final assembly i~ depicted in Fig. 4, where the assembled device i~ obtained by introducing the slide 42 into base plate 40, positioning transfer strip 52 and measurement ~trip 50 at their appropriate sites, introducing the transport solution pouch into well 62 or as indicated above, filling well 62 with transport solution and sealing with a Poly-foil seal, assemhling the cover plAte and base plate and then sealing, conuRniQntLy by sonic welding, the base plate ~~ and the CQver plate. This procedure locates the sample i25 rece$ving ~ite of the slide directly beneath the filtra-tLon media of the cover plate, as well as locating the ~hearing points o~ ~he slide beneath the foiLed sQaled pouch located in the cover plate.
In order to carry out a cholQsterol mQasure-ment, the user lances a finger and applies a hanging drop of blood to the applicatLon site, which is a white central wQll with;a red or black border. When the white center is no longer vi~ible, a sufficient amount of blood ha~ been applied. A fill to line may also be included which is located about 0.5 to lmm above the filters around the bottom of the sample well. When the ~ample covers the line, a sufficient amount of blood has been applied. The user then waits about 30 seconds to 2 0127gO 20.
2~3~
minutes or more to allow adequate filtration and recovery of plasma onto the sample receiving pad.
The slide is then pulled until it snaps into place. At this point the sample receiving pad contain-ing the plasma sample has been metered by the squeegee metering bar and is brought into contact and fluid transferring relationship pith the transfer region and the measurement region or the reagent region bridging under the two mm gap. The shearing points of the slide have also pierced the foil seal of the pouch in the well of the cover plate, releasing the transport solution into the receiving well in the base plate. The transport ~olution iq 0.10 to 2 ml of an aqueous buffer f which may contain horseradish peroxidase in sufficient - 15 amount to insure rapid reaction of the hydrogen peroxide and dye or the horseradish peroxidase may be present diffusibly bound on the strip either upstream or down-stream of the ~ample receiving region. Upon contact of the strip with the transport solution, the strip assembly begins wicking up the transport solution which washes the cholesterol esterase and cholesterol oxidase enzyme~ into the sampl~, or-~ice versa depending-upon the re~ative placement of enzymea and aample, and -~ further washe~ the hydrogen peroxide reaction product of -'25 th~ enzym~a snd chole~terol, as well a~ the hor~eradish peroxidase, into the measurement region of the strip.
The measurement region ia impregnated with a peroxidase substrste, particularly a modified N~,N-dimethylaniline. (See U.S. patent application Serial No. 195,881, filed May 19, 1988.) The reaction of the reagents results in a colored region with a defined boundary, thereby giving the user a preci~e reading of the cholesterol level above a threshold level. This reading is made when the color indicator site above the viewing slot shows the test is complete. Normally, it will take fewer than about 15 minutes for the assay to be complete, reading the peak of a colored area in the viewing slot.
012790 21.
2 ~ 7 '~
The following examples are offered by way of illu~tration and not by way of limitation.
EXPERIMENTAL
This example illustrates the separation between cholesterol levels that can be obtained through placement of the cholesterol conversion reaqent on the strip in the transfer, in the region between the sample receiving and measurement regions on the sample receiv-ing or in the transport solution.
In carrying out the assay for cholesterol, a ~ device was prepared having an overall length of about ~ 15 95mm; 12mm of which was the transfer region, about 7 mm was for the sample receiving pad, and the remainder was the measurement region. The strip was made of Whatman 31ET paper, where a modified N,N-dimethyIaniline was covalently linked to the paper with MBTH being immobilized passively in the measurement region. This strip configuration was used when the cholesterol con-version roagent~ are on the ~ample receiving pAd, th~
tran~fer region or in the transport ~olution. When the -~~ conversion re~gent~ are position~d between the sample recei~}ng and measurement regions, an add$t$onal strip area is included directly upstream from the sample rQcQivi~g area. Thi~ ~trip area ia 7mm long as seen in Fig. 8 and i8 called the conversion pad.
The conver~ion solution comprised 5.33 g sodium phosphate dibasic, 1.25 g sucrose, 1 ml Nonidet P-40, 1.0 g cholic acid, 0.83 g Mega-8, 0.71 g ~odium pota~ium tartrate, 0.65 g sodium nitroprus~ide, O.S77 g sodium stannate, and 0.01 g sodium azide, after the addition of which the solution was ad~u~ted to pH 7.0, and 1800 units of cholesterol esterase and 5000 units of cholesterol oxidase added thereto, and the volume brought to 100 ml. The strip was wetted with this solution in the conver~ion region and dried. Each test 012790 22.
2~3~6~'~
contains about 0.18 units cholesterol esterase and 0.60 units of cholesterol oxida~e.
The wicking buffer or transport solution was comprised of O.05 M sodium phosphate, 2 mg/ml BGG, S 0.005 mg/ml HRP, 0.01% gentamycin, and 0.01% Nonidet P-40.
The resulting device was tested u~ing three calibrators at different levels provided by the College of American Pathologists (CAP), with chole~terol levels at 137, 234 and 333 mg/dl which use lyophilized human serum. Calibrators were reconstituted fresh each day.
Four modifications of the above device were tested for separation (in mm) between the tri-level CA*
calibrators. The device modifications involved the location of the conversion reagents: (A) ~ample receiving region, (~) transport solution, (C) transfer region, and (D) region between the sample receiving and measurement regions.
In carrying out the assay using the above four device configurations, a 10 '"m"'l serum sample was placed on the sample receiving pad, and the tran~port solution allowed to wick up the strip. The cholesterol react~ w~th the con~ersiQn reagents, specifically the ~~ chole~terol esterase and cholesterol oxidase to form -25 hydrogen perox~d~, which ln turn reacts with the MBT~
in the presQnce of the horseradl~h peroxidase solution to produce a blue color. After the wicking was com-plete, the color front height was measured, which coIor front height i~ directly correlatable with the serum cholesterol level.
Wlth the above modified designs, separatlon impro~ed to provlde from 6.6 to 8.7 mm between each 100 mg/dl change. Thi~ compared with a 4.5 to 7.2 mm separation between every 100 mg/dl change using the configuration in which the conversion reagents were placed in the sample receiving region. Overall, there was observed a 27.5% increase in separation over the full assay range of about 200 mg/dl with the configura-012790 23.
2~3~67 L'J~
tion~ in which the conversion reagent wa~ placed in the transfer region or in the region between the sample receiving and measurement regions, as compared to the configuration in which the conversion reagent wa~ placed S in the ~ample receiving region. The following table summarizes the results.
,f, .
012790 24.
2o3rj~7 n m ~ n . . . . o _ ~ ~ ~ ~3 ~ cn ~D rt 11 ~--O
o o ~-o ~ ~ o U~ ~- tD
X ~:~
~S ~ ~ ~ O1-- ~D
~- ~ tD ~
n ~ ~ ~o ~ ,t 0~ ~ ~D o o ~ ~
~.
D n~
t1 ~ ~ ~~ l- o O~ I ~ ~
o o V~ ~ ~
~n 1l ~
,~. ~ ~ ~W ~ o t~
~J ~ ~ ~P ~ l--Il . . .. ~
p ~ O O 3 ~0 ~q ~P
U~ ~ ~ ~_ ~D . .. .
Ul ~ ,_ CO ~ ~ ~ ~ ~
~ I ~
~ ~ ~ ~D
I ~
,~ ~ ~ ~.
W ~ o ~o ~0 ~ ~ 3 `~7~
This example illustrates the separation between cholesterol levels that can be obtained through S placement of an additional reagent of the detectable signal reagent system, the horseradish peroxidase which participates in the reaction of the hydrogen peroxide intermediate product with the peroxidase substrate to produce a detectable signal, on the strip in the transfer region or in the region between the sample receiving and measurement regions, as compared to in the transport solution. This example illustrates that the results vary depending on the location of the peroxidase enzyme.
~_ 15 In carrying out the assay for cholesterol, the same device and general protocol was used as in Example 1 and illustrated in Fig. 8. The cholesterol conversion reagents were placed on the strip in the sample receiving region.
Three modifications of the device were tested for separation (in mm) between the tri-level CAP
calibratars~ The design modifications involved the location of the horseradish peroxidase: (A) transport --~ solution (cantrol), (Bj transfer region, and (C) region '25 between the ~ample recei~ing and~mea~ur~ment regions.
With the above modified design, curve separation ~etween the 137 ~nd 333 mg/dl CAP calibrators was 12.0mm when the peroxidase is in the transport solution; 23.3mm when the peroxidase is in the region between the sample receiving and measurement regions.
This compared with a 4.4 to 7.5 mm, ~eparation between every 100 mg/dl change using the configuration Ln which the horseradish peroxidase was placed in the transport solution. Overall, there was observed 95.8% and 41.2%
increases in separation, respectively, over the full assay range of about 200 mg~dl with the configuration in which the horseradish peroxidase was placed in the transfer region or in the region between the ~ample 012790 26.
2~3~7~1 receiving and measurement region~, as compared to the configuration in which the horseradish peroxidase was placed in the transport solution. The following table summarizes the results.
o 012790 27.
2 ~ 7 ~ n ~ ~
o . . . ~ 8 ~ c ~
u~ O D~ U~ O
1~ ~ -O 1'- 0Ul r~ tn O
O ~ C ~ ~ ~ O
~ ~ t ~
D~ ~ ~
~ ~D
,.. r~
O ~~s ~ w ~ 1--o ~D
~s o ~J
~ o o o ~ Ul~ ~P.
u~ ~ cn ~ I--~ o o ~n ~_ ~~ . ~ ~
I
U~
. . .
O O ~ ~~~
1~ ~ 1- ~
O~ ~ ~ ~0 . . . W ~ ~
co ~ o 3 2~3~
It is ev-ident :frorn the above resul-ts that a number o:f substan-t1al advantages accrue wi.th proper placement of the cholestero]. conversion reagent and horseradish peroxidase.
By providing for placement of the choles-terol conversion reagent and/or horseradish pero~idase on the strip in -the -transfer region or in the region be-tween the sample receiving and measurement regions, the di.stance traversed for an incremental value of cholesterol is increased.
Thus, higher sensitivity is achieved, while beneficial 1n performance characteristics are obtained.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skil]. in the art in light of the teachings of this invention that certain changes and modifications may he made thereto without departing from the spirit or scope of -the appended claims.
` A more sophisticated device i8 shown in Figures 2 to 4. The invention may be fabricated from three in~ection molded parts or by any other convenient process. The parts comprise a base plate 40, a slide 42 and a cover plate 44, as shown in Pig. 2 and 3. The base plate 40 consists of a cutout to accept the glide 42, a slot 46 with locating pins 48 into which the quantitation strip 50 and bibulous strip 52 are precisely positioned, maintaining about a 2 mm gap 54 between them, and a well 5* de~igned to capture the J released tran~port ~olution, e.g., wicking buffer.
Th~ slide 42 consist~ of a vented receptor site 58 into which the sample receiving pad 59 is inserted, an ~nm 60 with she~ring action designed~to facilitate the relqase of the transport soLution from a pouch which i~ housed in well 62 of cover plate 44, and a snap 61 to lock the slide in place, once pulled. The sample receiving pad may have antibodie~ which react with the chole~terol to prevent the chole~terol from reacting with detectable signal reagent system compo-nents. Thus, for example, a predetermined amount of cholesterol can be inhibited from reacting with enzyme to produce hydrogen peroxide.
The cover plate 44 consi~ts of a well 62, which houses a sealed foil pouch (not shown) containing 012790 19.
2 ~ 7 4 the transport ~olution or the well 62 may be filled with transport ~olution and covered with a peelable foil seal. The cover plate has an orifice 64 for the introduction of the sample. Underneath orifice 64 are filters 66, for separating cells from blood sample~. The filtration system may comprise dual glass fiber disks and a final filtration membrane in order to deliver cell free plasma to the ~ample receiving element. The cover plate also comprises the squeegee metering bar 68, which serves to control the volume of ~ample absorbed by the sample receiving element, as well as a viewing slot 70.
At the top of the viewing slot 70 is an indicator hole 72, which changes color when the test is complete to ! inform the user that a reading may be taken.
- 15 The final assembly i~ depicted in Fig. 4, where the assembled device i~ obtained by introducing the slide 42 into base plate 40, positioning transfer strip 52 and measurement ~trip 50 at their appropriate sites, introducing the transport solution pouch into well 62 or as indicated above, filling well 62 with transport solution and sealing with a Poly-foil seal, assemhling the cover plAte and base plate and then sealing, conuRniQntLy by sonic welding, the base plate ~~ and the CQver plate. This procedure locates the sample i25 rece$ving ~ite of the slide directly beneath the filtra-tLon media of the cover plate, as well as locating the ~hearing points o~ ~he slide beneath the foiLed sQaled pouch located in the cover plate.
In order to carry out a cholQsterol mQasure-ment, the user lances a finger and applies a hanging drop of blood to the applicatLon site, which is a white central wQll with;a red or black border. When the white center is no longer vi~ible, a sufficient amount of blood ha~ been applied. A fill to line may also be included which is located about 0.5 to lmm above the filters around the bottom of the sample well. When the ~ample covers the line, a sufficient amount of blood has been applied. The user then waits about 30 seconds to 2 0127gO 20.
2~3~
minutes or more to allow adequate filtration and recovery of plasma onto the sample receiving pad.
The slide is then pulled until it snaps into place. At this point the sample receiving pad contain-ing the plasma sample has been metered by the squeegee metering bar and is brought into contact and fluid transferring relationship pith the transfer region and the measurement region or the reagent region bridging under the two mm gap. The shearing points of the slide have also pierced the foil seal of the pouch in the well of the cover plate, releasing the transport solution into the receiving well in the base plate. The transport ~olution iq 0.10 to 2 ml of an aqueous buffer f which may contain horseradish peroxidase in sufficient - 15 amount to insure rapid reaction of the hydrogen peroxide and dye or the horseradish peroxidase may be present diffusibly bound on the strip either upstream or down-stream of the ~ample receiving region. Upon contact of the strip with the transport solution, the strip assembly begins wicking up the transport solution which washes the cholesterol esterase and cholesterol oxidase enzyme~ into the sampl~, or-~ice versa depending-upon the re~ative placement of enzymea and aample, and -~ further washe~ the hydrogen peroxide reaction product of -'25 th~ enzym~a snd chole~terol, as well a~ the hor~eradish peroxidase, into the measurement region of the strip.
The measurement region ia impregnated with a peroxidase substrste, particularly a modified N~,N-dimethylaniline. (See U.S. patent application Serial No. 195,881, filed May 19, 1988.) The reaction of the reagents results in a colored region with a defined boundary, thereby giving the user a preci~e reading of the cholesterol level above a threshold level. This reading is made when the color indicator site above the viewing slot shows the test is complete. Normally, it will take fewer than about 15 minutes for the assay to be complete, reading the peak of a colored area in the viewing slot.
012790 21.
2 ~ 7 '~
The following examples are offered by way of illu~tration and not by way of limitation.
EXPERIMENTAL
This example illustrates the separation between cholesterol levels that can be obtained through placement of the cholesterol conversion reaqent on the strip in the transfer, in the region between the sample receiving and measurement regions on the sample receiv-ing or in the transport solution.
In carrying out the assay for cholesterol, a ~ device was prepared having an overall length of about ~ 15 95mm; 12mm of which was the transfer region, about 7 mm was for the sample receiving pad, and the remainder was the measurement region. The strip was made of Whatman 31ET paper, where a modified N,N-dimethyIaniline was covalently linked to the paper with MBTH being immobilized passively in the measurement region. This strip configuration was used when the cholesterol con-version roagent~ are on the ~ample receiving pAd, th~
tran~fer region or in the transport ~olution. When the -~~ conversion re~gent~ are position~d between the sample recei~}ng and measurement regions, an add$t$onal strip area is included directly upstream from the sample rQcQivi~g area. Thi~ ~trip area ia 7mm long as seen in Fig. 8 and i8 called the conversion pad.
The conver~ion solution comprised 5.33 g sodium phosphate dibasic, 1.25 g sucrose, 1 ml Nonidet P-40, 1.0 g cholic acid, 0.83 g Mega-8, 0.71 g ~odium pota~ium tartrate, 0.65 g sodium nitroprus~ide, O.S77 g sodium stannate, and 0.01 g sodium azide, after the addition of which the solution was ad~u~ted to pH 7.0, and 1800 units of cholesterol esterase and 5000 units of cholesterol oxidase added thereto, and the volume brought to 100 ml. The strip was wetted with this solution in the conver~ion region and dried. Each test 012790 22.
2~3~6~'~
contains about 0.18 units cholesterol esterase and 0.60 units of cholesterol oxida~e.
The wicking buffer or transport solution was comprised of O.05 M sodium phosphate, 2 mg/ml BGG, S 0.005 mg/ml HRP, 0.01% gentamycin, and 0.01% Nonidet P-40.
The resulting device was tested u~ing three calibrators at different levels provided by the College of American Pathologists (CAP), with chole~terol levels at 137, 234 and 333 mg/dl which use lyophilized human serum. Calibrators were reconstituted fresh each day.
Four modifications of the above device were tested for separation (in mm) between the tri-level CA*
calibrators. The device modifications involved the location of the conversion reagents: (A) ~ample receiving region, (~) transport solution, (C) transfer region, and (D) region between the sample receiving and measurement regions.
In carrying out the assay using the above four device configurations, a 10 '"m"'l serum sample was placed on the sample receiving pad, and the tran~port solution allowed to wick up the strip. The cholesterol react~ w~th the con~ersiQn reagents, specifically the ~~ chole~terol esterase and cholesterol oxidase to form -25 hydrogen perox~d~, which ln turn reacts with the MBT~
in the presQnce of the horseradl~h peroxidase solution to produce a blue color. After the wicking was com-plete, the color front height was measured, which coIor front height i~ directly correlatable with the serum cholesterol level.
Wlth the above modified designs, separatlon impro~ed to provlde from 6.6 to 8.7 mm between each 100 mg/dl change. Thi~ compared with a 4.5 to 7.2 mm separation between every 100 mg/dl change using the configuration in which the conversion reagents were placed in the sample receiving region. Overall, there was observed a 27.5% increase in separation over the full assay range of about 200 mg/dl with the configura-012790 23.
2~3~67 L'J~
tion~ in which the conversion reagent wa~ placed in the transfer region or in the region between the sample receiving and measurement regions, as compared to the configuration in which the conversion reagent wa~ placed S in the ~ample receiving region. The following table summarizes the results.
,f, .
012790 24.
2o3rj~7 n m ~ n . . . . o _ ~ ~ ~ ~3 ~ cn ~D rt 11 ~--O
o o ~-o ~ ~ o U~ ~- tD
X ~:~
~S ~ ~ ~ O1-- ~D
~- ~ tD ~
n ~ ~ ~o ~ ,t 0~ ~ ~D o o ~ ~
~.
D n~
t1 ~ ~ ~~ l- o O~ I ~ ~
o o V~ ~ ~
~n 1l ~
,~. ~ ~ ~W ~ o t~
~J ~ ~ ~P ~ l--Il . . .. ~
p ~ O O 3 ~0 ~q ~P
U~ ~ ~ ~_ ~D . .. .
Ul ~ ,_ CO ~ ~ ~ ~ ~
~ I ~
~ ~ ~ ~D
I ~
,~ ~ ~ ~.
W ~ o ~o ~0 ~ ~ 3 `~7~
This example illustrates the separation between cholesterol levels that can be obtained through S placement of an additional reagent of the detectable signal reagent system, the horseradish peroxidase which participates in the reaction of the hydrogen peroxide intermediate product with the peroxidase substrate to produce a detectable signal, on the strip in the transfer region or in the region between the sample receiving and measurement regions, as compared to in the transport solution. This example illustrates that the results vary depending on the location of the peroxidase enzyme.
~_ 15 In carrying out the assay for cholesterol, the same device and general protocol was used as in Example 1 and illustrated in Fig. 8. The cholesterol conversion reagents were placed on the strip in the sample receiving region.
Three modifications of the device were tested for separation (in mm) between the tri-level CAP
calibratars~ The design modifications involved the location of the horseradish peroxidase: (A) transport --~ solution (cantrol), (Bj transfer region, and (C) region '25 between the ~ample recei~ing and~mea~ur~ment regions.
With the above modified design, curve separation ~etween the 137 ~nd 333 mg/dl CAP calibrators was 12.0mm when the peroxidase is in the transport solution; 23.3mm when the peroxidase is in the region between the sample receiving and measurement regions.
This compared with a 4.4 to 7.5 mm, ~eparation between every 100 mg/dl change using the configuration Ln which the horseradish peroxidase was placed in the transport solution. Overall, there was observed 95.8% and 41.2%
increases in separation, respectively, over the full assay range of about 200 mg~dl with the configuration in which the horseradish peroxidase was placed in the transfer region or in the region between the ~ample 012790 26.
2~3~7~1 receiving and measurement region~, as compared to the configuration in which the horseradish peroxidase was placed in the transport solution. The following table summarizes the results.
o 012790 27.
2 ~ 7 ~ n ~ ~
o . . . ~ 8 ~ c ~
u~ O D~ U~ O
1~ ~ -O 1'- 0Ul r~ tn O
O ~ C ~ ~ ~ O
~ ~ t ~
D~ ~ ~
~ ~D
,.. r~
O ~~s ~ w ~ 1--o ~D
~s o ~J
~ o o o ~ Ul~ ~P.
u~ ~ cn ~ I--~ o o ~n ~_ ~~ . ~ ~
I
U~
. . .
O O ~ ~~~
1~ ~ 1- ~
O~ ~ ~ ~0 . . . W ~ ~
co ~ o 3 2~3~
It is ev-ident :frorn the above resul-ts that a number o:f substan-t1al advantages accrue wi.th proper placement of the cholestero]. conversion reagent and horseradish peroxidase.
By providing for placement of the choles-terol conversion reagent and/or horseradish pero~idase on the strip in -the -transfer region or in the region be-tween the sample receiving and measurement regions, the di.stance traversed for an incremental value of cholesterol is increased.
Thus, higher sensitivity is achieved, while beneficial 1n performance characteristics are obtained.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skil]. in the art in light of the teachings of this invention that certain changes and modifications may he made thereto without departing from the spirit or scope of -the appended claims.
Claims (12)
1. A device for determining the cholesterol level in a sample, said device comprising:
(a) a first bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, (b) a bibulous member comprising a sample receiving region;
(c) a second bibulous strip comprising a measurement region;
wherein said sample receiving region is in fluid communication with said transport and measurement regions, or can be moved into fluid transfer relationship with said transport and measurement regions; with the proviso that when said sample receiving region is moved into fluid transfer relation-ship with said transport and measurement regions, said device further comprises means for moving said sample receiving region from a first site for receiving said sample to fluid transfer relationship with said transport and measurement regions;
(d) conversion reagent capable of reacting with cholesterol and cholesterol ester to form an intermediate product present on said strip in the transfer region or a region between the sample receiving and measurement regions such that movement of the transport medium through said strip will bring the conversion reagent and cholesterol together to react to form said intermediate product; and (e) reagent non-diffusively bound in the measurement region which reacts in the presence of said intermediate product to produce a detectable border related to the cholesterol level in said sample.
(a) a first bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, (b) a bibulous member comprising a sample receiving region;
(c) a second bibulous strip comprising a measurement region;
wherein said sample receiving region is in fluid communication with said transport and measurement regions, or can be moved into fluid transfer relationship with said transport and measurement regions; with the proviso that when said sample receiving region is moved into fluid transfer relation-ship with said transport and measurement regions, said device further comprises means for moving said sample receiving region from a first site for receiving said sample to fluid transfer relationship with said transport and measurement regions;
(d) conversion reagent capable of reacting with cholesterol and cholesterol ester to form an intermediate product present on said strip in the transfer region or a region between the sample receiving and measurement regions such that movement of the transport medium through said strip will bring the conversion reagent and cholesterol together to react to form said intermediate product; and (e) reagent non-diffusively bound in the measurement region which reacts in the presence of said intermediate product to produce a detectable border related to the cholesterol level in said sample.
2. The device of Claim 1, wherein said conversion reagent comprises cholesterol esterase and cholesterol oxidase and said intermediate product is hydrogen peroxide.
012790 30.
012790 30.
3. The device of Claim 1, wherein horse-radish peroxidase is present on the strip downstream from said measurement region.
4. The device of Claim 1, wherein said conversion reagent is in a region between the sample receiving and measurement regions, when said sample receiving region is in fluid receiving relationship with said transport and measurement regions.
5. The device of Claim 1, wherein said device further comprises a capture region on or upstream from the sample region and the measurement region, which capture region diminishes a predetermined portion of reaction of the bound reagent in the measurement region.
6. The device of Claim 1, wherein said peroxidase substrate is a leuco dye.
7. The device of Claim 1, wherein said device is a single bibulous strip.
8. The device of Claim 1, wherein said device comprises a single bibulous strip comprising the transport region and the measurement region separate from said sample receiving region.
9. A device for determining the cholesterol level in a sample, said device comprising:
(a) a first bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, (b) a bibulous member comprising a sample receiving region;
(c) a second bibulous strip comprising a measurement region;
wherein said sample receiving region is in fluid communication with said transport and measurement regions, or can be moved into fluid transfer relationship with said transport and measurement regions; with the proviso that when said sample receiv-ing region is moved into fluid transfer relationship with said transport and measurement regions, said device further comprises means for moving said sample receiving 012790 31.
region from a first site for receiving said sample to fluid transfer relationship with said transport and measurement regions;
(d) conversion reagent comprising cholesterol esterase and cholesterol oxidase capable of reacting with cholesterol and cholesterol ester to form an intermediate product present on said strip in a region between the sample receiving and measurement regions such that movement of the transport medium through said strip will bring the conversion reagent and cholesterol together to react to form said intermediate product; and (e) reagent non-diffusively bound in the measurement region which reacts in the presence of said intermediate product to produce a detectable border related to the cholesterol level in said sample.
(a) a first bibulous strip comprising a transfer region for transporting a transport medium from a transport medium source, (b) a bibulous member comprising a sample receiving region;
(c) a second bibulous strip comprising a measurement region;
wherein said sample receiving region is in fluid communication with said transport and measurement regions, or can be moved into fluid transfer relationship with said transport and measurement regions; with the proviso that when said sample receiv-ing region is moved into fluid transfer relationship with said transport and measurement regions, said device further comprises means for moving said sample receiving 012790 31.
region from a first site for receiving said sample to fluid transfer relationship with said transport and measurement regions;
(d) conversion reagent comprising cholesterol esterase and cholesterol oxidase capable of reacting with cholesterol and cholesterol ester to form an intermediate product present on said strip in a region between the sample receiving and measurement regions such that movement of the transport medium through said strip will bring the conversion reagent and cholesterol together to react to form said intermediate product; and (e) reagent non-diffusively bound in the measurement region which reacts in the presence of said intermediate product to produce a detectable border related to the cholesterol level in said sample.
10. The device of Claim 9, wherein said device further comprises a capture region on or upstream from the sample region and the measurement region, which capture region diminishes a predetermined portion of reaction of the bound reagent in the measurement region.
11. The device of Claim 10, wherein said capture region is upstream from said conversion region and comprises a reactant capable of reacting with hydrogen peroxide.
12. The device of Claim 9, wherein said sample receiving region is out of fluid transfer rela-tionship with said transport and measurement regions and further comprising means for moving said sample receiving region from a first site for receiving said sample to fluid transfer relationship with said transport and measurement regions.
012790 32.
012790 32.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/474,991 US5132086A (en) | 1990-02-06 | 1990-02-06 | Non-instrumented cholesterol assay |
| US474,991 | 1990-02-06 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2035674A1 true CA2035674A1 (en) | 1991-08-07 |
Family
ID=23885790
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002035674A Abandoned CA2035674A1 (en) | 1990-02-06 | 1991-02-05 | Non-instrumented cholesterol assay |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5132086A (en) |
| EP (1) | EP0441325B1 (en) |
| JP (1) | JPH0643170A (en) |
| AT (1) | ATE116445T1 (en) |
| AU (1) | AU634339B2 (en) |
| CA (1) | CA2035674A1 (en) |
| DE (1) | DE69106156T2 (en) |
| ES (1) | ES2069105T3 (en) |
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| JPS5670460A (en) * | 1979-11-13 | 1981-06-12 | Fuji Photo Film Co Ltd | Multilayer chemical analysis material for analysis of water liquid |
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-
1990
- 1990-02-06 US US07/474,991 patent/US5132086A/en not_active Expired - Lifetime
-
1991
- 1991-02-05 CA CA002035674A patent/CA2035674A1/en not_active Abandoned
- 1991-02-05 AT AT91101530T patent/ATE116445T1/en not_active IP Right Cessation
- 1991-02-05 EP EP91101530A patent/EP0441325B1/en not_active Expired - Lifetime
- 1991-02-05 DE DE69106156T patent/DE69106156T2/en not_active Expired - Fee Related
- 1991-02-05 ES ES91101530T patent/ES2069105T3/en not_active Expired - Lifetime
- 1991-02-05 AU AU70287/91A patent/AU634339B2/en not_active Ceased
- 1991-02-06 JP JP3100596A patent/JPH0643170A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0643170A (en) | 1994-02-18 |
| EP0441325A2 (en) | 1991-08-14 |
| ATE116445T1 (en) | 1995-01-15 |
| DE69106156D1 (en) | 1995-02-09 |
| DE69106156T2 (en) | 1995-06-22 |
| AU634339B2 (en) | 1993-02-18 |
| EP0441325B1 (en) | 1994-12-28 |
| ES2069105T3 (en) | 1995-05-01 |
| US5132086A (en) | 1992-07-21 |
| EP0441325A3 (en) | 1993-03-24 |
| AU7028791A (en) | 1991-08-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |