CA2045682A1 - 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
- CA2045682A1 CA2045682A1 CA 2045682 CA2045682A CA2045682A1 CA 2045682 A1 CA2045682 A1 CA 2045682A1 CA 2045682 CA2045682 CA 2045682 CA 2045682 A CA2045682 A CA 2045682A CA 2045682 A1 CA2045682 A1 CA 2045682A1
- Authority
- CA
- Canada
- 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.)
- Abandoned
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
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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (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
W O 91/08311 2 ~ 2 PCr/US90/06922 Methods of Coating Viral Antigens in the Presence of Nonionic Detergents and Acidic pH onto a Solid Phase S BACKGROUN~ 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 ennance the activity of bound viral antigens.
1~ 2. ~escription of the Prior Art AntiDodies are immobilized on solid phases, such as microtiter - ~lates and latex beads, for use in research and diagnostic assays.
Detection of monoclonal anti~odies to membrane-associated proteins, however, presents a problem, since nonionic detergents used to 1~ solubilize membrane antigens retard or prevent protein binding to latex microtiter wells. G. ~rexler et al., pld and Simple Method for Efficient Coating of Microtiter Plates Using Low Amr,unts of Antigen in tne ~resence of Detergent, 95 J. Immuno. Metn. 117 ~ .
(19~). Garaas et al., Coating of Proteins in the Presence of 2u ~etergents, 10~ J. Immuno. Meth. ~51 (198B). Tnis problem nas been addressed in the past with a variety of solutions. One investigator suyyested using glutoraldehyde to immobilize antigen in the presence of detergent. ~. Evans, 73 J. Immuno. 427 (lY84). Another investigator suggested the use of Bouin's fluid to increase the binding of antigen solubili~e with a nonionic detergent. Noteboo~
et al., 7~ J. lmmuno. Meth. 141 (19~4). ~till another investigator ! aisclosed a process to increase the binding of antigen to a microtiter plate using beads to adsorb the residual nonionic aeter~ent. ~ee Drexler, supra. One author, however, suggested that ! ~U proteins can De effectively bound to latex surfaces provided that a detergent witn a nigh critical micelle concentration (CMC), i.e. the concentration ot a detergent wnich allows the formation of micelles, is used. ~ee ~araas, supra. This investigator tested the nonionic :. , ' - .
:
.
' `: ' ' , :: .
WO 91/08311 PCI~/US90/06922 2~Llai~
detergent, Triton X-lOO, among other detergents, at a concentration of .~ or .bP 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-lUU, inhioits binding at one of the lowest concentrations and thus would appear to be a poor choice for use in an immunoassay.
SUM~ARY OF THE INVENTION
This invention relates to a method to increase the concentration of viral antigen on a solid phase ~y coating the solid phase with lU viral antigen in the presence of nonionic detergents, sucn as Triton X-lOU or NP4~, in acidic pH.
~ETAILEU ~ESCRIPTION OF rH~ INVENTI~N
Antiyen was diluted into a coating buffer and added to l~ particles. Tne coatin~ buffer was preferably comprised of 0.1 ~l acetate, pH ~.U with 5~ NP4U, however, other nonionic detergents, sucn as Triton X-lUO, 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 deter~ent can range from .2% to lO~, with the optimu~
concentration from between about l and lO~b. It was observed that p~
can range from 4.U to 6.U, with the optimum pH at 5Ø
; Exa~ple 1 - Coating of HIV antigen onto magnetic microparticles using NP4U and acid pH.
l ml of 4 X 108 particles/ml of 3 - 4 micron carboxylated magnetic particles were pelleted and the supernatent was carefully removea and discarded. Tne particles were resuspended in l ml of U.l M acetate buffer, pH S.U, containing ~% NP40 and 250 ug/ml of HIV antigen. The particles were tumbled end over end at S - 6 rpm ~U o~ernignt at room temperature. The particles were pelleted and ;~ supernatent was discaraed. The particles were resuspended in 1 ml ot PBS, pelleted and tne su~ernatent was discarded. This was re-peatea two ~imes. The particles were resuspended in l ml of diluen~
(0.1~ acetate, pH ~.0, U.~M NACl, 1% NaN3) and stored at 2 - ~.
" .
- - , - - . . ~ . , :
WO 91/08311 2 ~ ~ ~ & ~ 2 PCI~/US90/06922 ... ;,................................................................ .
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 5Ø Three different coating buffers were assessed: Buffer A was comprised of ù.1 M acetate, at a pH SØ Buffer B was comprised of 0.1 M
acetate, 0.1 M NaCl, at a pH 5Ø Buffer C was comprised of 0.1 M
acetate, 0.1 M NaCl, 0.2~ NP4~, at a pH 5Ø
HIV negative serum was designated "617", positive was designated "61~", and high positive serum was designated "R-J 1825". These 1~ sera were diluted 1/100 in sample dilution buffer (SDB) (32% calf serum, ~.02M phosphate, pH 7.4, 0.5M NACl, 1% NP40, and 0.1% NaN3).
- The dialyzed preparation was coated at 1/2 dilution with coating ~uffers A, B, and C on to 1 ml of 2.5% carboxylated paramagnetic particles (3.~ um). Particles were incubated overnight at 2 Q 8 13 C. The particles were pelleted, the supernatent was discarded, and tne particles were resuspended in 1% normal goat serum (NGS) in 0.1 M acetate, at a pH 5Ø
, Tne coated particles were incubated for two hours at room temperature, then washed tnree times with 1 mL isotonic buffered 2u saline (IBS).
; 2uul of particles (1 X 107 parts/mL) were added to 50 ul of sample (1/1~0 in SBD). Tnis mixture was incubated for 30 minutes at ~ 37C. Tne solid phase was washed to remove unbound material. 50 ul -1 of goat anti-human beta glactosidase labeled antibody was added to ` 2~ the mixture. This mixture was then incubated for 15 minutes at37C. 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.
, :
, ' ', . ! , .
'. . : . ~ . '' ~ ' . ' . ' W O 91/083tl PcT/US9o/o6922 2 ~ j ~ 8 2 Table _l S ~ B 617 618 1825 Buffer A U.S 601.3 + 111.7 4329 t7.2) 16191 ~26.9) ~uffer B 3 750.3 + 98.5 4287 (5.7) 19059 t25.4) Buffer C 0 473 + 36.34096.3 (8.7) 12669 (26.8) Channel Report: 4U0/450 Gain Setting: lU
Read Time: lUU ms 1U Table 2 s u e ~17 618 1825 _ _ Buffer A 8 7~1.3 + 145.5 5449.7 (7.U) 19738 (2S.3) ~uffer ~ 7.5 970.7 + 130.6 52~7.7 (5.4) 23434 (24.2) ~utfer C U 611.7 + 5Y.5 5136.7 (8.4) 15434 (2~.2) 1~ Cnannel ~eporl: 4UU/4~0 ~ain Setting: lU . .
Read Time: lUU ms Table 3 ~U S D B 617 61~ 1825 ~uffer A 7.5 1~01120.7 (6.2) 3517 (19.7) ~uffer ~ 4.~ 22u.41000.7 (4.5) 4425 (20.1) Buffer C U 13B.71040.4 (7.5) 2765 (19.9) . :
Uelta ~eaa (Ta~le 2 - Table 1) .
' .
Conclusions The coating Duffer "C" comprised of 0.1 ~ acetate, û.1 M NACl, ~U and U.~ 40, and resulted in a greater concentration of viral antiyens on the solia phase. AIDS antigens coated in the presence of nonionic aetergents and acidic pH resultea in viral coated ~articles witn enhancea activity over particles coated using other buffers.
:
,: . - -- : . . - - : , . : : : -': ' '': ~ - . ' : ~ : ' : ' :
., - - , . . .
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 ennance the activity of bound viral antigens.
1~ 2. ~escription of the Prior Art AntiDodies are immobilized on solid phases, such as microtiter - ~lates and latex beads, for use in research and diagnostic assays.
Detection of monoclonal anti~odies to membrane-associated proteins, however, presents a problem, since nonionic detergents used to 1~ solubilize membrane antigens retard or prevent protein binding to latex microtiter wells. G. ~rexler et al., pld and Simple Method for Efficient Coating of Microtiter Plates Using Low Amr,unts of Antigen in tne ~resence of Detergent, 95 J. Immuno. Metn. 117 ~ .
(19~). Garaas et al., Coating of Proteins in the Presence of 2u ~etergents, 10~ J. Immuno. Meth. ~51 (198B). Tnis problem nas been addressed in the past with a variety of solutions. One investigator suyyested using glutoraldehyde to immobilize antigen in the presence of detergent. ~. Evans, 73 J. Immuno. 427 (lY84). Another investigator suggested the use of Bouin's fluid to increase the binding of antigen solubili~e with a nonionic detergent. Noteboo~
et al., 7~ J. lmmuno. Meth. 141 (19~4). ~till another investigator ! aisclosed a process to increase the binding of antigen to a microtiter plate using beads to adsorb the residual nonionic aeter~ent. ~ee Drexler, supra. One author, however, suggested that ! ~U proteins can De effectively bound to latex surfaces provided that a detergent witn a nigh critical micelle concentration (CMC), i.e. the concentration ot a detergent wnich allows the formation of micelles, is used. ~ee ~araas, supra. This investigator tested the nonionic :. , ' - .
:
.
' `: ' ' , :: .
WO 91/08311 PCI~/US90/06922 2~Llai~
detergent, Triton X-lOO, among other detergents, at a concentration of .~ or .bP 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-lUU, inhioits binding at one of the lowest concentrations and thus would appear to be a poor choice for use in an immunoassay.
SUM~ARY OF THE INVENTION
This invention relates to a method to increase the concentration of viral antigen on a solid phase ~y coating the solid phase with lU viral antigen in the presence of nonionic detergents, sucn as Triton X-lOU or NP4~, in acidic pH.
~ETAILEU ~ESCRIPTION OF rH~ INVENTI~N
Antiyen was diluted into a coating buffer and added to l~ particles. Tne coatin~ buffer was preferably comprised of 0.1 ~l acetate, pH ~.U with 5~ NP4U, however, other nonionic detergents, sucn as Triton X-lUO, 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 deter~ent can range from .2% to lO~, with the optimu~
concentration from between about l and lO~b. It was observed that p~
can range from 4.U to 6.U, with the optimum pH at 5Ø
; Exa~ple 1 - Coating of HIV antigen onto magnetic microparticles using NP4U and acid pH.
l ml of 4 X 108 particles/ml of 3 - 4 micron carboxylated magnetic particles were pelleted and the supernatent was carefully removea and discarded. Tne particles were resuspended in l ml of U.l M acetate buffer, pH S.U, containing ~% NP40 and 250 ug/ml of HIV antigen. The particles were tumbled end over end at S - 6 rpm ~U o~ernignt at room temperature. The particles were pelleted and ;~ supernatent was discaraed. The particles were resuspended in 1 ml ot PBS, pelleted and tne su~ernatent was discarded. This was re-peatea two ~imes. The particles were resuspended in l ml of diluen~
(0.1~ acetate, pH ~.0, U.~M NACl, 1% NaN3) and stored at 2 - ~.
" .
- - , - - . . ~ . , :
WO 91/08311 2 ~ ~ ~ & ~ 2 PCI~/US90/06922 ... ;,................................................................ .
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 5Ø Three different coating buffers were assessed: Buffer A was comprised of ù.1 M acetate, at a pH SØ Buffer B was comprised of 0.1 M
acetate, 0.1 M NaCl, at a pH 5Ø Buffer C was comprised of 0.1 M
acetate, 0.1 M NaCl, 0.2~ NP4~, at a pH 5Ø
HIV negative serum was designated "617", positive was designated "61~", and high positive serum was designated "R-J 1825". These 1~ sera were diluted 1/100 in sample dilution buffer (SDB) (32% calf serum, ~.02M phosphate, pH 7.4, 0.5M NACl, 1% NP40, and 0.1% NaN3).
- The dialyzed preparation was coated at 1/2 dilution with coating ~uffers A, B, and C on to 1 ml of 2.5% carboxylated paramagnetic particles (3.~ um). Particles were incubated overnight at 2 Q 8 13 C. The particles were pelleted, the supernatent was discarded, and tne particles were resuspended in 1% normal goat serum (NGS) in 0.1 M acetate, at a pH 5Ø
, Tne coated particles were incubated for two hours at room temperature, then washed tnree times with 1 mL isotonic buffered 2u saline (IBS).
; 2uul of particles (1 X 107 parts/mL) were added to 50 ul of sample (1/1~0 in SBD). Tnis mixture was incubated for 30 minutes at ~ 37C. Tne solid phase was washed to remove unbound material. 50 ul -1 of goat anti-human beta glactosidase labeled antibody was added to ` 2~ the mixture. This mixture was then incubated for 15 minutes at37C. 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.
, :
, ' ', . ! , .
'. . : . ~ . '' ~ ' . ' . ' W O 91/083tl PcT/US9o/o6922 2 ~ j ~ 8 2 Table _l S ~ B 617 618 1825 Buffer A U.S 601.3 + 111.7 4329 t7.2) 16191 ~26.9) ~uffer B 3 750.3 + 98.5 4287 (5.7) 19059 t25.4) Buffer C 0 473 + 36.34096.3 (8.7) 12669 (26.8) Channel Report: 4U0/450 Gain Setting: lU
Read Time: lUU ms 1U Table 2 s u e ~17 618 1825 _ _ Buffer A 8 7~1.3 + 145.5 5449.7 (7.U) 19738 (2S.3) ~uffer ~ 7.5 970.7 + 130.6 52~7.7 (5.4) 23434 (24.2) ~utfer C U 611.7 + 5Y.5 5136.7 (8.4) 15434 (2~.2) 1~ Cnannel ~eporl: 4UU/4~0 ~ain Setting: lU . .
Read Time: lUU ms Table 3 ~U S D B 617 61~ 1825 ~uffer A 7.5 1~01120.7 (6.2) 3517 (19.7) ~uffer ~ 4.~ 22u.41000.7 (4.5) 4425 (20.1) Buffer C U 13B.71040.4 (7.5) 2765 (19.9) . :
Uelta ~eaa (Ta~le 2 - Table 1) .
' .
Conclusions The coating Duffer "C" comprised of 0.1 ~ acetate, û.1 M NACl, ~U and U.~ 40, and resulted in a greater concentration of viral antiyens on the solia phase. AIDS antigens coated in the presence of nonionic aetergents and acidic pH resultea in viral coated ~articles witn enhancea activity over particles coated using other buffers.
:
,: . - -- : . . - - : , . : : : -': ' '': ~ - . ' : ~ : ' : ' :
., - - , . . .
Claims (6)
1. An active solid phase coated with viral antigen made by the process comprising:
Coating a solid phase with viral antigen in an acidic suspension of a nonionic detergent, and a buffer.
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 paramagnetic microparticle.
4. The active solid phase of Claim 1 wherein said detergent is NP40 or Triton X-100.
5. The active solid phase of Claim 1 wherein the pH of the suspension ranges from about 4.0 to 6Ø
6. The active solid phase of Claim 1 wherein the pH is about 5Ø
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US44363589A | 1989-11-29 | 1989-11-29 | |
| US443,635 | 1989-11-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2045682A1 true CA2045682A1 (en) | 1991-05-30 |
Family
ID=23761588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2045682 Abandoned CA2045682A1 (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 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0136798A3 (en) * | 1983-08-25 | 1986-02-19 | Biotech Research Laboratories Inc. | Titre plate and assay kit for detection of antibodies in human serum and their production and use |
| US4870003A (en) * | 1987-06-15 | 1989-09-26 | Coulter Corporation | Simultaneous enzyme immunoassay for detecting antigen and/or antibody in humans |
| 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 |
| US5077198A (en) * | 1988-04-14 | 1991-12-31 | Eastman Kodak Company | Diagnostic kit and method for rapid detection of antibodies |
-
1990
- 1990-11-28 CA CA 2045682 patent/CA2045682A1/en not_active Abandoned
- 1990-11-28 EP EP19910902536 patent/EP0455807A4/en not_active Withdrawn
- 1990-11-28 JP JP50267491A patent/JPH04507003A/en active Pending
- 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
Also Published As
| Publication number | Publication date |
|---|---|
| EP0455807A4 (en) | 1992-07-08 |
| JPH04507003A (en) | 1992-12-03 |
| WO1991008311A1 (en) | 1991-06-13 |
| AU7160491A (en) | 1991-06-26 |
| EP0455807A1 (en) | 1991-11-13 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Dead |