EP0455807A4 - Methods of coating viral antigens in the presence of nonionic detergents and acidic ph onto a solid phase - Google Patents
Methods of coating viral antigens in the presence of nonionic detergents and acidic ph onto a solid phaseInfo
- Publication number
- EP0455807A4 EP0455807A4 EP19910902536 EP91902536A EP0455807A4 EP 0455807 A4 EP0455807 A4 EP 0455807A4 EP 19910902536 EP19910902536 EP 19910902536 EP 91902536 A EP91902536 A EP 91902536A EP 0455807 A4 EP0455807 A4 EP 0455807A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- solid phase
- acidic
- coating
- viral antigens
- onto
- 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.)
- Withdrawn
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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56988—HIV or HTLV
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Definitions
- This invention relates to a process to coat viral antigens in the presence of nonionic detergent and acidic pH, onto a solid phase, to enhance the activity of bound viral antigens.
- Antibodies are immobilized on solid phases, such as microtiter plates and latex beads, for use in research and diagnostic assays. Detection of monoclonal antibodies to membrane-associated proteins, however, presents a problem, since nonionic detergents used to
- This invention relates to a method to increase the concentration of viral antigen on a solid phase by coating the solid phase with lu viral antigen in the presence of nonionic detergents, such as Triton X-l ⁇ U or NP4U, in acidic pH.
- nonionic detergents such as Triton X-l ⁇ U or NP4U
- Antigen was diluted into a coating buffer and added to lb particles.
- the coating buffer was preferably comprised of 0.1 M acetate, pH b.O with 5* NP40, however, other nonionic detergents, such as Triton X-100, can be used. It was observed, however, that one nonionic detergent, Tween, did not result in a sufficiently high concentration of antigen to conduct an immunoassay.
- 2U tion of detergent can range from .2% to 10 , with the optimum concentration from between about 1 and 10%. It was observed that pH can range from 4.U to 6.U, with the optimum pH at 5.0.
- Buffer A was comprised of b 0.1 M acetate, at a pH 5.0.
- Buffer B was comprised of 0.1 M acetate, 0.1 M NaCl, at a pH 5.0.
- Buffer C was comprised of 0.1 M acetate, 0.1 M NaCl, 0.2% NP40, at a pH 5.0.
- HIV negative serum was designated "617", positive was designated “618", and high positive serum was designated “R-J 1825”.
- SDB sample dilution buffer
- the dialyzed preparation was coated at 1/2 dilution with coating buffers A, B, and C on to 1 ml of 2.5% carboxylated paramagnetic particles (3.5 urn). Particles were incubated overnight at 2 Q 8 lb C. The particles were pelleted, the supernatent was discarded, and the particles were resuspended in 1% normal goat serum (NGS) in 0.1 M acetate, at a pH 5.0.
- NGS normal goat serum
- the coated particles were incubated for two hours at room temperature, then washed three times with 1 L isotonic buffered U saline (IBS).
- IBS isotonic buffered U saline
- the coating buffer "C” comprised of 0.1 M acetate, 0.1 M NAC1 , 30 and 0.2A NP40, and resulted in a greater concentration of viral antigens on the solid phase.
- AIDS antigens coated in the presence of nonionic detergents and acidic pH resulted in viral coated particles witn enhanced activity over particles coated using other buffers.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- AIDS & HIV (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Detergent Compositions (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
This invention relates to a process to enhance the activity of viral antigens that are bound to a solid phase. The viral antigens are coated to a solid phase in the presence of a nonionic detergeent, such as Triton X-100 or NP40, in a solution having an acidic pH.
Description
Methods of Coating Viral Antigens in the Presence of Nonionic Detergents and Acidic pH onto a Solid Phase
5 BACKGROUND OF THE INVENTION 1. Field of the Invention
This invention relates to a process to coat viral antigens in the presence of nonionic detergent and acidic pH, onto a solid phase, to enhance the activity of bound viral antigens.
1U 2. Description of the Prior Art
Antibodies are immobilized on solid phases, such as microtiter plates and latex beads, for use in research and diagnostic assays. Detection of monoclonal antibodies to membrane-associated proteins, however, presents a problem, since nonionic detergents used to
ID solubilize membrane antigens retard or prevent protein binding to latex microtiter wells. G. Urexler et al . , A Rapid and Simple Method for Efficient Coating of Microtiter Plates Using Low Amounts of Antigen in the Presence of Detergent, 95 J. Immuno. Metn. 117 (1986). Gardas et al., Coating of Proteins in the Presence of
2ϋ Detergents, 10b J. Immuno. Meth. 251 (1988). This problem nas been addressed in the past with a variety of solutions. One investigator suggested using glutoraldehyde to immobilize antigen in the presence of detergent. G. Evans, 73 J. Immuno. 427 (1984). Another investigator suggested the use of Bouin's fluid to increase the
2b binding of antigen -solubilize with a nonionic detergent. Notebooπ et al., 7b J. Immuno. Meth. 141 (1984). Still another investigator disclosed a process to increase the binding of antigen to a microtiter plate using beads to adsorb the residual nonionic detergent. See Drexler, supra. One author, however, suggested that iu proteins can oe effectively bound to latex surfaces provided that a detergent witn a hign critical micelle concentration (CMC), i.e. the concentration ot a detergent which allows the formation of micelles, is used. See Garαas, supra. This investigator tested the nonionic
detergent, Triton X-100, among other detergents, at a concentration of .2 or .b% and a pH of 9.6. From this experiment, one skilled in the relevant art would conclude that this nonionic detergent, i.e. Triton X-100, inhibits binding at one of the lowest concentrations b and thus would appear to be a poor choice for use in an immunoassay.
SUMMARY OF THE INVENTION
This invention relates to a method to increase the concentration of viral antigen on a solid phase by coating the solid phase with lu viral antigen in the presence of nonionic detergents, such as Triton X-lύU or NP4U, in acidic pH.
DETAILED DESCRIPTION OF THE INVENTION
Antigen was diluted into a coating buffer and added to lb particles. The coating buffer was preferably comprised of 0.1 M acetate, pH b.O with 5* NP40, however, other nonionic detergents, such as Triton X-100, can be used. It was observed, however, that one nonionic detergent, Tween, did not result in a sufficiently high concentration of antigen to conduct an immunoassay. The concentra-
2U tion of detergent can range from .2% to 10 , with the optimum concentration from between about 1 and 10%. It was observed that pH can range from 4.U to 6.U, with the optimum pH at 5.0. Example 1 - Coating of HIV antigen onto magnetic microparticles using NP4D and acid pH.
2b 1 ml of 4 X 10**-* particles/ml of 3 - 4 micron carboxylated magnetic particles were pelleted and the supernatent was carefully removed and discarded. Tne particles were resuspended in 1 ml of 0.1 M acetate buffer, pH 5.0, containing b% NP40 and 250 ug/ml of HIV antigen. The particles were tumbled end over end at 5 - 6 rpm
3U overnignt at room temperature. The particles were pelleted and supernatent was discarded. The particles were resuspended in 1 ml oτ PBS, pelleted and tne supernatent was discarded. This was re- peated two times. The particles were resuspended in 1 ml of diluent (0.1M acetate, pH b.O, U.5M NAC1 , 1% NaN ) and stored at 2 - 8°.
Example 2 - Comparison of coating conditions.
5 ml of solution containing HIV antigen, was dialyzed against three changes of four liters of 0.1 M acetate pH 5.0. Three different coating buffers were assessed: Buffer A was comprised of b 0.1 M acetate, at a pH 5.0. Buffer B was comprised of 0.1 M acetate, 0.1 M NaCl, at a pH 5.0. Buffer C was comprised of 0.1 M acetate, 0.1 M NaCl, 0.2% NP40, at a pH 5.0.
HIV negative serum was designated "617", positive was designated "618", and high positive serum was designated "R-J 1825". These 10 sera were diluted 1/100 in sample dilution buffer (SDB) (32% calf serum, 0.02M phosphate, pH 7.4, O.faM NAC1 , 1% NP40, and 0.1% NaN3).
The dialyzed preparation was coated at 1/2 dilution with coating buffers A, B, and C on to 1 ml of 2.5% carboxylated paramagnetic particles (3.5 urn). Particles were incubated overnight at 2 Q 8 lb C. The particles were pelleted, the supernatent was discarded, and the particles were resuspended in 1% normal goat serum (NGS) in 0.1 M acetate, at a pH 5.0.
The coated particles were incubated for two hours at room temperature, then washed three times with 1 L isotonic buffered U saline (IBS).
20ul of particles (1 X 107 parts/mL) were added to 50 ul of sample (1/lUO in SBD). This mixture was incubated for 30 minutes at 37°C. The solid phase was washed to remove unbound material. 50 ul of goat anti-human beta glactosidase labeled antibody was added to 5 the mixture. This mixture was then incubated for 15 minutes at 37°C. The mixture was washed to remove unbound antibody and 50 ul substrate (B-D-galactopyranoside) was added. The fluorescence was read at 2 and 14 minutes using a Pandex" FCA instrument. The results are reported below. D
Tabl e 1
S D B 617 618 1825
10 Tabl e 2
S D B 617 618 1825
Buffer A 8 781.3 + 145.5 5449.7 (7.0) 19738 (25.3) Buffer B 7.5 970.7 +_ 130.6 5287.7 (5.4) 23484 (24.2) Buffer C 0 611.7 + 5y .5 5136.7 (8.4) 15434 (25.2) lb Cnannel Report : 400/450 Gai n Setti ng : 10 Read Time : 100 ms
Table 3
2U
25 Delta Read (Table 2 - Table 1)
Conclusions
The coating buffer "C" comprised of 0.1 M acetate, 0.1 M NAC1 , 30 and 0.2A NP40, and resulted in a greater concentration of viral antigens on the solid phase. AIDS antigens coated in the presence of nonionic detergents and acidic pH resulted in viral coated particles witn enhanced activity over particles coated using other buffers.
Claims
I Cl ai m:
1. An active solid phase coated with viral antigen made by the process comprising:
5 Coating a solid phase with viral antigen in an acidic suspension of a nonionic detergent, and a buffer.
2. The active solid phase of Claim 1 wherein the concentration of said detergent ranges from .2 to 10%.
3. The active solid phase of Claim 1 wherein said solid phase is a 10 paramagnetic microparticle.
4. The active solid phase of Claim 1 wherein said detergent is NP40 or Triton X-100. b. The active solid phase of Claim 1 wherein the pH of the suspension ranges from about 4.0 to 6.0. lb 6. Tne active solid phase of Claim 1 wherein the pH is about 5.0.
2U
b
3D
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US44363589A | 1989-11-29 | 1989-11-29 | |
US443635 | 1989-11-29 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0455807A1 EP0455807A1 (en) | 1991-11-13 |
EP0455807A4 true EP0455807A4 (en) | 1992-07-08 |
Family
ID=23761588
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19910902536 Withdrawn EP0455807A4 (en) | 1989-11-29 | 1990-11-28 | Methods of coating viral antigens in the presence of nonionic detergents and acidic ph onto a solid phase |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0455807A4 (en) |
JP (1) | JPH04507003A (en) |
AU (1) | AU7160491A (en) |
CA (1) | CA2045682A1 (en) |
WO (1) | WO1991008311A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110596374A (en) * | 2019-09-19 | 2019-12-20 | 潍坊市康华生物技术有限公司 | Method for coupling magnetic particles and antigen |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0136798A2 (en) * | 1983-08-25 | 1985-04-10 | Biotech Research Laboratories Inc. | Titre plate and assay kit for detection of antibodies in human serum and their production and use |
EP0316495A1 (en) * | 1987-11-19 | 1989-05-24 | Iaf Biochem International Inc. | Synthetic peptides useful for the detection of aids-related antibodies in human sera |
EP0337785A1 (en) * | 1988-04-14 | 1989-10-18 | Johnson & Johnson Clinical Diagnostics, Inc. | Diagnostic kit and method for rapid detection of antibodies |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4870003A (en) * | 1987-06-15 | 1989-09-26 | Coulter Corporation | Simultaneous enzyme immunoassay for detecting antigen and/or antibody in humans |
-
1990
- 1990-11-28 CA CA 2045682 patent/CA2045682A1/en not_active Abandoned
- 1990-11-28 AU AU71604/91A patent/AU7160491A/en not_active Abandoned
- 1990-11-28 WO PCT/US1990/006922 patent/WO1991008311A1/en not_active Application Discontinuation
- 1990-11-28 JP JP50267491A patent/JPH04507003A/en active Pending
- 1990-11-28 EP EP19910902536 patent/EP0455807A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0136798A2 (en) * | 1983-08-25 | 1985-04-10 | Biotech Research Laboratories Inc. | Titre plate and assay kit for detection of antibodies in human serum and their production and use |
EP0316495A1 (en) * | 1987-11-19 | 1989-05-24 | Iaf Biochem International Inc. | Synthetic peptides useful for the detection of aids-related antibodies in human sera |
EP0337785A1 (en) * | 1988-04-14 | 1989-10-18 | Johnson & Johnson Clinical Diagnostics, Inc. | Diagnostic kit and method for rapid detection of antibodies |
Non-Patent Citations (2)
Title |
---|
BERGMEYER H.U. 'METHODS OF ENZYMATIC ANALYSIS, VOL. 10' 1986 , VCH , WEINHEIM,DE PAGES 469-483 " Human T-lymphotropic virus III / Lymphadenopathy-associated virus antibodies " V. Erfle et al. * |
See also references of WO9108311A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP0455807A1 (en) | 1991-11-13 |
CA2045682A1 (en) | 1991-05-30 |
WO1991008311A1 (en) | 1991-06-13 |
JPH04507003A (en) | 1992-12-03 |
AU7160491A (en) | 1991-06-26 |
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