CA1050867A - Method and apparatus for assaying liquid materials - Google Patents
Method and apparatus for assaying liquid materialsInfo
- Publication number
- CA1050867A CA1050867A CA225,325A CA225325A CA1050867A CA 1050867 A CA1050867 A CA 1050867A CA 225325 A CA225325 A CA 225325A CA 1050867 A CA1050867 A CA 1050867A
- Authority
- CA
- Canada
- Prior art keywords
- cavities
- liquid
- separating means
- phase separating
- liquid phase
- 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.)
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
- B04B—CENTRIFUGES
- B04B5/00—Other centrifuges
- B04B5/04—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers
- B04B5/0407—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles
- B04B5/0414—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles comprising test tubes
- B04B5/0421—Radial chamber apparatus for separating predominantly liquid mixtures, e.g. butyrometers for liquids contained in receptacles comprising test tubes pivotably mounted
Abstract
METHOD AND APPARATUS FOR
ASSAYING LIQUID MATERIALS
ABSTRACT OF THE DISCLOSURE
Assaying Or fluids to determine the level of a substance in a fluid sample such as blood serum involving the reacting of fluid samples with one or more reagents and centrifugally separating reaction constituents and measuring a property of a reaction constituent.
Individual mixing, reaction, separation and measurement steps required in previous techniques have been eliminated in that fluids are reacted in a set of cavities in a rotatable disc, the reacted fluids are centrifugally transferred from such cavities to a separate liquid phase separating means and the fluids thus transferred are separated into at least two separate phases to permit measurement of a thus separated phase.
S P E C I F I C A T I O N
1.
ASSAYING LIQUID MATERIALS
ABSTRACT OF THE DISCLOSURE
Assaying Or fluids to determine the level of a substance in a fluid sample such as blood serum involving the reacting of fluid samples with one or more reagents and centrifugally separating reaction constituents and measuring a property of a reaction constituent.
Individual mixing, reaction, separation and measurement steps required in previous techniques have been eliminated in that fluids are reacted in a set of cavities in a rotatable disc, the reacted fluids are centrifugally transferred from such cavities to a separate liquid phase separating means and the fluids thus transferred are separated into at least two separate phases to permit measurement of a thus separated phase.
S P E C I F I C A T I O N
1.
Description
:1~51)~367 The present lnvention is directed to the assaylng of 1ulds. More particularly, ~he present invention ls directed to the determination of the level of a substance in a fluid sample, e.g. serum, by reacting a fluid sample with one or mor~ reagents, and centrifugally separating reaction constituents to accurately obtain an indication of the level o the substance of interest.
In ~he analysis of fluids, e.g. serum, it is frequently important to determine:in a fluid sample, the level of substances sach as thyroid hormones, sex hormones, cardiac glycosides, vitamins, and cancer antigens. It is further ~xtremely important that such ; levels be determined accurately and~ rapidlyO
i Previous techn~ques have invoIved time consuming, ~ individual mixing, reaction, separation and meaæur~men~
;:~ steps.
It is an object of the present invention to provide a m~thod for rapidly and accurately assaying the level of substances in fluids.
Other objec~s will b~ apparent from t~e ` following description and claims taken in conjunction . with the drawing w~Lerein Figure 1 is an elevation Vi8W of an apparatus :~ suitable for use in the paractice of an embodiment of the presen~ inve~tion, ^ 2.
.
-. ~ .
36~ ~
Figure 2 ls a partial plan vlew of the apparatus Or Figure 1, : :
Figure 2(a) is a ~ragmented YieW of a portion ~. :
. .
Or the apparatus shown ln Figure 2, Figure 3 is a somewhat schema~ic .representation~ :
of a measuring arrangement for use in accordance with the present invention, ~ ~ ~
Figure 4 is a representation of a graph of -~ ;
the type which can be used as a re~erence standard in accordance with the present invention, ~ ~:
Figures 5(a), (b) and (c~, appearing on sheet 3 of 5 o~ the drawing below Figure 3, illustra~e sche-~ matically the ~unctioning of liquid phase separating : media in a particular embodiment of the present in- :
vention, and Figures 6 and 6(a) are a partial plan and elevation vièws of apparatus sultable for use in a further embodiment of the present invention.
A method in accordance with a par~icular embodiment o~ the present invention for assaying a plurality of ~::
liquid samples comprises (i) reacting ak least two liquid materials in a plurality of cavities (ii) providing li~
quid phase separating means in communication with said ;
; cavities (iii) subjecting the cavitles and liquid phase separating means to centrifugal force sufficient to transfer the liquid contents of the cavities to the communicating liquid phase separating means and to .~
,, ~, r~; r~
: ~ ~
g50 ~L11)5~67 provide separatlon of the llquid transferred thereto into at least two phases and (iv) measuring at least one property o~ a separated phase.
The present invention will be more fully under-~tood with reference to the drawing wherein Figure 1 ~how~
an apparatus suitable ~or use in the practice o~ the pre3ent invention comprising a rotatable ~upport member 15 af~ixed to sha~t 20 adapted to be driven by motor 30 which is coupled to shaft 20 as indicated at 18 over a range o~ speed~. The above-noted members are supported by base plate 40 and enclosed within housing 50 which is provlded with a removable cover - 60. Ring 70 ls removably attached to rotating member 15 and supports a plurality o~ removable tubes 65 which are engaged with rlng 70 by way o~ ball seat arrangements 80 such that the tubes are freely movable ~rom rest position 90 to rotational position 100 upon suitable rotation of member 15. Disc 110 is removably mounted on member 15 and indexed thereon by means of pin 112 such thatg with reference to Figure 2J
each row 120 of radially allgned cavities 130 and 140 are 20 in substantial alignment with tube 65. Tubes 65 are slightly `
displaced ~rom exact radial alignment with opposite cavitles 130 and 140 to compensate for inertia o~ liquld during transfer to tubes 65 and ~or a given apparatus can be routinely - determined and ad~usted.
By way o~ general description, in the practice o~ the present invention, for example for the purpose of obtaining the level of a substance in serum or serum-llke samples, a precise amoun~ Or reagent 150 ls placed . , .
., .
.; , :. 1~. ' 8 ~ 7 in cavities 130 ~nd a precise a~ount of serum 160 is placed in cavities 140, the reagent being such a~ to react with the substance ln the sample, the :level of which is sought, to produce a physically separatable reactlon produc~
The ca~itles 130 and 140 can be thus loaded by manual pipetting or by use of the apparatus disclosed in U.S.
Patent 3,801,283 - S. Shapiro and T. Picunko issuad April 2, 1974. m~ motor 30 is accelerated to a first speed such that the centrifugal force developed causes reagen~ 150 from cavities 130 to be transferred to ~ .`
cavitLes 140 and mix with and interact~with samples 160 in cavities 140.` The ~peed of motor 30 is controlled such that the contents o~ cav~ties 140 arè not forced out of cavities 140 by centri~ugal force. Reagent 150 and ~amples 160 interact in cavitLes 140 and, wlth time, and an increasing amount of reactIon product is formed in cavities L40 and~uItimàtely~~an equilibrium condition :~
.
would occur and after such time an analysis of the : , contents of cavities I40 could be u~ed to determine by ' . 20 known techniques the level in samples 160 of the substance o i~terest. Such a practice would however be tediu~
at best and take an extended period of time~ up ~o an : hour or more for many application~. In tha practice of ~he present invention, however, it is not ~ece~sary that .1 .
the interaction in cavities 140 proceed ~o equilLbrium, : :
. but only tha~ a mea~urable amount of reaction product, `~ : or change in reactant amo~nt~be produce~ in cavit~es 140, whereupon the speed o~ motor 30 is increased to that at which the con~enes of cavitles 140 are transferred by .
... . .. . .
867 :~:
centrifugal force via channels 700 into llquid phase separation media devices 190 shown as chromatographic gel oolumns 200 contained in open topped glass envelopes 210 which are removably seated in tubes 65. The liquid material con-tacting the gel columns 200 is chromatographlcally separated thereby upon the application of eluent thereto. This i9 accomplished with the apparatus of Figure 1 by dispensing a stream of a suitable liquld, e.g. burfer solution, from reservoir 222 via eluent pump 220 through conduit 230 and dispenser 240 into the cavities 130 promptly after the contents 160 of cavities 140 are transferred to gel columns 200. With reference to Figure 1, pump 220 is actuated by -way of a conventional timer arrangement 212 at a convenient time, e.g. 15 seconds, after the lncreased second speed is reached. Pump 220 provides a ~ixed ~low rate of eluent for a fixed period o~ t~me and the total eluent quantity is automatically divided into the cavities 130. The eluent is transferred to gel columns 200 by centrifugal force via cavities 130 and 140. Upon transfer of eluent to gel columns 200, centrifugal force causes chromatographic separtion of constituents of the liquid transferred from cavities 140, With appropriate selection of gel column 200, and with reference to the exemplary procedure herelnafter discussed~ a reaction constituent in the material in the gel column can be rapidly separated by elution and transferred by centrifugal force via outlets 215 of envelope 210 to tubes 65 as shown at 152. In the :::
.
~502 . ~ ~
~0S~i~367 : ~:
lnstance where a reactant employed was radloactive, each tube 65 can be removed from ring 70 and the radioactivlty ;~
of the contents 152 measured using the conventional arrange-ment of Figure 3 comprising a gamm~ ray detector 2309 eOg.
a sodium iodide crystal/photomultiplier tube combination, ~ ;
ampllfier 240J pulse heigh~ analyzer 245J counter ~50 and a display device 260, e.g. a digital printer, The count thus obtained for each tube 65 can be related to the level in the sample of the substance of lnterest by computation or by ;~
10 comparison with a standard. ~, As shown ln the particular embodiment of Figure
In ~he analysis of fluids, e.g. serum, it is frequently important to determine:in a fluid sample, the level of substances sach as thyroid hormones, sex hormones, cardiac glycosides, vitamins, and cancer antigens. It is further ~xtremely important that such ; levels be determined accurately and~ rapidlyO
i Previous techn~ques have invoIved time consuming, ~ individual mixing, reaction, separation and meaæur~men~
;:~ steps.
It is an object of the present invention to provide a m~thod for rapidly and accurately assaying the level of substances in fluids.
Other objec~s will b~ apparent from t~e ` following description and claims taken in conjunction . with the drawing w~Lerein Figure 1 is an elevation Vi8W of an apparatus :~ suitable for use in the paractice of an embodiment of the presen~ inve~tion, ^ 2.
.
-. ~ .
36~ ~
Figure 2 ls a partial plan vlew of the apparatus Or Figure 1, : :
Figure 2(a) is a ~ragmented YieW of a portion ~. :
. .
Or the apparatus shown ln Figure 2, Figure 3 is a somewhat schema~ic .representation~ :
of a measuring arrangement for use in accordance with the present invention, ~ ~ ~
Figure 4 is a representation of a graph of -~ ;
the type which can be used as a re~erence standard in accordance with the present invention, ~ ~:
Figures 5(a), (b) and (c~, appearing on sheet 3 of 5 o~ the drawing below Figure 3, illustra~e sche-~ matically the ~unctioning of liquid phase separating : media in a particular embodiment of the present in- :
vention, and Figures 6 and 6(a) are a partial plan and elevation vièws of apparatus sultable for use in a further embodiment of the present invention.
A method in accordance with a par~icular embodiment o~ the present invention for assaying a plurality of ~::
liquid samples comprises (i) reacting ak least two liquid materials in a plurality of cavities (ii) providing li~
quid phase separating means in communication with said ;
; cavities (iii) subjecting the cavitles and liquid phase separating means to centrifugal force sufficient to transfer the liquid contents of the cavities to the communicating liquid phase separating means and to .~
,, ~, r~; r~
: ~ ~
g50 ~L11)5~67 provide separatlon of the llquid transferred thereto into at least two phases and (iv) measuring at least one property o~ a separated phase.
The present invention will be more fully under-~tood with reference to the drawing wherein Figure 1 ~how~
an apparatus suitable ~or use in the practice o~ the pre3ent invention comprising a rotatable ~upport member 15 af~ixed to sha~t 20 adapted to be driven by motor 30 which is coupled to shaft 20 as indicated at 18 over a range o~ speed~. The above-noted members are supported by base plate 40 and enclosed within housing 50 which is provlded with a removable cover - 60. Ring 70 ls removably attached to rotating member 15 and supports a plurality o~ removable tubes 65 which are engaged with rlng 70 by way o~ ball seat arrangements 80 such that the tubes are freely movable ~rom rest position 90 to rotational position 100 upon suitable rotation of member 15. Disc 110 is removably mounted on member 15 and indexed thereon by means of pin 112 such thatg with reference to Figure 2J
each row 120 of radially allgned cavities 130 and 140 are 20 in substantial alignment with tube 65. Tubes 65 are slightly `
displaced ~rom exact radial alignment with opposite cavitles 130 and 140 to compensate for inertia o~ liquld during transfer to tubes 65 and ~or a given apparatus can be routinely - determined and ad~usted.
By way o~ general description, in the practice o~ the present invention, for example for the purpose of obtaining the level of a substance in serum or serum-llke samples, a precise amoun~ Or reagent 150 ls placed . , .
., .
.; , :. 1~. ' 8 ~ 7 in cavities 130 ~nd a precise a~ount of serum 160 is placed in cavities 140, the reagent being such a~ to react with the substance ln the sample, the :level of which is sought, to produce a physically separatable reactlon produc~
The ca~itles 130 and 140 can be thus loaded by manual pipetting or by use of the apparatus disclosed in U.S.
Patent 3,801,283 - S. Shapiro and T. Picunko issuad April 2, 1974. m~ motor 30 is accelerated to a first speed such that the centrifugal force developed causes reagen~ 150 from cavities 130 to be transferred to ~ .`
cavitLes 140 and mix with and interact~with samples 160 in cavities 140.` The ~peed of motor 30 is controlled such that the contents o~ cav~ties 140 arè not forced out of cavities 140 by centri~ugal force. Reagent 150 and ~amples 160 interact in cavitLes 140 and, wlth time, and an increasing amount of reactIon product is formed in cavities L40 and~uItimàtely~~an equilibrium condition :~
.
would occur and after such time an analysis of the : , contents of cavities I40 could be u~ed to determine by ' . 20 known techniques the level in samples 160 of the substance o i~terest. Such a practice would however be tediu~
at best and take an extended period of time~ up ~o an : hour or more for many application~. In tha practice of ~he present invention, however, it is not ~ece~sary that .1 .
the interaction in cavities 140 proceed ~o equilLbrium, : :
. but only tha~ a mea~urable amount of reaction product, `~ : or change in reactant amo~nt~be produce~ in cavit~es 140, whereupon the speed o~ motor 30 is increased to that at which the con~enes of cavitles 140 are transferred by .
... . .. . .
867 :~:
centrifugal force via channels 700 into llquid phase separation media devices 190 shown as chromatographic gel oolumns 200 contained in open topped glass envelopes 210 which are removably seated in tubes 65. The liquid material con-tacting the gel columns 200 is chromatographlcally separated thereby upon the application of eluent thereto. This i9 accomplished with the apparatus of Figure 1 by dispensing a stream of a suitable liquld, e.g. burfer solution, from reservoir 222 via eluent pump 220 through conduit 230 and dispenser 240 into the cavities 130 promptly after the contents 160 of cavities 140 are transferred to gel columns 200. With reference to Figure 1, pump 220 is actuated by -way of a conventional timer arrangement 212 at a convenient time, e.g. 15 seconds, after the lncreased second speed is reached. Pump 220 provides a ~ixed ~low rate of eluent for a fixed period o~ t~me and the total eluent quantity is automatically divided into the cavities 130. The eluent is transferred to gel columns 200 by centrifugal force via cavities 130 and 140. Upon transfer of eluent to gel columns 200, centrifugal force causes chromatographic separtion of constituents of the liquid transferred from cavities 140, With appropriate selection of gel column 200, and with reference to the exemplary procedure herelnafter discussed~ a reaction constituent in the material in the gel column can be rapidly separated by elution and transferred by centrifugal force via outlets 215 of envelope 210 to tubes 65 as shown at 152. In the :::
.
~502 . ~ ~
~0S~i~367 : ~:
lnstance where a reactant employed was radloactive, each tube 65 can be removed from ring 70 and the radioactivlty ;~
of the contents 152 measured using the conventional arrange-ment of Figure 3 comprising a gamm~ ray detector 2309 eOg.
a sodium iodide crystal/photomultiplier tube combination, ~ ;
ampllfier 240J pulse heigh~ analyzer 245J counter ~50 and a display device 260, e.g. a digital printer, The count thus obtained for each tube 65 can be related to the level in the sample of the substance of lnterest by computation or by ;~
10 comparison with a standard. ~, As shown ln the particular embodiment of Figure
2(a) a cavity 130 of the inner row communicates with the cavity 140 of the outer row with which is aligned by way o~ a trough-like passage means indicated at 500 which is formed by the side surfaces and ri~ing bottom surface of an inner cavity 130. With sufficient rotation and centri-fugal force, liquid in a cavity 130 overflows raised portlon 800 lnto an aligned outer cavity 140. Also, a cavity 140 of the outer row communicates with a liquid phase separation means 190 (not shown ln Figure 2a) which is aligned therewith by way of an extended trough-like means indicated at 600 which ... .
is formed by the side surfaces and rising bottom surface of an outer cavity 140, and channel 700. With su~ficient rotation ~ -and centrifugal force liquid ln an outer cavity 140 is overflowed and transferred into an aligned separating means, However, the slope 145 of outer cavitles 140 is steeper ,~ , ' , :,~
....
;' 7.
~ , ,~ , ~ " " , than the ~lopes 147 of inner cavltie~ 130, as shown in Figure 1J SO that liquld will be confined ln the outer ;
cavity 140, rai8ed port~on 600 forming a dam-llke barrler, until an increased centrifugal force i9 applied which i~
:greater than the centrifugal force requlred to over~low -liquid ~rom an inner cavity 130 to an outer cavity 140.
In the practice of the pre~ent invention, a above described, it is theoretically posslble, for a given ;~reaction and for glven particular concentrations o~ reactants, ~.
10 to calculate the concentration of a reaction product, or -reactant, at any given time after the start o~ the reaction, ~:
and a plot o~ concentration vs time obtalned, wlth respect ~ ::
to which mea~ured concentrations at particular times can be compared. For a simple case the procedure can be a~ ~ollows: `
; For a hypothetlcalg bimolecular, irrever~ible . .
reaction A ~ B ~ 3 C, where equal concentrations oP
A and B are mixed at time t=0, and at t=0, the concentration of C=0, it can be shown that the concentration at any time a~ter t=0 is given by:
C = Ao2Klt 1 + AoKlt Where:
~: Ao is the starting concentration Or reactants A and B.
Kl=A ~-Ea/RT) - Arrhenius Equation where A is the frequency factor, Ea is the activation : ' ; -''' ' 8. : ~
, .. . . . . , . , , , - . ,, .. . ,, :
. 9502 ~ ~5~D8 ~ 7 cnergy of the reaction, T i8 tha temperatura of ~-~
the raaction and R i~ the unlversal ~a~ constant.
Wlth such a calculation, ~md a plot darived . ~
thereform, ~or the given reaction, the concentratlon measured ater a r~latively short r~action time, could be routinely converted into the total~concen~ration . ~
or level o~ the substance of interest.
In a particular embodiment of the present ~;~
invention, a standard i8 used ;~hich avoids the inconvenience ~ ;~
of the above described Approach. In this embodiment, with re'ference to Figures 1 and'2`,''a''g'e'n'er'al procedure illustra-'' tlv'e o~ this'embodiment``'i~ t'o placë~'antibody, as a reactane, ``in innermost'~cavities I30''o~''the''`disc ~1109 a~d se~um ~a~ples '~
,. . .
containing'an'~-ungnown ~mount'''of 'a~"~'subs'ë'aDce, e.g. thyroxine~
~ T-4), togëthë'r`wIth 'rad~o'a`ct'ivë' T-'4~re'agen~'and' '~ a dispIace`ment 'reagent, are-''plac'ed in the outer "~ ~:
cavities 140. Th~ disc i~ rspidly aecelerat~d to a ~ ;~
firBt rotational speed in the course o whi~h ~ntibody re ceant rom~the inner cav`itles 130 ls cau~ed to move by centrifugal force to t~e outer caYitey 140 wherein the ;;~
antibody and T-4 mix and react. In the cour~e of the I ~ re~ction, T-4 in the ~e ~ ~ample8 i8 displac~d rom it~s carrier ~nd is ree to ccmpet~ wleh'the radiolabeled ; T-4 for a llmlted number o~ binding sLe~s on the antibody ¦ reactant. At a~ time aft~r mixing an~ during the ongoing reaction in~cavities 1409 ~he raeio o~ehe ~ntiLbody-bound , g -~
., ~.~, ~ . .
radiolabeled T-4, to the free radiolabeled T-4 in cavities 140 provides a measure of the initial level of T~4 in the serum samples. Thus, when tha reaction has proceeded at the intial speed for a short time suf~ic~ent to provide meaningful radioactive coun~ing data and well before reaction equilibrium is reached, ~he rotation of disc 110 is increased to a higher value at which contents of outer cavitles 140 are thrown by centrifugal force into communicating scparating media 200 wherein the T-4 antibody complex (containing both radioacti~e and nonradioactive T-4) is pas~ed, together with unreacted antibody through the separating media 200 wlth the uncomplexed T-4 (both radioa¢tive and non-radioactive) beihg adsorbed by the separating media 200.
is action halts the complexing~reaction by removing at leas t one reac~ant (antibody) from the reaction .
environment (gel columns 200) and consequent~y a count of the radioactivity of the separated antibody T-4 complex, :~ when compared to a standard, pxovides a measure of the init~al T-4 content of the ~ample under test. me ~tandard can be provided by using serum or serum-like materials of known bu~ different T~4 levels and~ using ~h~ same .. ~ .
reaction conditions, as for the ~e~t sample3 above, plotting the radioactive counts (or ratio of counts) . ! . ' obtained for each material vs its known level o~ T-4.
In a preferred practice, the "standard" materials are placed in appropriate cavitie$ 140 in the same dL~c 110 ~:. used for the test samples o unkno~n T~4 level and the 10.
. .
~, . . .
g502 ;i0~367 standard ~ata and test data, are obtained concurrently.
Figure 4, which is directly related to ~he ; specific example presented hereinbelowg illu3~rates a standard graph obtainable or use in the foregoing manner and shows the radioactive counts per minute ob~ained using "standard'l starting materials cont~ining a known amount of T-4. By way of example, Figure 4 indicatesg ~or the particular conditions employed, ~ha~
when a count of 4000 is obtained from a tes~ serum sample r~n concurrently with the "star.~ar~'materials? the initial level o~ T-4 in a serum sample is 6.2 ~grams o~ T-4 per 100 ml o sample. It is,of course, understood that in ., .
I practicing the present invention, appropriate~and precisely .. controlled amounts o~ reac~tants are employed.a~d the i :~
~ present invention is generally applicable to all liquid ; -liquid reactions, particularly those employed in the well-known clinical as~aying techniques for blood serum or serum like materials using reagents known to the art.
The method of the present invention is particularly appllcable to the assaying of a wide range oP physiologically - - important molecules ~or example as disclosed in Clinical Chemistry~` Vol. 19, No~ 2, 1973 (Article by D. S. Kell`ey, L. ~. Brown and .1 .
P. K. Besch at page 146).
, '1 ,. .
11.
~` 9~02 ~5~)867 The ~ollowing example wil.l ~urther illustra~e ~ .
the presen~ inven~ion.
EXA~PLE :1 Clinical serum samples were analyzed to determine the level of thyrox~ne (T-4) ~herein using ~' apparatus of the type illustrated in Figure 1, 2 and 3 and a standard curve as shown in Figure 4. Nine clin~cal serum samples of ~nknown T-4 level, in duplicate~ and ive "standard" solutions each o~ di~ferent~ but knawn T-4 levels, in duplicate, were processed simulta- :
neously. Thi8 was accomplished by loading two outer cavities 140 o~ disc~ 10 with 35 microliters each of a particular clinical serum sample, thus loading eighteen outer cavities in convenienly designated 1, 1'; 2, 2';
is formed by the side surfaces and rising bottom surface of an outer cavity 140, and channel 700. With su~ficient rotation ~ -and centrifugal force liquid ln an outer cavity 140 is overflowed and transferred into an aligned separating means, However, the slope 145 of outer cavitles 140 is steeper ,~ , ' , :,~
....
;' 7.
~ , ,~ , ~ " " , than the ~lopes 147 of inner cavltie~ 130, as shown in Figure 1J SO that liquld will be confined ln the outer ;
cavity 140, rai8ed port~on 600 forming a dam-llke barrler, until an increased centrifugal force i9 applied which i~
:greater than the centrifugal force requlred to over~low -liquid ~rom an inner cavity 130 to an outer cavity 140.
In the practice of the pre~ent invention, a above described, it is theoretically posslble, for a given ;~reaction and for glven particular concentrations o~ reactants, ~.
10 to calculate the concentration of a reaction product, or -reactant, at any given time after the start o~ the reaction, ~:
and a plot o~ concentration vs time obtalned, wlth respect ~ ::
to which mea~ured concentrations at particular times can be compared. For a simple case the procedure can be a~ ~ollows: `
; For a hypothetlcalg bimolecular, irrever~ible . .
reaction A ~ B ~ 3 C, where equal concentrations oP
A and B are mixed at time t=0, and at t=0, the concentration of C=0, it can be shown that the concentration at any time a~ter t=0 is given by:
C = Ao2Klt 1 + AoKlt Where:
~: Ao is the starting concentration Or reactants A and B.
Kl=A ~-Ea/RT) - Arrhenius Equation where A is the frequency factor, Ea is the activation : ' ; -''' ' 8. : ~
, .. . . . . , . , , , - . ,, .. . ,, :
. 9502 ~ ~5~D8 ~ 7 cnergy of the reaction, T i8 tha temperatura of ~-~
the raaction and R i~ the unlversal ~a~ constant.
Wlth such a calculation, ~md a plot darived . ~
thereform, ~or the given reaction, the concentratlon measured ater a r~latively short r~action time, could be routinely converted into the total~concen~ration . ~
or level o~ the substance of interest.
In a particular embodiment of the present ~;~
invention, a standard i8 used ;~hich avoids the inconvenience ~ ;~
of the above described Approach. In this embodiment, with re'ference to Figures 1 and'2`,''a''g'e'n'er'al procedure illustra-'' tlv'e o~ this'embodiment``'i~ t'o placë~'antibody, as a reactane, ``in innermost'~cavities I30''o~''the''`disc ~1109 a~d se~um ~a~ples '~
,. . .
containing'an'~-ungnown ~mount'''of 'a~"~'subs'ë'aDce, e.g. thyroxine~
~ T-4), togëthë'r`wIth 'rad~o'a`ct'ivë' T-'4~re'agen~'and' '~ a dispIace`ment 'reagent, are-''plac'ed in the outer "~ ~:
cavities 140. Th~ disc i~ rspidly aecelerat~d to a ~ ;~
firBt rotational speed in the course o whi~h ~ntibody re ceant rom~the inner cav`itles 130 ls cau~ed to move by centrifugal force to t~e outer caYitey 140 wherein the ;;~
antibody and T-4 mix and react. In the cour~e of the I ~ re~ction, T-4 in the ~e ~ ~ample8 i8 displac~d rom it~s carrier ~nd is ree to ccmpet~ wleh'the radiolabeled ; T-4 for a llmlted number o~ binding sLe~s on the antibody ¦ reactant. At a~ time aft~r mixing an~ during the ongoing reaction in~cavities 1409 ~he raeio o~ehe ~ntiLbody-bound , g -~
., ~.~, ~ . .
radiolabeled T-4, to the free radiolabeled T-4 in cavities 140 provides a measure of the initial level of T~4 in the serum samples. Thus, when tha reaction has proceeded at the intial speed for a short time suf~ic~ent to provide meaningful radioactive coun~ing data and well before reaction equilibrium is reached, ~he rotation of disc 110 is increased to a higher value at which contents of outer cavitles 140 are thrown by centrifugal force into communicating scparating media 200 wherein the T-4 antibody complex (containing both radioacti~e and nonradioactive T-4) is pas~ed, together with unreacted antibody through the separating media 200 wlth the uncomplexed T-4 (both radioa¢tive and non-radioactive) beihg adsorbed by the separating media 200.
is action halts the complexing~reaction by removing at leas t one reac~ant (antibody) from the reaction .
environment (gel columns 200) and consequent~y a count of the radioactivity of the separated antibody T-4 complex, :~ when compared to a standard, pxovides a measure of the init~al T-4 content of the ~ample under test. me ~tandard can be provided by using serum or serum-like materials of known bu~ different T~4 levels and~ using ~h~ same .. ~ .
reaction conditions, as for the ~e~t sample3 above, plotting the radioactive counts (or ratio of counts) . ! . ' obtained for each material vs its known level o~ T-4.
In a preferred practice, the "standard" materials are placed in appropriate cavitie$ 140 in the same dL~c 110 ~:. used for the test samples o unkno~n T~4 level and the 10.
. .
~, . . .
g502 ;i0~367 standard ~ata and test data, are obtained concurrently.
Figure 4, which is directly related to ~he ; specific example presented hereinbelowg illu3~rates a standard graph obtainable or use in the foregoing manner and shows the radioactive counts per minute ob~ained using "standard'l starting materials cont~ining a known amount of T-4. By way of example, Figure 4 indicatesg ~or the particular conditions employed, ~ha~
when a count of 4000 is obtained from a tes~ serum sample r~n concurrently with the "star.~ar~'materials? the initial level o~ T-4 in a serum sample is 6.2 ~grams o~ T-4 per 100 ml o sample. It is,of course, understood that in ., .
I practicing the present invention, appropriate~and precisely .. controlled amounts o~ reac~tants are employed.a~d the i :~
~ present invention is generally applicable to all liquid ; -liquid reactions, particularly those employed in the well-known clinical as~aying techniques for blood serum or serum like materials using reagents known to the art.
The method of the present invention is particularly appllcable to the assaying of a wide range oP physiologically - - important molecules ~or example as disclosed in Clinical Chemistry~` Vol. 19, No~ 2, 1973 (Article by D. S. Kell`ey, L. ~. Brown and .1 .
P. K. Besch at page 146).
, '1 ,. .
11.
~` 9~02 ~5~)867 The ~ollowing example wil.l ~urther illustra~e ~ .
the presen~ inven~ion.
EXA~PLE :1 Clinical serum samples were analyzed to determine the level of thyrox~ne (T-4) ~herein using ~' apparatus of the type illustrated in Figure 1, 2 and 3 and a standard curve as shown in Figure 4. Nine clin~cal serum samples of ~nknown T-4 level, in duplicate~ and ive "standard" solutions each o~ di~ferent~ but knawn T-4 levels, in duplicate, were processed simulta- :
neously. Thi8 was accomplished by loading two outer cavities 140 o~ disc~ 10 with 35 microliters each of a particular clinical serum sample, thus loading eighteen outer cavities in convenienly designated 1, 1'; 2, 2';
3, 3'; ----- 9, 9' in Figure 2. Also, two ou~er cavities 140 of disc 110 were loaded with 35 mierolite~s each of a particular one of five-~fstand~rd" solutions thus loading : -:~ ten outer cavIties 140 conveniënki~ designa~ed a, ar; b, b';
~ e, e' in Figure 2. m e T-4 levels in the"standard" ;:
--20 solùtions prëparëd a~ herein~elow dèscribed, were . as follows:
Standard , a 0 b 2 c :6 j d 12 12.
~105~8~
In the course of loading of the outer cavities as described above each 35 microliter quantity was mixed with 65 micro-liters of dlstilled water. Additionally, to each of the outer cavities 140 loaded as above describecl 50 microliters of a radioactive T-4-125I solution (prepared as herelnbelow described) were added. Each o~ inner caviti.es 10, 10' ----90, 90' and Ag A' ---E, E' were loaded with antibody reagent (hereinbelow described). Disc llQ~ loaded as above described was rapldly accelerated in the apparatus of Figure 1 to a ~irst speed such that the contents of inner cavities 10, 10' -- 90, 90' and A, A' --- E, E' were transferred, within about 3 seconds, due to centrifugal force, to the respectlve communicating outer cavities 1, 1' --- 9, 9' and : a, a'---- e, e' wherein reaction commenced and proceeded ~or thirty minutes.
`~ T4 * AB ~ ~ AB T4 T4R ~ AB ~ AB T4R
` After the elapse of thirty minutes the rotatlonal speed of the disc 110 was rapidly ac~celerated to a second ;~
speed and the centrifugal force developed caused the contents of cavities 1, 1' --- 9, 9' and a, a' --- e, e' to be ~: .
transferred to respective communicating chromatographic columns 200.
Fifteen seconds after acceleration of disc 110 to this second ` speed the eluent pump shown at 220 in Figure 1 was activated by way of a conventional timer arran~ement ~12 to dispenæe 2 milliliters of buffer solution (hereinbelow described) from container 222 into each of the inner cavities 130 by 13.
1(315C~ ;7 way of dispenser 240. A kotal flow of 60 ml i~ provlded by dispen~lng 30 ml per minute ~or two minut~es, the total flow being "chopped" by the thirty cavitie~ 'Lnto 2 ml per cavity The solution thus added by way of dlspenser ~40 ls caused by centrifugal force to be transferred from cavitles 10, 10'~ 90, 90' and A, A' - -E, ]3' to 1, 1'--9, 9' and a, a' --- e, e' to the respective chromatographic columns 200 wherein the reactants and reactlon products are sub~ect to elution due to the rotationaIly developed ;~
10 centrifugal force acting upon the eluent, and as a result .:
T-4 antlbody complex (containlng both radioactive and non-radioactive T-4) together wlth unreacted antibody are rapidly*
eluted from the individual chromatographlc columns 200 and, caused by centrifugal force, to be thrown to tubes 65 as indicated at 152 in Figures 1 and 2. Unreacted T 4-125I
solution and serum components o~ low molecular weight remain in the chromatographic columns 200 In the present example, -~
upon separation of antibody by elution, the above noted reaction is essentially halted at the time of separation 20 in each of the chromatographic columns 200. At any convenient -~
tlme arter elution, tubeæ 65 are transferred to an arrangement of the type shown in Figure 3 and each tube 65, with its contents 152 counted for one minute as shown in the Table below " , ~'.
*e.g. wlthin one minute : :
14.
~ ~ , , . . . . . , . : . - , 050~ 7 9502 TABLE
Tube Corresding C~ L~CACL~n CountslMinute gm%T4 a O 7623 O
a ' O ' 7468 O
"Standard7' b 1 5398 2 levels plot~ed in Figure: 4. ::
b' 1' 5496 2 c 2 3194 6 : ~
c' 2t 3307 6 . .
d 3 2235 12 a' 3 ' 2325 12 e 4 1299 30 e ' 4 ' 1336 30 ; 1 5 4544 4.7 1 ' 5 ' 4408 5. :L
2 6 3009 8 . 9 UnknOWn Clinical 2 ' 6 ' 2956 9 . û Sample Levels de termined ~ :
~: 3 7 3919 6.4 from plot of 3 q 7 ' 4051 ~ 6 .1 Figure 4 :1: : .-; 4 8 4092 . 6.0 `:
~ e, e' in Figure 2. m e T-4 levels in the"standard" ;:
--20 solùtions prëparëd a~ herein~elow dèscribed, were . as follows:
Standard , a 0 b 2 c :6 j d 12 12.
~105~8~
In the course of loading of the outer cavities as described above each 35 microliter quantity was mixed with 65 micro-liters of dlstilled water. Additionally, to each of the outer cavities 140 loaded as above describecl 50 microliters of a radioactive T-4-125I solution (prepared as herelnbelow described) were added. Each o~ inner caviti.es 10, 10' ----90, 90' and Ag A' ---E, E' were loaded with antibody reagent (hereinbelow described). Disc llQ~ loaded as above described was rapldly accelerated in the apparatus of Figure 1 to a ~irst speed such that the contents of inner cavities 10, 10' -- 90, 90' and A, A' --- E, E' were transferred, within about 3 seconds, due to centrifugal force, to the respectlve communicating outer cavities 1, 1' --- 9, 9' and : a, a'---- e, e' wherein reaction commenced and proceeded ~or thirty minutes.
`~ T4 * AB ~ ~ AB T4 T4R ~ AB ~ AB T4R
` After the elapse of thirty minutes the rotatlonal speed of the disc 110 was rapidly ac~celerated to a second ;~
speed and the centrifugal force developed caused the contents of cavities 1, 1' --- 9, 9' and a, a' --- e, e' to be ~: .
transferred to respective communicating chromatographic columns 200.
Fifteen seconds after acceleration of disc 110 to this second ` speed the eluent pump shown at 220 in Figure 1 was activated by way of a conventional timer arran~ement ~12 to dispenæe 2 milliliters of buffer solution (hereinbelow described) from container 222 into each of the inner cavities 130 by 13.
1(315C~ ;7 way of dispenser 240. A kotal flow of 60 ml i~ provlded by dispen~lng 30 ml per minute ~or two minut~es, the total flow being "chopped" by the thirty cavitie~ 'Lnto 2 ml per cavity The solution thus added by way of dlspenser ~40 ls caused by centrifugal force to be transferred from cavitles 10, 10'~ 90, 90' and A, A' - -E, ]3' to 1, 1'--9, 9' and a, a' --- e, e' to the respective chromatographic columns 200 wherein the reactants and reactlon products are sub~ect to elution due to the rotationaIly developed ;~
10 centrifugal force acting upon the eluent, and as a result .:
T-4 antlbody complex (containlng both radioactive and non-radioactive T-4) together wlth unreacted antibody are rapidly*
eluted from the individual chromatographlc columns 200 and, caused by centrifugal force, to be thrown to tubes 65 as indicated at 152 in Figures 1 and 2. Unreacted T 4-125I
solution and serum components o~ low molecular weight remain in the chromatographic columns 200 In the present example, -~
upon separation of antibody by elution, the above noted reaction is essentially halted at the time of separation 20 in each of the chromatographic columns 200. At any convenient -~
tlme arter elution, tubeæ 65 are transferred to an arrangement of the type shown in Figure 3 and each tube 65, with its contents 152 counted for one minute as shown in the Table below " , ~'.
*e.g. wlthin one minute : :
14.
~ ~ , , . . . . . , . : . - , 050~ 7 9502 TABLE
Tube Corresding C~ L~CACL~n CountslMinute gm%T4 a O 7623 O
a ' O ' 7468 O
"Standard7' b 1 5398 2 levels plot~ed in Figure: 4. ::
b' 1' 5496 2 c 2 3194 6 : ~
c' 2t 3307 6 . .
d 3 2235 12 a' 3 ' 2325 12 e 4 1299 30 e ' 4 ' 1336 30 ; 1 5 4544 4.7 1 ' 5 ' 4408 5. :L
2 6 3009 8 . 9 UnknOWn Clinical 2 ' 6 ' 2956 9 . û Sample Levels de termined ~ :
~: 3 7 3919 6.4 from plot of 3 q 7 ' 4051 ~ 6 .1 Figure 4 :1: : .-; 4 8 4092 . 6.0 `:
4' 8' 3899 6.4 9 3415 7. 7 ~:
5' 9' 3627 7. 1 1
6 10 4500 4.8 . .
6 ' 10 ' 4389 5, 2 ~
':
-.' 7 11 4092 6 . 0 ~; 7 ' 11 ' 3~g9 6 . 4 ~.
12 3225 8 .
' 12 ' 3192 8. 2 -~ 9 13 3S01 7.4 /
~` 9' . 13' 4052 6.0/
15 .
. . .
,. . . ;; - , .. .. ..
~1~5~8~7 ~
The known T-4 levels in ugm of T-4 per 100 ml of sample of the "standards" a, al -- e3 el, were plotted vs the obtained counts per minute to provid~ the plot of Figure 4 uslng the measured counts in the Table corres- -ponding to the samples. me determined le~els in the Table for cllnical samples were obtained from the plot o~ Figure 4~ The following is a ~etalled description the materials and procedure of the example described above.
I. Substance_und Clinical Serum Samples.
II._ Materials Used 1~ Thyroxine (T-4 stock), free acid: Cat. No. 2376 Sigma Chemical Co., St. Louis, ~o.
2. Thyroxine-125I (T-4-125): Cat. No. 6751 "Tetramet~
-125", Abbott Labs, North Chicago, Illinois.
3. Anti-thyroxine serum (rabbit): Wien Labs., Succa-sunna~ New Jersey.
4. ~ydrochloric Acid: Cat. No. A-144, Fisher Scienti~ic, New York, New York.
5. Sodium ~ydroxide, 0.lN: Cat. No. S0-S-276~ Fisher Scientific.
6. Sodium Barbital: Cat~ No. B 22, Fisher Scientific~
6 ' 10 ' 4389 5, 2 ~
':
-.' 7 11 4092 6 . 0 ~; 7 ' 11 ' 3~g9 6 . 4 ~.
12 3225 8 .
' 12 ' 3192 8. 2 -~ 9 13 3S01 7.4 /
~` 9' . 13' 4052 6.0/
15 .
. . .
,. . . ;; - , .. .. ..
~1~5~8~7 ~
The known T-4 levels in ugm of T-4 per 100 ml of sample of the "standards" a, al -- e3 el, were plotted vs the obtained counts per minute to provid~ the plot of Figure 4 uslng the measured counts in the Table corres- -ponding to the samples. me determined le~els in the Table for cllnical samples were obtained from the plot o~ Figure 4~ The following is a ~etalled description the materials and procedure of the example described above.
I. Substance_und Clinical Serum Samples.
II._ Materials Used 1~ Thyroxine (T-4 stock), free acid: Cat. No. 2376 Sigma Chemical Co., St. Louis, ~o.
2. Thyroxine-125I (T-4-125): Cat. No. 6751 "Tetramet~
-125", Abbott Labs, North Chicago, Illinois.
3. Anti-thyroxine serum (rabbit): Wien Labs., Succa-sunna~ New Jersey.
4. ~ydrochloric Acid: Cat. No. A-144, Fisher Scienti~ic, New York, New York.
5. Sodium ~ydroxide, 0.lN: Cat. No. S0-S-276~ Fisher Scientific.
6. Sodium Barbital: Cat~ No. B 22, Fisher Scientific~
7. Sodium Azlde: Cat. I~o. S 227, Fisher Scienti~ic. `~-
8. Normal rabbit serum.
*'Trademark of Abbott Labs ~ 9502 ~L~S1~8~7 : `
*'Trademark of Abbott Labs ~ 9502 ~L~S1~8~7 : `
9. 8-Anilino-l-naphthal~ne sulfonic acid, sodium ;~
salt (ANS): Cat. No. go4l~ K&K Labs~ Plainvlew~
New York.
lO. Normal pooled human plasma: Plasma Products.
ll. Activated charcoal: Darco G 60, Matheson~ Coleman & Bell, Rutherford, New Jersey.
120 Sephadex~ ~-25, rine (chromatographic gel)~
Pharmacia Fine Chemicals, Piscataway, I~.J.
13. Columns (for chromatographic gel): Cat. No. 102/2, Walter Sarstedt~ Inc.~ Princeton9 N.J.
14. Tubes: 12 x 75 mm and 17 x lO0 mm test tubes.
;
III. Reagents Used ~; A. 6~ ~iydrochloric Acid, l llter.
B. arbital Buffer, Q.075M, pH 8.6; l liter.
. ~. .~i ~issolve 15.54 ~m sodium barbital and lO0 mg sodium azide in 800 ml distilled water. Using ~`~
a standardized pH meter, bring the pH of the solution to 8. 6 by the dropwise addition of 6~J HCl, mixlng the barbital thoroughly throughout.
(Approximately 2 ml of 6N HCl is needed.) Fill up to l liter with distilled water. This buffer is good for one month with refrigeration. ~`~
C. ?% Normal_Rabbit Serum-O~rblt-1 Bu':e7, lO0 ml Add 2 ml of 1~10rmal Rabbit Serum to 98 ml of Barbital Buffer, mlx thoroughly. Good for 2 weeks with refrigeration.
, *Trademark of Pharmacia Fine Chemicals 17.
~, .,~; ;;
;
D5~6 D. ~ , 2Om:L.
m orou~hly mix 3 gm of actlvated charcoal in~o ~0 ml of pooled human pla~ma in ~1 di~po~able 50 ml conlcal centrifuge tube, taking care to wet all the charcoalO Cover the mixture and place in refrigerator overn$ght. On the next ~ ;
; day, centrifuge the mixture at about 8~000 rpm ~
. ~ .
for 10 minutes. men, using a 20 ml syringe fltted with a 25 mm Milllpore rilter holder with a Swlnnex adaptor, ~llter the supernatant ~uce~
slvely with (1) filter paper~ (2) a 0.45 mLcron filter, (3) a 0.22 micron rilter. Prepare weekly and refrigerate, or, if frozen, good for at least 3 months. ; ;~;
E. Thyroxine (T-4) Standards Preparatlon ~
,; ~, 1. T 4 Stock (o.6 mg~ml)O
` Dissolve 6.00 mg o~ T-4 Stook in a minimum volume of 0.1 N sodium hydroxide. Fill to
salt (ANS): Cat. No. go4l~ K&K Labs~ Plainvlew~
New York.
lO. Normal pooled human plasma: Plasma Products.
ll. Activated charcoal: Darco G 60, Matheson~ Coleman & Bell, Rutherford, New Jersey.
120 Sephadex~ ~-25, rine (chromatographic gel)~
Pharmacia Fine Chemicals, Piscataway, I~.J.
13. Columns (for chromatographic gel): Cat. No. 102/2, Walter Sarstedt~ Inc.~ Princeton9 N.J.
14. Tubes: 12 x 75 mm and 17 x lO0 mm test tubes.
;
III. Reagents Used ~; A. 6~ ~iydrochloric Acid, l llter.
B. arbital Buffer, Q.075M, pH 8.6; l liter.
. ~. .~i ~issolve 15.54 ~m sodium barbital and lO0 mg sodium azide in 800 ml distilled water. Using ~`~
a standardized pH meter, bring the pH of the solution to 8. 6 by the dropwise addition of 6~J HCl, mixlng the barbital thoroughly throughout.
(Approximately 2 ml of 6N HCl is needed.) Fill up to l liter with distilled water. This buffer is good for one month with refrigeration. ~`~
C. ?% Normal_Rabbit Serum-O~rblt-1 Bu':e7, lO0 ml Add 2 ml of 1~10rmal Rabbit Serum to 98 ml of Barbital Buffer, mlx thoroughly. Good for 2 weeks with refrigeration.
, *Trademark of Pharmacia Fine Chemicals 17.
~, .,~; ;;
;
D5~6 D. ~ , 2Om:L.
m orou~hly mix 3 gm of actlvated charcoal in~o ~0 ml of pooled human pla~ma in ~1 di~po~able 50 ml conlcal centrifuge tube, taking care to wet all the charcoalO Cover the mixture and place in refrigerator overn$ght. On the next ~ ;
; day, centrifuge the mixture at about 8~000 rpm ~
. ~ .
for 10 minutes. men, using a 20 ml syringe fltted with a 25 mm Milllpore rilter holder with a Swlnnex adaptor, ~llter the supernatant ~uce~
slvely with (1) filter paper~ (2) a 0.45 mLcron filter, (3) a 0.22 micron rilter. Prepare weekly and refrigerate, or, if frozen, good for at least 3 months. ; ;~;
E. Thyroxine (T-4) Standards Preparatlon ~
,; ~, 1. T 4 Stock (o.6 mg~ml)O
` Dissolve 6.00 mg o~ T-4 Stook in a minimum volume of 0.1 N sodium hydroxide. Fill to
10 ml with distilled water. This may be allquoted into 0.2 ml vlals and stored ~rozen ~or 3 months.
2. "Working" Standards Prepar~ 12 x 75 mm test tubes and label 1-5.
.~
Prepare the T-4 Working St~ndards according ` to the ~ollowing scheme: ~
` .. ,: ,.' ~ ', . .
:.
*Trademark of Millipore Fllter Corporation , "~, :~, :
': ''~ ;;. ' . , ., ; . . ., ~, . . . , ,... . ~ ~ , ,. - . .. . . .. . . . .. . . . . .. ...
g502 1635U 8~;7 Add Barbital Remove Tube No. Buffer Bu~fer hdd T-4 S~tock Final Conc.
1 1.0 ml 0 ul 0 ul0 ug/ml 2 1.0 ml 10 ul 10 ul T-4 Sltock2 ug~ml dilut~ n 3 3 1.0 ml 10 ul 10 ul6 ug/ml 4 1.0 ml 20 ul 20 ul12 ug/ml 1.0 ml 50 ul 50 ul30 ul/ml 3. Actual Standards Used.
Label ~ive 17 x 100 mm test tubes 1-5; adcl 5.0 ml Or T-4 Free Plasma to each. ~emo~e 50 microliters of the corresponding "Worklng"
Standard shown above. Mlx well. The final results will be:
Tube No. ng/ml ng/35 ulugm/100 ml 1 a 0 0 0 2 b 20 0.7 2 3 c 60 2.1 6 4 ~ 120 4.2 12 e 300 10.5 30 Freeze in 0.5 ml aliquots.
~:-F. Thyroxlne I125 Solution 1. ANS Solution Dissolve 60 mg o~ 8-anilino--1-naphthanlene sulfonic acid in 10 ml of Reagent C. -~
2. Isotope Solution A minimum order o~ Tetramet~-125 ls 500 microcuries~ The solution is good for six ~ Trademark of Abbott Labs .
19 .
~'1'"~
.` ~' '.
~. .. . . . ., , ~- . . . . . : .. ' ' ' 867 ~ ~`
weeks. The expiration date is glven ~he Abbott label. The activity~ i.e. 9 the micro-curies per milliliter, wlll vary ~rom lot to lot; ~hls is also given for ~each lot on the label. 14~000 counts per minute (CPM~ is to be added to each assay tube in 50 micro-llters; 14,000 cpm ls approximately 0.014 microcuries. Determine the total number of ~ ~
assay tubes, standards and unknowns, increase ~`
by 10% as a safety factor and multiply the final number by 0.014; this is the ~otal number of microcurles needed. Nextg multiply `;
the total number of tubes 9 including the extra 10~ 3 by 50. This is the total number ~ ~-,.
of milliliters of AI~S needed. Withdraw the number of microllters of Tetramet*-125 corres- ;
ponding to the number of microcuries and add to the correct volume of ~S.
Prepare on day of use. ;
~, :
G. Antibody Reagent - ~
.
'~he antibody comes from Wien Labs lyophilized ; in ~ials labeled "100 Test". Each vial is reconstituted with 14.0 ml of Reagent C.
Good for 2 weeks, with refrigeration.
.
':
~ , *Trademark of Abbot Labs , 20.
~5~ 7 IV. Protocol A. Reactlon Conditions: ~;
; 50 microliters T-4-125I solution 35 microliters Standard or Serum Sample, and ; 65 microllters distilled water rlllsh are mixed together~
200 microliters Antibody Reagent are added next.
The react~on is perm~tted to run ~or 30 minutes at room temperature at the first speed of the incubator/separator and when the speed ls increased to the second level, the total reaction volume is trans~erred to a column of Sephadex* G-25, ~ine.
The complex is eluted with 2.0 ml of Barbital Buffer. The complex is counted ~or 1 minute in ; a gamma counter. -~
~ach sample and standard is run in duplica~e Standards 1-5 occupy 10 positions.
B. Treatment of data:
The Standards are plotted as follows: counts per minute on the y-axis vs the log o~ ugm~100 ml on the x-axis. The standards as prepared above are:
0, 2a 6, 12, 30 ugm thyroxine per 100 mlO Unknowns are determined from the standard curve by finding the ugm thyroxine per 100 ml value corresponding ; to the unknown sample's counts. ~
, '':
~Trademark Or Pharmacia Fine Chemicals .
21.
~ '~' ~
--~ 9502 1C~S0867 V. Substance under ~nalysis Clinlcal Serum Samples.
In the embodiment of the present in~ention illustrated by the ~oregoing specific example3 particular advantages are obtained by essentiaLly ha]ting the described complex Eorming reaction upon rapid seParation of the reaction media constituents under controlled conditions in the chroma-tographic columns 200. To consider a general case where reactants designated A and B are Placed in inner and outer cavities 130 and 140, respectively7 and caused to mix an~
proceed to react in the outer cavities to ~roduce increasing amoun~s of a reactioD product C, by separating the mixture of A, B and C on the chromatographic columns 200, into phases, one of whlch 1s collected ln tubes 65 and contains at least one reactant, e.g. either A or B, the C producing reaction is eQsentially halted in the collecting chromatograph columns, and9 even though rotation contlnues, no further amount of C will be produced, and hence eluted, and the parameter of the elu~ed phase which is to be measured, e.g. radioactivity, color, fluorescence, enzyme label? is "fixed1' at essentially the same time for all ~, .
of the samples being analyzed. This embodiment is of particular advantage in such applications as kinetic assays involving the dete~nination in a sample o~
thyroid hormones, sex hormones, cardiac glycosides, vitamin~ cancer antig~ns using standard radioin~unoassay reagents.
\
~2.
~ . . . .. . .
~L05(91~67 ~
The foregoing will be more rully under~tood with re~erence to Figure 5(a) wh~ch schematically represents a point in time at which the unreacted portion o~ reactants A and BJ and reaction product C have been transferred to chromatograph gel 200', but before trans~er of eluent to chromatograph gel 200'. Under ~uch conditlons A and B
can continue to react and produce additional amounts o~ C.
However, upon trans~er of eluent to chromatograph gel 200', which is selected in this instance to separate reactant 10 B together with reaction product C, B and CJ are rapidly .
separated ~rom A into a phase which is moved along chromatograph gel 200' as indicated in Figure 5(b), .; thus halking the formation o~ additional reaction product C, The phase 152' comprising fixed proportions of B and C ls trans~erred by centri~ugal force to tube 65' as indicated .~
in Figure 5~c) and the fixed value of the parameter of ~:
: interest of either B or C can be measured in due course.
In other applications involving the process of :~
the present invention where it is not o~ critical importance . 20 to halt the reaction in the chromatograph gel 20Q, wlth ; all samples and standards being sub~ected to essentially -~
the same reaction and separation conditions, the chroma- ;
~; tograph gel can be selected so as to elute and separate the reaction product ~rom the reactants, particularly in `~ -; the instances where any further ~ormation of reaction : product in the gel, and elution thereo~ will be compensated ~;' :;' , ,,~ , ; , -''. .
. ., .~ 23.
. .. .
, , ~
' ~ .
:... - - . , .. . - , . . . .
~502 .~
~ 8~ 7 when using a simultaneously processed standard.
In the practice of the present invention the parameter of interest can be radioactivity, as particularly described hereinabove, color, fluorescence or any other suitable physical or chemical property. Accordingly, instead of a radioactive counter arrangement other conventional sansing d~vices known to the art can also be utilized.
In a further embodiment of the present inventîon, with reference to Figure 6, a disc 510, is employed having a plurality of single cavities 520 instead of a pair of radiaLly aligned cavities 130 and 140, as in the device of Figure 1 In the practice of the invention using the : apparatus of Figure 6, precise amounts of two or more reactant~
e.g. serum and reagent indicated at 525 are~placed in cavities -~
520~wherein a reaction occurs to provide a physically separatable reaction product. Loading of the cavities 520 can be accomplished by pipetting as previously disclosed. On or more of cavities 520 can be loaded ~ 20 with standard reactants in the manner previously described .
i~ and the thus loaded disc 510 can be positioned on support .,~ .
:member 15 in the same manner as disc 110 in Flgure 1, and rota~ed at a speed suflcient to cause the contents : of the cavities 520 to be ~ransferred by centrifugal force into communicating separating media 190. E'rom this point on the process proceeds in ~he same manner : as pxeviously described in connection with the apparatus - arrangement of Figure 1 and a standard as exemplified in 2~- :
950~
. .
~ 3~ 7 in Figure 4. In practicing the foregoing embodiment as disc S10 containing cavities 5~0 can be loaded with reactants and the reaction permitted to go t~ equilibrium.
That is to say, the discs 510 can be loaded~and stored ~ .
for extended periods o~ time, e.g. for hours or more after which the discs 510 can be arranged in place o~ discs 110 in ~he device of Figure 1 and an assay performed as previously described.~ mls embodiment can be effectively employed with slow reactions, e.g. the deeermination in blood serum o , 10 human growth hormone, which i~ uslng the previously described dual cavity embodiment would entail impracticaliy long rotation at the higher speeds, e.g. 1 hour , . ~ or more. Alternatively, where the discs 510 are loaded : . .
;l~ in a period of time such that for the particular slow -l reaction involved, it can be considered as a practical matter that~the reactions~ih the different single ~ cavities have all started at the same time, the loaded disc 510 can be rotated and the contents o~ the cavities 520 trans~erred to communicating separating media 190 ~ ~ at any time ~that a measurable amount of separatable ~`~ cons~ituent has bePn produced in cavities 520. This procedure is;~effective in ins~ances where any loss of l ~ :
assay accuracy which might result from~ the different ; reaction times in the various cavities is not significant a~ compared with the time~saved.
Particular advantages of ~he mechani ally and chemically continuQus tandem method of the present . . .
inven~ion include the essen~ial eliminatio~ of manual ~ 9502 ~ 6~
or mechanical interventlon in the course of performing an assay which minimizes the significance of variables other than those of interest, and the abillty to utilize short reaction times and permit si.multaneous kinetic studies under controlled time conditions.
For purposes of the present inventivn reactive :~ constituents include substances which will react chemically to provide a chemically different reaction product or products and~also~substances which can be ~ .
considered to react physicaLly (e.g. certain physical adsorption phenomena) to produce one or more physical dif~erent materials.
, . . .
The liquid phase separating medium in the ;~
practice of the present inven~tion can be conventional chromatographic arrangements, for example, which provide separation on the basis of molecular size~ physical ~ :
adsorption phenomena, chemisorption, ion exchange properties, specific molecular affinities (af~inity chromatography)-and other k.nown techniques utilizing , ~
~or example, gels,solids,~and resins. .~
~..................................................... , ,: ~ ~ : : .
~, ' . l :
~ 26.
2. "Working" Standards Prepar~ 12 x 75 mm test tubes and label 1-5.
.~
Prepare the T-4 Working St~ndards according ` to the ~ollowing scheme: ~
` .. ,: ,.' ~ ', . .
:.
*Trademark of Millipore Fllter Corporation , "~, :~, :
': ''~ ;;. ' . , ., ; . . ., ~, . . . , ,... . ~ ~ , ,. - . .. . . .. . . . .. . . . . .. ...
g502 1635U 8~;7 Add Barbital Remove Tube No. Buffer Bu~fer hdd T-4 S~tock Final Conc.
1 1.0 ml 0 ul 0 ul0 ug/ml 2 1.0 ml 10 ul 10 ul T-4 Sltock2 ug~ml dilut~ n 3 3 1.0 ml 10 ul 10 ul6 ug/ml 4 1.0 ml 20 ul 20 ul12 ug/ml 1.0 ml 50 ul 50 ul30 ul/ml 3. Actual Standards Used.
Label ~ive 17 x 100 mm test tubes 1-5; adcl 5.0 ml Or T-4 Free Plasma to each. ~emo~e 50 microliters of the corresponding "Worklng"
Standard shown above. Mlx well. The final results will be:
Tube No. ng/ml ng/35 ulugm/100 ml 1 a 0 0 0 2 b 20 0.7 2 3 c 60 2.1 6 4 ~ 120 4.2 12 e 300 10.5 30 Freeze in 0.5 ml aliquots.
~:-F. Thyroxlne I125 Solution 1. ANS Solution Dissolve 60 mg o~ 8-anilino--1-naphthanlene sulfonic acid in 10 ml of Reagent C. -~
2. Isotope Solution A minimum order o~ Tetramet~-125 ls 500 microcuries~ The solution is good for six ~ Trademark of Abbott Labs .
19 .
~'1'"~
.` ~' '.
~. .. . . . ., , ~- . . . . . : .. ' ' ' 867 ~ ~`
weeks. The expiration date is glven ~he Abbott label. The activity~ i.e. 9 the micro-curies per milliliter, wlll vary ~rom lot to lot; ~hls is also given for ~each lot on the label. 14~000 counts per minute (CPM~ is to be added to each assay tube in 50 micro-llters; 14,000 cpm ls approximately 0.014 microcuries. Determine the total number of ~ ~
assay tubes, standards and unknowns, increase ~`
by 10% as a safety factor and multiply the final number by 0.014; this is the ~otal number of microcurles needed. Nextg multiply `;
the total number of tubes 9 including the extra 10~ 3 by 50. This is the total number ~ ~-,.
of milliliters of AI~S needed. Withdraw the number of microllters of Tetramet*-125 corres- ;
ponding to the number of microcuries and add to the correct volume of ~S.
Prepare on day of use. ;
~, :
G. Antibody Reagent - ~
.
'~he antibody comes from Wien Labs lyophilized ; in ~ials labeled "100 Test". Each vial is reconstituted with 14.0 ml of Reagent C.
Good for 2 weeks, with refrigeration.
.
':
~ , *Trademark of Abbot Labs , 20.
~5~ 7 IV. Protocol A. Reactlon Conditions: ~;
; 50 microliters T-4-125I solution 35 microliters Standard or Serum Sample, and ; 65 microllters distilled water rlllsh are mixed together~
200 microliters Antibody Reagent are added next.
The react~on is perm~tted to run ~or 30 minutes at room temperature at the first speed of the incubator/separator and when the speed ls increased to the second level, the total reaction volume is trans~erred to a column of Sephadex* G-25, ~ine.
The complex is eluted with 2.0 ml of Barbital Buffer. The complex is counted ~or 1 minute in ; a gamma counter. -~
~ach sample and standard is run in duplica~e Standards 1-5 occupy 10 positions.
B. Treatment of data:
The Standards are plotted as follows: counts per minute on the y-axis vs the log o~ ugm~100 ml on the x-axis. The standards as prepared above are:
0, 2a 6, 12, 30 ugm thyroxine per 100 mlO Unknowns are determined from the standard curve by finding the ugm thyroxine per 100 ml value corresponding ; to the unknown sample's counts. ~
, '':
~Trademark Or Pharmacia Fine Chemicals .
21.
~ '~' ~
--~ 9502 1C~S0867 V. Substance under ~nalysis Clinlcal Serum Samples.
In the embodiment of the present in~ention illustrated by the ~oregoing specific example3 particular advantages are obtained by essentiaLly ha]ting the described complex Eorming reaction upon rapid seParation of the reaction media constituents under controlled conditions in the chroma-tographic columns 200. To consider a general case where reactants designated A and B are Placed in inner and outer cavities 130 and 140, respectively7 and caused to mix an~
proceed to react in the outer cavities to ~roduce increasing amoun~s of a reactioD product C, by separating the mixture of A, B and C on the chromatographic columns 200, into phases, one of whlch 1s collected ln tubes 65 and contains at least one reactant, e.g. either A or B, the C producing reaction is eQsentially halted in the collecting chromatograph columns, and9 even though rotation contlnues, no further amount of C will be produced, and hence eluted, and the parameter of the elu~ed phase which is to be measured, e.g. radioactivity, color, fluorescence, enzyme label? is "fixed1' at essentially the same time for all ~, .
of the samples being analyzed. This embodiment is of particular advantage in such applications as kinetic assays involving the dete~nination in a sample o~
thyroid hormones, sex hormones, cardiac glycosides, vitamin~ cancer antig~ns using standard radioin~unoassay reagents.
\
~2.
~ . . . .. . .
~L05(91~67 ~
The foregoing will be more rully under~tood with re~erence to Figure 5(a) wh~ch schematically represents a point in time at which the unreacted portion o~ reactants A and BJ and reaction product C have been transferred to chromatograph gel 200', but before trans~er of eluent to chromatograph gel 200'. Under ~uch conditlons A and B
can continue to react and produce additional amounts o~ C.
However, upon trans~er of eluent to chromatograph gel 200', which is selected in this instance to separate reactant 10 B together with reaction product C, B and CJ are rapidly .
separated ~rom A into a phase which is moved along chromatograph gel 200' as indicated in Figure 5(b), .; thus halking the formation o~ additional reaction product C, The phase 152' comprising fixed proportions of B and C ls trans~erred by centri~ugal force to tube 65' as indicated .~
in Figure 5~c) and the fixed value of the parameter of ~:
: interest of either B or C can be measured in due course.
In other applications involving the process of :~
the present invention where it is not o~ critical importance . 20 to halt the reaction in the chromatograph gel 20Q, wlth ; all samples and standards being sub~ected to essentially -~
the same reaction and separation conditions, the chroma- ;
~; tograph gel can be selected so as to elute and separate the reaction product ~rom the reactants, particularly in `~ -; the instances where any further ~ormation of reaction : product in the gel, and elution thereo~ will be compensated ~;' :;' , ,,~ , ; , -''. .
. ., .~ 23.
. .. .
, , ~
' ~ .
:... - - . , .. . - , . . . .
~502 .~
~ 8~ 7 when using a simultaneously processed standard.
In the practice of the present invention the parameter of interest can be radioactivity, as particularly described hereinabove, color, fluorescence or any other suitable physical or chemical property. Accordingly, instead of a radioactive counter arrangement other conventional sansing d~vices known to the art can also be utilized.
In a further embodiment of the present inventîon, with reference to Figure 6, a disc 510, is employed having a plurality of single cavities 520 instead of a pair of radiaLly aligned cavities 130 and 140, as in the device of Figure 1 In the practice of the invention using the : apparatus of Figure 6, precise amounts of two or more reactant~
e.g. serum and reagent indicated at 525 are~placed in cavities -~
520~wherein a reaction occurs to provide a physically separatable reaction product. Loading of the cavities 520 can be accomplished by pipetting as previously disclosed. On or more of cavities 520 can be loaded ~ 20 with standard reactants in the manner previously described .
i~ and the thus loaded disc 510 can be positioned on support .,~ .
:member 15 in the same manner as disc 110 in Flgure 1, and rota~ed at a speed suflcient to cause the contents : of the cavities 520 to be ~ransferred by centrifugal force into communicating separating media 190. E'rom this point on the process proceeds in ~he same manner : as pxeviously described in connection with the apparatus - arrangement of Figure 1 and a standard as exemplified in 2~- :
950~
. .
~ 3~ 7 in Figure 4. In practicing the foregoing embodiment as disc S10 containing cavities 5~0 can be loaded with reactants and the reaction permitted to go t~ equilibrium.
That is to say, the discs 510 can be loaded~and stored ~ .
for extended periods o~ time, e.g. for hours or more after which the discs 510 can be arranged in place o~ discs 110 in ~he device of Figure 1 and an assay performed as previously described.~ mls embodiment can be effectively employed with slow reactions, e.g. the deeermination in blood serum o , 10 human growth hormone, which i~ uslng the previously described dual cavity embodiment would entail impracticaliy long rotation at the higher speeds, e.g. 1 hour , . ~ or more. Alternatively, where the discs 510 are loaded : . .
;l~ in a period of time such that for the particular slow -l reaction involved, it can be considered as a practical matter that~the reactions~ih the different single ~ cavities have all started at the same time, the loaded disc 510 can be rotated and the contents o~ the cavities 520 trans~erred to communicating separating media 190 ~ ~ at any time ~that a measurable amount of separatable ~`~ cons~ituent has bePn produced in cavities 520. This procedure is;~effective in ins~ances where any loss of l ~ :
assay accuracy which might result from~ the different ; reaction times in the various cavities is not significant a~ compared with the time~saved.
Particular advantages of ~he mechani ally and chemically continuQus tandem method of the present . . .
inven~ion include the essen~ial eliminatio~ of manual ~ 9502 ~ 6~
or mechanical interventlon in the course of performing an assay which minimizes the significance of variables other than those of interest, and the abillty to utilize short reaction times and permit si.multaneous kinetic studies under controlled time conditions.
For purposes of the present inventivn reactive :~ constituents include substances which will react chemically to provide a chemically different reaction product or products and~also~substances which can be ~ .
considered to react physicaLly (e.g. certain physical adsorption phenomena) to produce one or more physical dif~erent materials.
, . . .
The liquid phase separating medium in the ;~
practice of the present inven~tion can be conventional chromatographic arrangements, for example, which provide separation on the basis of molecular size~ physical ~ :
adsorption phenomena, chemisorption, ion exchange properties, specific molecular affinities (af~inity chromatography)-and other k.nown techniques utilizing , ~
~or example, gels,solids,~and resins. .~
~..................................................... , ,: ~ ~ : : .
~, ' . l :
~ 26.
Claims (31)
1. A method for assaying a plurality of liquid samples which comprises (i) reacting at least two liquid materials in a plurality of cavities to provide a liquid containing at least one reaction product (ii) providing liquid phase separating means in communication with said cavities (iii) subjecting said cavities and said liquid phase separating means to centrifugal force sufficient to transfer the liquid contents of said cavities to said communicating liquid phase separating means and provide sepa-ration of the liquid transferred thereto into at least two phases, and (iv) measuring at least one property of a separated phase.
2. A method in accordance with claim 1, wherein at least one of said liquid materials contains a radio-active constituent and the property measured in step (iv) is radioactivity.
3. A method in accordance with claim 1 wherein transfer of the contents of said cavities to said liquid phase separating means occurs at a time when increasing amounts of at least one reaction product are being formed.
27.
27.
4. A method in accordance with claim 1 wherein at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means by centrifugal force developed by rotation of said rotatable means.
5. A method in accordance with claim 1 wherein at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means to a vessel in communication therewith by centrifugal force.
6. A method in accordance with claim 5 wherein said phase transferred to said vessel is radioactive.
7. A method in accordance with claim 5 wherein at least one property of said phase transferred to said vessel is measured.
8. A method in accordance with claim 1 wherein a liquid is introduced into said cavities subsequent to the transfer of liquid to said liquid phase separating means and during rotation thereof to provide an eluent in said liquid phase separating means upon transfer there-to by centrifugal force.
9. A method in accordance with claim 1 wherein at least one of said cavities contains liquid materials which provide upon reaction a definitive value of measurable property in at least one of said separated phases.
28.
28.
10. A method for assaying a plurality of liquid samples which comprises (i) providing liquid material in a plurality of substantially circularly disposed first cavities (ii) providing a different liquid material, reactable with said material in said first cavities, in a plurality of substantially circularly disposed second cavities (iii) providing a plurality of liquid phase separating means in a substantially circular arrangement, said first and second cavities and said liquid phase separating means being arranged substantially concentric about a common axis with said liquid phase separating means being at a further radial distance than said second cavities and said second cavities being at a further radial distance than said first cavities, said liquid phase separating means being in tandem communication with a second cavity which is in tandem communication with a first cavity;
(iv) causing rotation of said cavities about said common axis to develop a centrifugal force sufficient to cause liquid in said first cavities to be transferred to said second cavities to react with said liquid in said second cavities to produce at least one reaction product 29.
(v) subsequently causing rotation of said cavities and said liquid phase separating means at a speed to develop a centrifugal force sufficient to transfer the liquid contents of said second cavities to said liquid phase separating means (vi) continuing rotation of said liquid phase separating means to develop a centrifugal force sufficient to separate the liquid transferred thereto into at least two phases and (vii) measuring at least one property of at least one of said phases.
(iv) causing rotation of said cavities about said common axis to develop a centrifugal force sufficient to cause liquid in said first cavities to be transferred to said second cavities to react with said liquid in said second cavities to produce at least one reaction product 29.
(v) subsequently causing rotation of said cavities and said liquid phase separating means at a speed to develop a centrifugal force sufficient to transfer the liquid contents of said second cavities to said liquid phase separating means (vi) continuing rotation of said liquid phase separating means to develop a centrifugal force sufficient to separate the liquid transferred thereto into at least two phases and (vii) measuring at least one property of at least one of said phases.
11. A method in accordance with claim 10 wherein at least one of said liquid material contains a radioactive constituent and the property measured in step (vii) is radioactivity.
12. A method in accordance with claim 10 wherein transfer of the contents of said second cavities to said liquid phase separating means occurs at a time when increasing amounts of at least one reaction product arc being formed.
13. A method in accordance with claim 10 wherein at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means by centrifugal force developed by rotation of said rotatable means.
30.
30.
14. A method in accordance with clam 10 wherein at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means to a vessel in communication therewith by centrifugal force.
15. A method in accordance with claim 10 wherein a liquid is introduced into at least said second cavities subsequent to the transfer of liquid to said liquid phase separating means and during rotation thereof to provide an eluent in said liquid phase separating means upon transfer thereto by centrifugal force.
16. A method in accordance with claim 10 wherein at least one of said first cavities and one of said second cavities contain liquid materials which provide upon reaction a definitive value of a measurable property in at least one of said separated phases.
17. A method for assaying a plurality of liquid samples which comprises (i) providing rotatable means having rotatable therewith a plurality of first and second cavities each of said cavities being adapted to contain liquid and said cavities being arranged in communication with each other such that upon development of a sufficient 31.
centrifugal force by rotation of said rotatable means, liquid in a said first cavity is transferred to said second cavity, and upon development of a sufficient centrifugal force by rotation of said rotatable means liquid in a said second cavity is transferred out of said second cavity (ii) providing at least one liquid material in a plurality of said first cavities and at least one different liquid material in a plurality of said second cavities said materials being such that upon contact therebetween interaction occurs to provide in said second cavities a mixture having at least one separatable constituent capable of being separated from said mixture by liquid phase separating means (iii) causing rotation of said rotatable member to develop a centrifugal force sufficient to transfer the liquid contents of said first cavities to the second cavities to contact the liquid contents of the second cvaties to provide a mixture confined in said second cavity containing at least one separatable constituent capable of being separated from said mixture by liquid phase separating means 32.
(iv) providing liquid phase separating means in communication with said second cavities and arranged to be rotatable therewith and be subjected to centrifugal force developed by rotation of said rotatable means (v) adjusting the speed of the rotatable means at a time subsequent to the formation of said separatable constituent in said second cavities to the extent that there is provided a centrifugal force sufficient to transfer the mixture in said second cavities to said liquid phase separating means, (vi) continuing rotation of said rotatable means to provide a centrifugal force acting upon the mixture transferred to said liquid phase separating means sufficient to cause said mixture to be separated into at least two phases, one of said phases containing at least one of said separatable constituent of said mixture and (vii) measuring at least one property of a said phase containing said at least one separatable constituent.
centrifugal force by rotation of said rotatable means, liquid in a said first cavity is transferred to said second cavity, and upon development of a sufficient centrifugal force by rotation of said rotatable means liquid in a said second cavity is transferred out of said second cavity (ii) providing at least one liquid material in a plurality of said first cavities and at least one different liquid material in a plurality of said second cavities said materials being such that upon contact therebetween interaction occurs to provide in said second cavities a mixture having at least one separatable constituent capable of being separated from said mixture by liquid phase separating means (iii) causing rotation of said rotatable member to develop a centrifugal force sufficient to transfer the liquid contents of said first cavities to the second cavities to contact the liquid contents of the second cvaties to provide a mixture confined in said second cavity containing at least one separatable constituent capable of being separated from said mixture by liquid phase separating means 32.
(iv) providing liquid phase separating means in communication with said second cavities and arranged to be rotatable therewith and be subjected to centrifugal force developed by rotation of said rotatable means (v) adjusting the speed of the rotatable means at a time subsequent to the formation of said separatable constituent in said second cavities to the extent that there is provided a centrifugal force sufficient to transfer the mixture in said second cavities to said liquid phase separating means, (vi) continuing rotation of said rotatable means to provide a centrifugal force acting upon the mixture transferred to said liquid phase separating means sufficient to cause said mixture to be separated into at least two phases, one of said phases containing at least one of said separatable constituent of said mixture and (vii) measuring at least one property of a said phase containing said at least one separatable constituent.
18. A method in accordance with claim 17 wherein (i) said at least one liquid material provided in said first cavity contains at least one reactive constituent;
33.
(ii) said at least one liquid material provided in said second cavity contains at least one reactive constituent, (iii) said reactive constituents, upon transfer of the liquid contents of said first cavity to said second cavity, proceed to react in said second cavity with the formation of a reaction product (iv) one of said at least two phases separated in said liquid phase separating means contains substantially all of at least one, but not all of said reactive constituents in said mixture transferred to said liquid phase separating means.
33.
(ii) said at least one liquid material provided in said second cavity contains at least one reactive constituent, (iii) said reactive constituents, upon transfer of the liquid contents of said first cavity to said second cavity, proceed to react in said second cavity with the formation of a reaction product (iv) one of said at least two phases separated in said liquid phase separating means contains substantially all of at least one, but not all of said reactive constituents in said mixture transferred to said liquid phase separating means.
19. A method in accordance with claim 17 wherein at least one of said liquids contains a radio-active constituent and the property measured in step (vii) is radioactivity.
20. A method in accordance with claim 18 wherein at least one of said reactable constituents is radioactive.
21. A method in accordance with claim 18 wherein transfer of the contents of the second cavity to said liquid phase separating means occurs at a time when the reactive constituents are reacting with the formation of increasing amounts of reaction product.
34.
34.
22. A method in accordance with claim 17 where-in at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means by centrifugal force developed by rotation of said rotatable means.
23. A method in accordance with claim 17 where-in at least one of said phases separated in said liquid phase separating means is transferred from said liquid phase separating means to a vessel in communication therewith by centrifugal force developed by rotation of said rotatable means.
24. A method in accordance with claim 23 where-in said phase transferred to said vessel is radioactive.
25. A method in accordance with claim 23 wherein at least one property of said phase transferred to said vessel is measured.
26. A method in accordance with claim 17 where-in at least one of said first cavities and one of said second cavities contain liquid materials which provide upon reaction a definitive value of a measurable property in at least one of said separated phases.
35.
35.
27. A method in accordance with claim 17 wherein a liquid is introduced into at least said second cavities subsequent to the transfer of liquid to said liquid phase separating means and during rotation thereof to provide an eluent in said liquid phase separating means upon transfer thereto by centrifugal force.
28. Assay apparatus comprising a (i) rotatable member having a plurality of substantially circularly disposed first cavities adapted to contain liquid and a plurality of substantially circularly disposed second cavities adpated to contain liquid (ii) a plurality of liquid phase separating means arranged in a substantially circular arrangement and engaged to said rotatable member, said first and second cavities and said liquid phase separating means being arranged substantially concentric about a common axis with said liquid phase separating means being at a further radial distance than said second cavities and said second cavities being at a further radial distance than said first cavities, said liquid phase separating means being in tandem communi-cation with a second cavity which is in tandem 36.
communication with a first cavity and (iii) collecting means adapted to contain liquid engaged to said liquid phase separating means to receive liquid separated by said liquid phase separating means.
communication with a first cavity and (iii) collecting means adapted to contain liquid engaged to said liquid phase separating means to receive liquid separated by said liquid phase separating means.
29. Assay apparatus comprising (i) rotating means (ii) a disc shaped member adapted to be rotated in a substantially horizontal plane about its central axis by said rotating means, said disc member having a first row of a plurality of cavities adapted to contain liquid at a common radial distance from the central axis of said disc and a second row of a plurality of cavities adapted to contain liquid at a different and greater common radial distance from said first row, cavities of said first row being in substantial radial alignment with cavities of said second row;
(iii) first trough-like means having an upward slope between aligned cavities of said first row and said second row to provide communication therebetween for liquid over flowing from a cavity of said first row to a radially aligned cavity of said second row due to centrifugal force;
37.
(iv) a plurality of liquid phase separating means substantially in radial alignment with cavities of said second row and adapted to be rotated by said rotating means;
(v) second trough-like means having an upward slope between the aligned cavities of said second row and said liquid phase separating means to provide communication therebetween for liquid overflowing from a cavity of said second row due to centrifugal force, said second trough-like means having a steeper slope than said first trough like means such that a centrifugal force required to cause overflow of liquid from the cavities of the second row is greater than the centrifugal force required to cause overflow of liquid from the cavities of the first row.
(iii) first trough-like means having an upward slope between aligned cavities of said first row and said second row to provide communication therebetween for liquid over flowing from a cavity of said first row to a radially aligned cavity of said second row due to centrifugal force;
37.
(iv) a plurality of liquid phase separating means substantially in radial alignment with cavities of said second row and adapted to be rotated by said rotating means;
(v) second trough-like means having an upward slope between the aligned cavities of said second row and said liquid phase separating means to provide communication therebetween for liquid overflowing from a cavity of said second row due to centrifugal force, said second trough-like means having a steeper slope than said first trough like means such that a centrifugal force required to cause overflow of liquid from the cavities of the second row is greater than the centrifugal force required to cause overflow of liquid from the cavities of the first row.
30. Apparatus in accordance with claim 29 wherein said plurality of liquid phase separating means are in the form of individual pivotally mounted columns adapted to be displaced by centrifugal force to positions wherein the longitudinal axis of said columns extend substantially radially with respect to the central axis of said disc shaped member.
38.
38.
31. Apparatus for use with a centrifugal assay device comprising (i) a disc shaped member adapted to be rotated in a substantially horizontal plane about its central axis, said disc shaped member having a first row of a plurality of cavities adpated to contain liquid at a common radial distance from the central axis of said disc and a second row of a plurality of cavities adapted to contain liquid at a different and greater common radial distance from said first row, cavities of said first row being in substantial radial alignment with cavities of said second row;
(ii) first trough-like means having an upward slope between aligned cavities of said first row and said second row to provide communication therebetween for liquid overflowing from a cavity of said first row to a radially aligned cavity of said second row due to centrifugal force;
(iii) second trough-like means having an upward slope at the radially outermost portion of the aligned cavities of said second row to provide exit therefrom for liquid overflowing from a cavity of said second row due to centrifugal force, said second trough-like means having a steeper slope than said first trough-like means such that a centrifugal force required to cause overflow of liquid from the cavities of the second row is greater than the centrifugal force required to cause overflow of liquid from the cavities of the first row.
(ii) first trough-like means having an upward slope between aligned cavities of said first row and said second row to provide communication therebetween for liquid overflowing from a cavity of said first row to a radially aligned cavity of said second row due to centrifugal force;
(iii) second trough-like means having an upward slope at the radially outermost portion of the aligned cavities of said second row to provide exit therefrom for liquid overflowing from a cavity of said second row due to centrifugal force, said second trough-like means having a steeper slope than said first trough-like means such that a centrifugal force required to cause overflow of liquid from the cavities of the second row is greater than the centrifugal force required to cause overflow of liquid from the cavities of the first row.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/468,649 US3953172A (en) | 1974-05-10 | 1974-05-10 | Method and apparatus for assaying liquid materials |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1050867A true CA1050867A (en) | 1979-03-20 |
Family
ID=23860657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA225,325A Expired CA1050867A (en) | 1974-05-10 | 1975-04-18 | Method and apparatus for assaying liquid materials |
Country Status (21)
| Country | Link |
|---|---|
| US (1) | US3953172A (en) |
| JP (1) | JPS5436879B2 (en) |
| AR (1) | AR213072A1 (en) |
| BE (1) | BE828901A (en) |
| BR (1) | BR7502852A (en) |
| CA (1) | CA1050867A (en) |
| CH (1) | CH607013A5 (en) |
| CS (1) | CS190477B2 (en) |
| DD (1) | DD118468A5 (en) |
| DE (1) | DE2520714C3 (en) |
| DK (1) | DK205175A (en) |
| ES (3) | ES437523A1 (en) |
| FR (1) | FR2280084A1 (en) |
| GB (2) | GB1507933A (en) |
| HU (1) | HU172910B (en) |
| IL (2) | IL47264A0 (en) |
| NL (1) | NL7505497A (en) |
| NO (1) | NO751661L (en) |
| NZ (1) | NZ177467A (en) |
| PL (1) | PL96621B1 (en) |
| SE (1) | SE7505376L (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH587486A5 (en) * | 1974-11-29 | 1977-05-13 | Hoffmann La Roche | |
| US4151254A (en) * | 1975-06-16 | 1979-04-24 | Union Carbide Corporation | Adsorption columns for use in radioimmunoassays |
| GB1533272A (en) * | 1976-02-13 | 1978-11-22 | Radiochemical Centre Ltd | Centrifuge tube |
| US4112292A (en) * | 1976-08-17 | 1978-09-05 | Access Control Systems Proprietary Limited | Magnetic identification apparatus |
| US4086060A (en) * | 1976-10-22 | 1978-04-25 | Jocelyn Dickson | Disposable manipulative laboratory device for transferring biological fluids |
| US4208484A (en) * | 1977-03-22 | 1980-06-17 | Olympus Optical Co., Ltd. | Apparatus for handling centrifuge tubes in automatic culture system |
| IT1097442B (en) * | 1977-08-18 | 1985-08-31 | Guigan Jean | CONDITIONING DEVICE OF A LIQUID SAMPLE IN PREPARATION OF ITS ANALYSIS |
| US4157895A (en) * | 1977-10-07 | 1979-06-12 | Nuclear International Corporation | RIA reagents and processes |
| US4170454A (en) * | 1978-03-30 | 1979-10-09 | Union Carbide Corporation | Process for the preparation of a solid-phase radioimmunoassay support and use thereof |
| US4244694A (en) * | 1978-03-31 | 1981-01-13 | Union Carbide Corporation | Reactor/separator device for use in automated solid phase immunoassay |
| US4190530A (en) * | 1978-04-03 | 1980-02-26 | E. I. Du Pont De Nemours And Company | Centrifugal method and apparatus for processing fluid materials |
| JPS55500529A (en) * | 1978-07-25 | 1980-08-14 | ||
| US4234317A (en) * | 1979-05-24 | 1980-11-18 | Analytical Products, Inc. | Apparatus and method for fractionation of lipoproteins |
| US4270921A (en) * | 1979-09-24 | 1981-06-02 | Graas Joseph E | Microchromatographic device and method for rapid determination of a desired substance |
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| US3532470A (en) * | 1968-01-22 | 1970-10-06 | Beckman Instruments Inc | Sample holder with centrifugation means |
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| US3763374A (en) * | 1972-08-22 | 1973-10-02 | Atomic Energy Commission | Dynamic multistation photometer-fluorometer |
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-
1974
- 1974-05-10 US US05/468,649 patent/US3953172A/en not_active Expired - Lifetime
-
1975
- 1975-04-18 CA CA225,325A patent/CA1050867A/en not_active Expired
- 1975-05-06 HU HU75UI00000223A patent/HU172910B/en unknown
- 1975-05-07 JP JP5396475A patent/JPS5436879B2/ja not_active Expired
- 1975-05-08 CS CS753249A patent/CS190477B2/en unknown
- 1975-05-08 PL PL1975180253A patent/PL96621B1/en unknown
- 1975-05-09 FR FR7514545A patent/FR2280084A1/en active Granted
- 1975-05-09 BR BR2852/75A patent/BR7502852A/en unknown
- 1975-05-09 ES ES437523A patent/ES437523A1/en not_active Expired
- 1975-05-09 CH CH599575A patent/CH607013A5/xx not_active IP Right Cessation
- 1975-05-09 GB GB19647/75A patent/GB1507933A/en not_active Expired
- 1975-05-09 DE DE2520714A patent/DE2520714C3/en not_active Expired
- 1975-05-09 AR AR258706A patent/AR213072A1/en active
- 1975-05-09 NZ NZ177467A patent/NZ177467A/en unknown
- 1975-05-09 SE SE7505376A patent/SE7505376L/en unknown
- 1975-05-09 BE BE156207A patent/BE828901A/en unknown
- 1975-05-09 DK DK205175A patent/DK205175A/en unknown
- 1975-05-09 NO NO751661A patent/NO751661L/no unknown
- 1975-05-09 IL IL47264A patent/IL47264A0/en unknown
- 1975-05-09 GB GB42495/77A patent/GB1507934A/en not_active Expired
- 1975-05-09 NL NL7505497A patent/NL7505497A/en not_active Application Discontinuation
- 1975-05-12 DD DD185962A patent/DD118468A5/xx unknown
- 1975-12-18 ES ES443619A patent/ES443619A1/en not_active Expired
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1976
- 1976-12-11 ES ES454172A patent/ES454172A1/en not_active Expired
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1978
- 1978-07-06 IL IL55099A patent/IL55099A0/en unknown
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|---|---|
| DE2520714C3 (en) | 1979-05-03 |
| AU8050975A (en) | 1976-10-28 |
| JPS5116090A (en) | 1976-02-09 |
| DE2520714A1 (en) | 1975-11-13 |
| IL55099A0 (en) | 1978-09-29 |
| BR7502852A (en) | 1976-12-21 |
| ES437523A1 (en) | 1977-04-01 |
| SE7505376L (en) | 1975-11-11 |
| NL7505497A (en) | 1975-11-12 |
| GB1507933A (en) | 1978-04-19 |
| PL96621B1 (en) | 1978-01-31 |
| US3953172A (en) | 1976-04-27 |
| NO751661L (en) | 1975-11-11 |
| HU172910B (en) | 1978-12-28 |
| CH607013A5 (en) | 1978-11-30 |
| DK205175A (en) | 1975-11-11 |
| JPS5436879B2 (en) | 1979-11-12 |
| BE828901A (en) | 1975-11-10 |
| FR2280084B1 (en) | 1979-04-06 |
| DE2520714B2 (en) | 1978-08-31 |
| ES443619A1 (en) | 1977-10-01 |
| NZ177467A (en) | 1978-03-06 |
| ES454172A1 (en) | 1977-12-01 |
| CS190477B2 (en) | 1979-05-31 |
| DD118468A5 (en) | 1976-03-05 |
| IL47264A0 (en) | 1975-07-28 |
| AR213072A1 (en) | 1978-12-15 |
| FR2280084A1 (en) | 1976-02-20 |
| GB1507934A (en) | 1978-04-19 |
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