GB2124231A - Solid phase reagent for immunoassay - Google Patents
Solid phase reagent for immunoassay Download PDFInfo
- Publication number
- GB2124231A GB2124231A GB08215071A GB8215071A GB2124231A GB 2124231 A GB2124231 A GB 2124231A GB 08215071 A GB08215071 A GB 08215071A GB 8215071 A GB8215071 A GB 8215071A GB 2124231 A GB2124231 A GB 2124231A
- Authority
- GB
- United Kingdom
- Prior art keywords
- solid phase
- beads
- reagent
- antibody
- phase support
- 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.)
- Granted
Links
- 239000007790 solid phase Substances 0.000 title claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 12
- 238000003018 immunoassay Methods 0.000 title claims abstract description 9
- 238000000576 coating method Methods 0.000 claims abstract description 17
- 239000011248 coating agent Substances 0.000 claims abstract description 15
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 102000036639 antigens Human genes 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 10
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 10
- 239000011324 bead Substances 0.000 claims description 21
- 108010010803 Gelatin Proteins 0.000 claims description 9
- 229920000159 gelatin Polymers 0.000 claims description 9
- 239000008273 gelatin Substances 0.000 claims description 9
- 235000019322 gelatine Nutrition 0.000 claims description 9
- 235000011852 gelatine desserts Nutrition 0.000 claims description 9
- 239000004793 Polystyrene Substances 0.000 claims description 6
- 229920002223 polystyrene Polymers 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 238000000034 method Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 150000004756 silanes Chemical class 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
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
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/552—Glass or silica
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a reagent for use in immunoassays, comprising a solid phase support, a primary coating of antibody or antigen on the solid phase support, and a secondary coating of immunologically inert protein over the primary coating.
Description
SPECIFICATION
Solid phase reagent for immunoassay
This invention relates to a solid phase reagent for use in immunoassays.
Antibodies and antigens are commoniy coupled to solid phase supports for use in immunoassays.
Various water-insoluble carrier materials can be used as the solid phase support without substantial loss of biological activity. The solid phase immobilised reagent can be readily separated, e.g. by centrifugation, at an appropriate stage in the immunoassay procedure.
Any coupling procedure, for attaching the antibody or antigen to the solid phase support, is by necessity carried out in aqueous medium. However, for convenient handling and stability in storage, it is of value to present the chosen solid phase in a dry form. If a conventional solid phase immunological reagent is simply dried without any preliminary treatment, it is found that the immunological properties deteriorate to an unacceptable extent. An object of the present invention is accordingly to provide a stable, solid phase immunological reagent in dry form.
We have now found that stabilisation of antibody- or antigen-coated solid phase supports can be achieved by employing a proteinaceous coating procedure. Thus, the present invention provides a reagent for use in immunoassays, comprising a solid phase support, a primary coating of antibody or antigen on the solid phase support, and a secondary coating of immunologically inert protein over the primary coating.
In principle, any water-insoluble solid phase support can be used in the present invention.
Preferred examples are polystyrene and glass beads. Both covalent and non-covalent bonding methods are known for such solid phase supports and these include the use of silane derivatives as an example of covalent bonding, or simple incubation with a solution containing the appropriate concentration of antibody or antigen as an example of non-covalent bonding.
The protein employed in the secondary coating should have certain characteristics such as; it must be immunologically inert with respect to the antibody or antigen in the primary coating; it must contain no proteolytic enzymes and it must be cheap and readily available in large quantity. Suitable proteins which have been examined include bovine serum albumin, gelatin and degraded gelatin. Such proteins can be bonded to the primary coating (including surfaces which are uncoated during the primary coating process) by simple incubation at room temperature.
Once the secondary coating of protein has been applied, the reagent is finally dried, preferably in a current of warm air or under vacuum. The product can then be conveniently stored at 40C in a stoppered container and under such conditions it has been found to substantially retain its immunological properties.
The following examples illustrate the invention.
EXAMPLE 1
Anti serum containing antibody to hepatitis B surface antigen (anti HBs) was diluted 1:500 in 0.1 M phosphate buffer, pH 7.5. This was added to polystyrene beads of 6.4 mm diameter, previously washed with ethanol. The coating process was allowed to proceed for approximately 10 hours at 40C.
The beads were then washed 3 (three) times with deionised water (DIW).
To obtain stable beads, DlW washed beads were placed in 1% aqueous, protein solution and incubated at room temperature for approximately 1 5 minutes and finally air dried. Proteins examined included gelatin bovine serum albumin BSA (fraction V).
Assay Procedure: Beads dried in the described manner were utilised in a simultaneous sandwich assay with radio-labelled antibody.
TABLE 1 %Bound1 S/N2
Washed beads (wet) 14 44
BSA dried beads 15.1 47
Gelatin dried beads 1 3.5 43
Dried beads without addition 7.0 23
1. % bound refers to the percentage of total radioactivity
bound to a positive control in the simultaneous assay.
2. S/N: Signal to noise ratio is the number of counts of the
postive control divided by the counts of the negative control.
EXAMPLE 2
Beads of 6 mm diameter of semi-borosilicate glass had antibody to hepatitis B surface antigen coupled covalently via a known procedure involving silane derivatisation, glutaraldehyde activation and protein coupling. Glass beads having anti HBs thus coupled were dried in air following immersion in 1%
BSA in 0.1 M phosphate buffer pH 7.5 for approximately 16 hours at 40C.
TABLE II
% Bound S/N
Washed beads 11.76 22.8
BSA dried beads 12.24 22.8
Dried without addition 7.2 10.1
Beads, both glass and polystyrene, dried by the described procedure have been shown to be stable
for at least several months, i.e. their functionality in an immunoassay is retained, when stored at 40C.
EXAMPLE 3
Antibody (anti HBs) purified by chromatographic techniques was adsorbed onto polystyrene beads
previously washed with ethanol. The method of absorption was essentially that of Example 1. Antibody
solution was incubated with polystyrene beads for approximately 10 hours at 40C. The beads were then
washed 3 (three) times with deionised water. Beads were then immersed in BSA or partially degraded
gelatin for 15 minutes prior to drying in a current of air or under vacuum.
TABLE III
% Bound S/N Without addition 11 203 BSA 21 389 Partially degraded gelatin air dried 29 446 Partially degraded gelatin 34 502 vaccum dried
Claims (3)
1. A reagent for use in immunoassays, comprising a solid phase support, a primary coating of antibody or antigen on the solid phase support, and a secondary coating of immunologically inert protein over the primary coating.
2. A reagent as claimed in claim 1 , wherein the solid phase support comprises polystyrene beads or glass beads.
3. A reagent as claimed in claim 1 or 2, wherein the immunologically inert protein of the secondary coating is gelatin or bovine serum albumin or partially degraded gelatin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB08215071A GB2124231B (en) | 1982-05-24 | 1982-05-24 | Solid phase reagent for immunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB08215071A GB2124231B (en) | 1982-05-24 | 1982-05-24 | Solid phase reagent for immunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| GB2124231A true GB2124231A (en) | 1984-02-15 |
| GB2124231B GB2124231B (en) | 1985-10-02 |
Family
ID=10530574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB08215071A Expired GB2124231B (en) | 1982-05-24 | 1982-05-24 | Solid phase reagent for immunoassay |
Country Status (1)
| Country | Link |
|---|---|
| GB (1) | GB2124231B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985000664A1 (en) * | 1983-07-27 | 1985-02-14 | Cooper-Lipotech, Inc. | Large-liposome agglutination reagent and method |
| EP0140489A1 (en) * | 1983-08-05 | 1985-05-08 | Wako Pure Chemical Industries, Ltd. | Method for stabilizing immunologically active substances immobilized on an insoluble carrier and their use in the preparation of reagents for measuring physiologically active substances |
| EP0170746A1 (en) * | 1984-08-07 | 1986-02-12 | Covalent Technology Corporation | Biologically active material test |
| WO1986004095A1 (en) * | 1985-01-11 | 1986-07-17 | Unilever Nv | Preparation of reagents |
| DE3811659C1 (en) * | 1988-04-07 | 1989-11-16 | Henning Berlin Gmbh Chemie- Und Pharmawerk, 1000 Berlin, De | |
| EP0303980A3 (en) * | 1987-08-20 | 1989-11-29 | Hoechst Japan Limited | Carrier coated with antigen or antibody |
| DE10323901A1 (en) * | 2003-05-26 | 2005-01-05 | Institut Virion/Serion Gmbh | Method and test means for the examination and / or detection of biomolecules and / or active substances in liquid samples |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2016687A (en) * | 1978-03-20 | 1979-09-26 | Abbott Lab | Sugar Coated Reagents for Solid Phase Immunoassay |
| GB2099578A (en) * | 1981-04-29 | 1982-12-08 | Ciba Geigy Ag | Supported reagents and kits for immunological analysis |
-
1982
- 1982-05-24 GB GB08215071A patent/GB2124231B/en not_active Expired
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2016687A (en) * | 1978-03-20 | 1979-09-26 | Abbott Lab | Sugar Coated Reagents for Solid Phase Immunoassay |
| GB2099578A (en) * | 1981-04-29 | 1982-12-08 | Ciba Geigy Ag | Supported reagents and kits for immunological analysis |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1985000664A1 (en) * | 1983-07-27 | 1985-02-14 | Cooper-Lipotech, Inc. | Large-liposome agglutination reagent and method |
| EP0140489A1 (en) * | 1983-08-05 | 1985-05-08 | Wako Pure Chemical Industries, Ltd. | Method for stabilizing immunologically active substances immobilized on an insoluble carrier and their use in the preparation of reagents for measuring physiologically active substances |
| EP0170746A1 (en) * | 1984-08-07 | 1986-02-12 | Covalent Technology Corporation | Biologically active material test |
| WO1986004095A1 (en) * | 1985-01-11 | 1986-07-17 | Unilever Nv | Preparation of reagents |
| EP0192320A1 (en) * | 1985-01-11 | 1986-08-27 | Unilever Plc | Preparation of reagents |
| EP0303980A3 (en) * | 1987-08-20 | 1989-11-29 | Hoechst Japan Limited | Carrier coated with antigen or antibody |
| DE3811659C1 (en) * | 1988-04-07 | 1989-11-16 | Henning Berlin Gmbh Chemie- Und Pharmawerk, 1000 Berlin, De | |
| DE10323901A1 (en) * | 2003-05-26 | 2005-01-05 | Institut Virion/Serion Gmbh | Method and test means for the examination and / or detection of biomolecules and / or active substances in liquid samples |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2124231B (en) | 1985-10-02 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19920524 |