CA1074228A - Diagnostic devices and method for immuno-chemical quantification - Google Patents
Diagnostic devices and method for immuno-chemical quantificationInfo
- Publication number
- CA1074228A CA1074228A CA243,521A CA243521A CA1074228A CA 1074228 A CA1074228 A CA 1074228A CA 243521 A CA243521 A CA 243521A CA 1074228 A CA1074228 A CA 1074228A
- Authority
- CA
- Canada
- Prior art keywords
- capillary
- carrier
- immuno
- bound
- silica
- 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.)
- Expired
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/44—Antibodies bound to carriers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/97—Test strip or test slide
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/80—Fluorescent dyes, e.g. rhodamine
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/805—Optical property
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/81—Tube, bottle, or dipstick
Abstract
ABSTRACT OF THE DISCLOSURE
A diagnostic device for immuno-chemical quantification includes a capillary-containing, porous carrier, e.g., a strip of cellulose fibre, starch polyvinyl chloride or polyvinyl chloride-silica, with antibodies bound thereto by adsorption or covalent bonding. Immuno-chemical quantification is effected by a method involving adding a sample containing an antigen to be quantified to the device, initiating a capillary migration, and effecting an assay of antigen-containing areas of the device by the addition of antibodies bound to a color indicator, e.g. a fluorescent compound such as fluoresceine isocyanate or an enzyme capable of catalyzing a color-developing reaction.
A diagnostic device for immuno-chemical quantification includes a capillary-containing, porous carrier, e.g., a strip of cellulose fibre, starch polyvinyl chloride or polyvinyl chloride-silica, with antibodies bound thereto by adsorption or covalent bonding. Immuno-chemical quantification is effected by a method involving adding a sample containing an antigen to be quantified to the device, initiating a capillary migration, and effecting an assay of antigen-containing areas of the device by the addition of antibodies bound to a color indicator, e.g. a fluorescent compound such as fluoresceine isocyanate or an enzyme capable of catalyzing a color-developing reaction.
Description
1~74ZZ8 This invention relates to a diagnostic device for immuno-chemical quantification, and in particular to test strips in the form of a capillary-containing carrier material, and to the method for immuno-chemical quantification.
More specifically, the diagnostic device includes an antibody bound to a capillary-containing porous carrier material.
The assay method of immuno-chemical quantification is characteri-zed by the utilization of the capillary force of porous carrier materials to which are bound antibodies. The antibodies are bound to the carrier material in a known manner, e.g., by adsorp-tion or covalent bonding using compounds such as cyanogen bromide or glutaric aldehyde.
Samples containing the antigen to be assayed or quanti-fied are added to the capillary containing diagnostic device.
A capillary migration is allowed to take place, whereafter the antigen-containing areas of the diagnostic device are indicated by the addition of antibodies bound to a suitable color indicator such as a fluorescent compound or an enzyme catalyzing a color reaction.
The hitherto used quantitative immuno-chemical methods are based on immunodiffusion such as, for example, the Mancini or Ouchterlony methods named after their respective inventors, or on immunoelectrophoresis such as in Laurell's electroimmuno assay. Such methods, as well as their variants, bring about the necessary interaction between antigen and antibody by a diffusion process and/or an electrophoretic process, whereas the method described herein is based on the utilization of the capillary force of a porous carrier material with antibodies bound thereto.
Capillary-containing materials suitable for the realiza-tion of the invention are different types of cellulose fibre-containing material such as filter paper, chromatographic paper, ion exchange paper, cellulose acetate membrane, cellulose acetate discs, i -\ 107422~3 cellulose thin layer chromatography discs, and materials such as starch, Sephadex (trademark for synthetic, organic compounds derived from dextran) plastic materials such as polyvinyl chloride, ceramic materials and combinations such as polyvinyl chloride-silica. In particular, suitable carriers include polyvinyl chloride or a silica modified polymer in which finely divided silica is substantially uniformly embedded in a continu-ous matrix which is polyvinylchloride, vinyl chloride-propylene copolymer of vinyl chloride-vinyl acetate copolymer. Suitable antibodies are provided by immunization in a manner known to the art.
The invention is illustrated by the following examples:
Production of Diagnostic Devices Example 1 - Insolubilization of antibodies on capillary-containing ma-terial with CNBr: 10 g of cellulose-containing .
material is placed in distilled water for 5 minutes. Thereafter, a solution of 8g of CNBr in 300 ml of distilled water is added and the pH is adjusted to 10.5 with 1 M NaOH. While maintaining the pH value by pH adjustments, the reaction is carried out during a period of 20 minutes, and then the solution is decanted.
The cellulose material is washed with 2 litres of 0.005 M NaHCO3.
After removal of the washing solution, the cellulose material is incubated over night at 4C with a solution of 500 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 10 ml of 0.1 M NaHCO3, and then incubated again for 3 hours with 100 ml of 0.005 M ethanolamine solution, followed by another washing with 500 ml of 0.5 M NaHCO3, and then 200 ml of 0.1 M acetate buffer ~lith a pH of 4.0 and 500 ml of 0.075 M
sodium barbiturate buffer with 0.3% albumin are added at 4C.
Example 2 - Insolubilization of antibodies on capillary containing material with glutaric aldenyde: 0.6 g of cellulose acetate membrane is incubated with a solution of 100 ~ -2-- . : - - .:
'\ :
mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 10 ml of 0.1 M phosphate buffer with a pH of 6.8 for 30 minutes. When the solution has been decanted, the membrane is incubated in 10 ml of 1~ glutaric aldehyde solution during 30 minutes, followed by incubation in 10 ml of 1.0 M
methylamine for 15 -2a-.
~74Z28 minutes. Thereafter, the membrane is washed with 100 ml of sodium barbiturate and kept in the same buffer at 4C to which has been added 0.3% albumin.
Example 3 - Insolubilization of antibodies on capillary-containing carrier material by adsorption: 10 g of polyvinyl chloride - silica (Microporous Plastic Sheet, Amerace Esna Corp-oration, New York, N.Y.) is incubated over night at 4C with a solution of 500 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 100 ml of 0.1 M phosphate buffer at a pH of 6.8. After completed incubation, the material is washed with 1 litre of phosphate-buffered physiological NaCl at a pH of 7Ø The material is dried between filter papers.
Example 4 - Preparation of fluorescent antl dy_coniug-ates:
.. _ (a) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) 0.57 g of NaCl, 0.259 g of NaHCO3 and 0.049 g of Na2CO3-10H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 36 mg of fluoresceine isothiocyanate are added while stirring. The solution is then leftin the cool while stirring for 18 hours.
Thereafter, the mixture is dialyzed against phosphate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody conjugate is frozen.
(b) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment), 0.57 g of NaCI, 0.259 g of NaHCO3 and 0.049 g of Na2CO3 10H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 9 mg of rhodamine dissolved in 2 ml of acetone is added while stirring. The solution is leftin the cool while stirring for 18 hours. Thereafter, the mixture is dialyzed against phos-phate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody 1~74ZZ8 conjugate is frozen.
(c) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment), 0.57 g NaCl, 0.259 g NaHCO3 and 0.049 g of Na2CO3-10 H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 20 mg of dansyl chloride dissolved in 7 ml of acetone is added while stirring. The solution is left in the cool while stirring for 18 hours. Thereafter, the mixture is dialyzed against phosphate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody conjugate is frozen.
Example 5 - Preparation of LDH iso-enzyme H4 antibody conjugate:
A suspension of 4.5 mg of LDH iso-enzyme H4 in ammonium sulfate is dialyzed against 0.1 M phosphate buffer at a pH of 6.8. While stirring, 2.25 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) and phosphate buffer are added to make a total volume of 1.35 ml. When all of the rabbit gammaglobulin has been dissolved, 45 ~1 of 1~ glutaric aldehyde solution is added dropwise, and the reaction is left to stand at room temperature for 2 hours. The mixture is then dialyzed against a sodium barbiturate buffer and the conjugate stored at 4C. Before use, the conjugate is diluted with 10 ml of barbiturate buffer to which 2% albumin has been added.
Example 6 - Checking of the insolubilization:
Two strips of material treated with antibodies in accordance with a preceding example, and two untreated strips are used for the test. One strip of each kind is incubated in a solution of 1 mg of human gammaglobulin (IgG) per ml of sodium barbiturate buffer. All strips are then washed with 20 ml of sodium barbiturate buffer containing 0.15 M NaC1, ten times in all, whereafter they are incubated in fluoresceine 1~42ZB
isothiocyanate antibody conjugate for 10 minutes. After repeated washings as in the foregoing, the strips are dried and inspected in ultraviolet light at a wavelength of 340 nm to establish that fluorescence is present only in the strips that contain the insolubilized antibodies, and that have been incubated in the IgG-solution. When checking strips according to Example 3, strips treated with bovine albumin are used as controls.
Immuno-chemical quantification Example A Diagnostic devices produced according to the present invention are used for immuno-chemical quantification for diagnostic purposes in the following manner:
The material to be tested is placed - in a known quantity, preferably between 1 ~1 and 3 ~1 - on the capillary-containing carrier material at the same time as corresponding quantities of a standard solution with a known content of the antigen to be tested and in various dilutions are placed next to the test solution.
For a mobile phase to facilitate capillary migration there could be used, e.g. 0.075 M sodium barbiturate buffer with an additive of 0.3% albumin.
The capillary migration can be performed in both open and closed test recipients, and the mobile phase can be made to move upwards and downwards. Preferably, the combination closed recipient and rising phase should be applied. At an optional time after initiation of the capillary migration, the moist carrier material is transferred to an antibody solution containing either fluoresceine isothiocyanate - or lactatedehydro-genase (LDH)-bound antibodies. The preferred incubation time for either of the solutions is 10 minutes. After incubation, the carrier material is washed under running water at 37C for 5 minutes, followed by incubation for 2 x 5 minutes in 20 ml of sodium barbiturate buffer, containing 0.2~ albumin and 0.15 M NaCl.
, : .
1~742Z8 .
To the extent that fluorescent antibodies are used for the identification, the migration distance of the antibody is measured in ultraviolet light at 340 nm. If LDH antibody solution is used, the carrier material is treated in a dye bath prepared as follows:
30 mg of nitroblue tetrazolium and 40 mg of NAD are dissolved in 56 ml of 0.05 M TRIS buffer at a pH of 7.4. 1.60 ml of 3.6 M lithium lactate; 5 ml of 0.06 M calcium cyanide, and a few grains of methylfenazonium metasulfate dissolved in 2 ml of distilled water are added to the mixtu~e. The capillary-containing material is incubated in the dye bath at 37C until a distinct coloring can be observed. The co~or development is due to the fact that the LDH catalyzes the reaction of lactate + NAD ~ pyruvate + NADH2 and that NADH2 quantitatively reduces nitroblue tetrazolium to an insoluble lilac-colored formazan.
The indicated, antigen-covered areas of the diagnostic device will then increase in magnitude with the increasing content of antigen of the tested samples. The matrix-bound antibody molecules retard the migration of the antigen molecules by exchange reactions during the capillary migration. The higher the concentration of antigens, the less retarding effect because the average time of an antigen molecule being bound to an antibody will decrease with an increasing concentration of antigens.
Example B Strips of the antibody-containing carrier material with capillaries and prepared according to the present invention are lowered to a determined depth of, for example, 3 mm in a small volume of a solution of the material to be tested.
The thus initiated capillary migration is allowed to continue for 3 - 20 minutes and then interrupted by removal of the strip -from the solution. The migration distance of the antigen is then measured as mentioned in Example A, and the result is evaluated according to Example A.
More specifically, the diagnostic device includes an antibody bound to a capillary-containing porous carrier material.
The assay method of immuno-chemical quantification is characteri-zed by the utilization of the capillary force of porous carrier materials to which are bound antibodies. The antibodies are bound to the carrier material in a known manner, e.g., by adsorp-tion or covalent bonding using compounds such as cyanogen bromide or glutaric aldehyde.
Samples containing the antigen to be assayed or quanti-fied are added to the capillary containing diagnostic device.
A capillary migration is allowed to take place, whereafter the antigen-containing areas of the diagnostic device are indicated by the addition of antibodies bound to a suitable color indicator such as a fluorescent compound or an enzyme catalyzing a color reaction.
The hitherto used quantitative immuno-chemical methods are based on immunodiffusion such as, for example, the Mancini or Ouchterlony methods named after their respective inventors, or on immunoelectrophoresis such as in Laurell's electroimmuno assay. Such methods, as well as their variants, bring about the necessary interaction between antigen and antibody by a diffusion process and/or an electrophoretic process, whereas the method described herein is based on the utilization of the capillary force of a porous carrier material with antibodies bound thereto.
Capillary-containing materials suitable for the realiza-tion of the invention are different types of cellulose fibre-containing material such as filter paper, chromatographic paper, ion exchange paper, cellulose acetate membrane, cellulose acetate discs, i -\ 107422~3 cellulose thin layer chromatography discs, and materials such as starch, Sephadex (trademark for synthetic, organic compounds derived from dextran) plastic materials such as polyvinyl chloride, ceramic materials and combinations such as polyvinyl chloride-silica. In particular, suitable carriers include polyvinyl chloride or a silica modified polymer in which finely divided silica is substantially uniformly embedded in a continu-ous matrix which is polyvinylchloride, vinyl chloride-propylene copolymer of vinyl chloride-vinyl acetate copolymer. Suitable antibodies are provided by immunization in a manner known to the art.
The invention is illustrated by the following examples:
Production of Diagnostic Devices Example 1 - Insolubilization of antibodies on capillary-containing ma-terial with CNBr: 10 g of cellulose-containing .
material is placed in distilled water for 5 minutes. Thereafter, a solution of 8g of CNBr in 300 ml of distilled water is added and the pH is adjusted to 10.5 with 1 M NaOH. While maintaining the pH value by pH adjustments, the reaction is carried out during a period of 20 minutes, and then the solution is decanted.
The cellulose material is washed with 2 litres of 0.005 M NaHCO3.
After removal of the washing solution, the cellulose material is incubated over night at 4C with a solution of 500 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 10 ml of 0.1 M NaHCO3, and then incubated again for 3 hours with 100 ml of 0.005 M ethanolamine solution, followed by another washing with 500 ml of 0.5 M NaHCO3, and then 200 ml of 0.1 M acetate buffer ~lith a pH of 4.0 and 500 ml of 0.075 M
sodium barbiturate buffer with 0.3% albumin are added at 4C.
Example 2 - Insolubilization of antibodies on capillary containing material with glutaric aldenyde: 0.6 g of cellulose acetate membrane is incubated with a solution of 100 ~ -2-- . : - - .:
'\ :
mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 10 ml of 0.1 M phosphate buffer with a pH of 6.8 for 30 minutes. When the solution has been decanted, the membrane is incubated in 10 ml of 1~ glutaric aldehyde solution during 30 minutes, followed by incubation in 10 ml of 1.0 M
methylamine for 15 -2a-.
~74Z28 minutes. Thereafter, the membrane is washed with 100 ml of sodium barbiturate and kept in the same buffer at 4C to which has been added 0.3% albumin.
Example 3 - Insolubilization of antibodies on capillary-containing carrier material by adsorption: 10 g of polyvinyl chloride - silica (Microporous Plastic Sheet, Amerace Esna Corp-oration, New York, N.Y.) is incubated over night at 4C with a solution of 500 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) in 100 ml of 0.1 M phosphate buffer at a pH of 6.8. After completed incubation, the material is washed with 1 litre of phosphate-buffered physiological NaCl at a pH of 7Ø The material is dried between filter papers.
Example 4 - Preparation of fluorescent antl dy_coniug-ates:
.. _ (a) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) 0.57 g of NaCl, 0.259 g of NaHCO3 and 0.049 g of Na2CO3-10H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 36 mg of fluoresceine isothiocyanate are added while stirring. The solution is then leftin the cool while stirring for 18 hours.
Thereafter, the mixture is dialyzed against phosphate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody conjugate is frozen.
(b) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment), 0.57 g of NaCI, 0.259 g of NaHCO3 and 0.049 g of Na2CO3 10H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 9 mg of rhodamine dissolved in 2 ml of acetone is added while stirring. The solution is leftin the cool while stirring for 18 hours. Thereafter, the mixture is dialyzed against phos-phate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody 1~74ZZ8 conjugate is frozen.
(c) 720 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment), 0.57 g NaCl, 0.259 g NaHCO3 and 0.049 g of Na2CO3-10 H2O are dissolved in 72 ml of distilled water. The reagent solution is cooled in an icebath, and 20 mg of dansyl chloride dissolved in 7 ml of acetone is added while stirring. The solution is left in the cool while stirring for 18 hours. Thereafter, the mixture is dialyzed against phosphate buffered physiological NaCl at a pH of 7.0 until the dialyzing fluid stops fluorescing. The resulting antibody conjugate is frozen.
Example 5 - Preparation of LDH iso-enzyme H4 antibody conjugate:
A suspension of 4.5 mg of LDH iso-enzyme H4 in ammonium sulfate is dialyzed against 0.1 M phosphate buffer at a pH of 6.8. While stirring, 2.25 mg of rabbit gammaglobulin against human gammaglobulin (IgG Fc fragment) and phosphate buffer are added to make a total volume of 1.35 ml. When all of the rabbit gammaglobulin has been dissolved, 45 ~1 of 1~ glutaric aldehyde solution is added dropwise, and the reaction is left to stand at room temperature for 2 hours. The mixture is then dialyzed against a sodium barbiturate buffer and the conjugate stored at 4C. Before use, the conjugate is diluted with 10 ml of barbiturate buffer to which 2% albumin has been added.
Example 6 - Checking of the insolubilization:
Two strips of material treated with antibodies in accordance with a preceding example, and two untreated strips are used for the test. One strip of each kind is incubated in a solution of 1 mg of human gammaglobulin (IgG) per ml of sodium barbiturate buffer. All strips are then washed with 20 ml of sodium barbiturate buffer containing 0.15 M NaC1, ten times in all, whereafter they are incubated in fluoresceine 1~42ZB
isothiocyanate antibody conjugate for 10 minutes. After repeated washings as in the foregoing, the strips are dried and inspected in ultraviolet light at a wavelength of 340 nm to establish that fluorescence is present only in the strips that contain the insolubilized antibodies, and that have been incubated in the IgG-solution. When checking strips according to Example 3, strips treated with bovine albumin are used as controls.
Immuno-chemical quantification Example A Diagnostic devices produced according to the present invention are used for immuno-chemical quantification for diagnostic purposes in the following manner:
The material to be tested is placed - in a known quantity, preferably between 1 ~1 and 3 ~1 - on the capillary-containing carrier material at the same time as corresponding quantities of a standard solution with a known content of the antigen to be tested and in various dilutions are placed next to the test solution.
For a mobile phase to facilitate capillary migration there could be used, e.g. 0.075 M sodium barbiturate buffer with an additive of 0.3% albumin.
The capillary migration can be performed in both open and closed test recipients, and the mobile phase can be made to move upwards and downwards. Preferably, the combination closed recipient and rising phase should be applied. At an optional time after initiation of the capillary migration, the moist carrier material is transferred to an antibody solution containing either fluoresceine isothiocyanate - or lactatedehydro-genase (LDH)-bound antibodies. The preferred incubation time for either of the solutions is 10 minutes. After incubation, the carrier material is washed under running water at 37C for 5 minutes, followed by incubation for 2 x 5 minutes in 20 ml of sodium barbiturate buffer, containing 0.2~ albumin and 0.15 M NaCl.
, : .
1~742Z8 .
To the extent that fluorescent antibodies are used for the identification, the migration distance of the antibody is measured in ultraviolet light at 340 nm. If LDH antibody solution is used, the carrier material is treated in a dye bath prepared as follows:
30 mg of nitroblue tetrazolium and 40 mg of NAD are dissolved in 56 ml of 0.05 M TRIS buffer at a pH of 7.4. 1.60 ml of 3.6 M lithium lactate; 5 ml of 0.06 M calcium cyanide, and a few grains of methylfenazonium metasulfate dissolved in 2 ml of distilled water are added to the mixtu~e. The capillary-containing material is incubated in the dye bath at 37C until a distinct coloring can be observed. The co~or development is due to the fact that the LDH catalyzes the reaction of lactate + NAD ~ pyruvate + NADH2 and that NADH2 quantitatively reduces nitroblue tetrazolium to an insoluble lilac-colored formazan.
The indicated, antigen-covered areas of the diagnostic device will then increase in magnitude with the increasing content of antigen of the tested samples. The matrix-bound antibody molecules retard the migration of the antigen molecules by exchange reactions during the capillary migration. The higher the concentration of antigens, the less retarding effect because the average time of an antigen molecule being bound to an antibody will decrease with an increasing concentration of antigens.
Example B Strips of the antibody-containing carrier material with capillaries and prepared according to the present invention are lowered to a determined depth of, for example, 3 mm in a small volume of a solution of the material to be tested.
The thus initiated capillary migration is allowed to continue for 3 - 20 minutes and then interrupted by removal of the strip -from the solution. The migration distance of the antigen is then measured as mentioned in Example A, and the result is evaluated according to Example A.
Claims (10)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for immuno-chemical quantification, which comprises bringing a sample containing antigen to be quantified into contact with a device for immuno-chemical quantification, comprising a capillary-containing, porous carrier to which anti-bodies have been bound, allowing a capillary migration to occur and then assaying the antigen-containing areas of the sample-wetted device by the addition of antibodies bound to a color indi-cator.
2. A method according to Claim 1, wherein the anti-bodies are bound to the carrier by adsorption, absorption or covalent bonding.
3. A method according to Claim 2, wherein the anti-bodies are covalently bonded to the carrier via cyanogen bromide or glutaric aldehyde.
4. A method according to Claim 1, wherein the carrier comprises at least one capillary containing porous material which is cellulose fibre, starch, or a matrix of dextran chains, or a plastics material or ceramic material.
5. A method according to Claim 4, wherein the carrier is polyvinyl chloride or a silica modified polymer in which finely divided silica is substantially uniformly embedded in a continuous matrix which is polyvinylchloride, vinyl chloride-propylene copolymer of vinyl chloride-vinyl acetate copolymer.
6. A method according to Claim 1, wherein the device is in the form of a test strip or disc.
7. A method according to Claim 6, wherein the carrier is a test strip of a mixture of polyvinyl chloride and silica and the antibodies are antibodies against human gammaglobulin.
8. A method according to Claim 1, 2 or 3, wherein the color indicator is a fluorescent compound or an enzyme that catalyzes a color-developing reaction.
9. A method according to Claim 1, 2 or 3, wherein the color indicator is fluoresceine isothiocyanate, rhodamine or dansylchloride.
10. A method according to Claim 1, 2 or 3, wherein the device is a test strip of a polyvinylchloride-silica mixture onto which have been adsorbed antibodies against human gamma-globulin and the antibody-bound color indicator is fluoresceine-isothiocynanate.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE7500841A SE388694B (en) | 1975-01-27 | 1975-01-27 | WAY TO PROVIDE AN ANTIGEN EXV IN SAMPLES OF BODY WHEATS, USING POROST BERAR MATERIAL BONDED OR ADSORBING ANTIBODIES |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1074228A true CA1074228A (en) | 1980-03-25 |
Family
ID=20323501
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA243,521A Expired CA1074228A (en) | 1975-01-27 | 1976-01-14 | Diagnostic devices and method for immuno-chemical quantification |
Country Status (18)
Country | Link |
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US (1) | US4168146A (en) |
JP (2) | JPS51101122A (en) |
AT (1) | AT347601B (en) |
AU (1) | AU508642B2 (en) |
BE (1) | BE837896A (en) |
CA (1) | CA1074228A (en) |
DE (1) | DE2603004C2 (en) |
DK (1) | DK149968C (en) |
FI (1) | FI760074A (en) |
FR (1) | FR2298798A1 (en) |
GB (1) | GB1502563A (en) |
IL (1) | IL48722A (en) |
IT (1) | IT1123602B (en) |
NL (1) | NL7600128A (en) |
NO (1) | NO760233L (en) |
NZ (1) | NZ179830A (en) |
SE (1) | SE388694B (en) |
ZA (1) | ZA76421B (en) |
Families Citing this family (278)
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US5622871A (en) * | 1987-04-27 | 1997-04-22 | Unilever Patent Holdings B.V. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US4233402A (en) * | 1978-04-05 | 1980-11-11 | Syva Company | Reagents and method employing channeling |
AU531777B2 (en) * | 1978-04-05 | 1983-09-08 | Syva Co. | Label/solid conjugate immunoassay system |
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- 1975-12-24 IL IL48722A patent/IL48722A/en unknown
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1976
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- 1976-01-14 FI FI760074A patent/FI760074A/fi not_active Application Discontinuation
- 1976-01-14 CA CA243,521A patent/CA1074228A/en not_active Expired
- 1976-01-15 US US05/649,248 patent/US4168146A/en not_active Expired - Lifetime
- 1976-01-23 AU AU10563/76A patent/AU508642B2/en not_active Expired
- 1976-01-23 IT IT19527/76A patent/IT1123602B/en active
- 1976-01-26 ZA ZA421A patent/ZA76421B/en unknown
- 1976-01-26 DK DK029176A patent/DK149968C/en not_active IP Right Cessation
- 1976-01-26 BE BE163789A patent/BE837896A/en unknown
- 1976-01-26 GB GB2936/76A patent/GB1502563A/en not_active Expired
- 1976-01-26 NO NO760233A patent/NO760233L/no unknown
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- 1976-01-26 NZ NZ179830A patent/NZ179830A/en unknown
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IT1123602B (en) | 1986-04-30 |
ZA76421B (en) | 1977-01-26 |
AU508642B2 (en) | 1980-03-27 |
NL7600128A (en) | 1976-07-29 |
IL48722A (en) | 1978-07-31 |
AT347601B (en) | 1979-01-10 |
FI760074A (en) | 1976-07-28 |
DK29176A (en) | 1976-07-28 |
JPS6134097B2 (en) | 1986-08-06 |
DK149968B (en) | 1986-11-03 |
DK149968C (en) | 1987-06-29 |
IL48722A0 (en) | 1976-02-29 |
DE2603004A1 (en) | 1976-07-29 |
BE837896A (en) | 1976-05-14 |
NO760233L (en) | 1976-07-28 |
US4168146A (en) | 1979-09-18 |
JPS5990056A (en) | 1984-05-24 |
GB1502563A (en) | 1978-03-01 |
NZ179830A (en) | 1978-12-18 |
FR2298798A1 (en) | 1976-08-20 |
SE388694B (en) | 1976-10-11 |
DE2603004C2 (en) | 1985-10-10 |
AU1056376A (en) | 1977-07-28 |
FR2298798B1 (en) | 1981-12-24 |
SE7500841L (en) | 1976-07-28 |
ATA49076A (en) | 1978-05-15 |
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