METHODES D'ENROBAGE D'ANTIGENES VIRAUX A UNE PHASE SOLIDE EN PRESENCE DE DETERGENTS NON IONIQUES DANS UNE SOLUTION AYANT UN PH ACIDE

METHODS OF COATING VIRAL ANTIGENS IN THE PRESENCE OF NONIONIC DETERGENTS AND ACIDIC PH ONTO A SOLID PHASE

Abrégé anglais

2045682 9108311 PCTABS00005
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.

Revendications

WO 91/08311 PCT/US90/06922
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I Claim:
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.
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

Description

W O 91/08311 2 ~ 2 PCr/US90/06922
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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
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WO 91/08311 PCI~/US90/06922
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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 - ~.
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WO 91/08311 2 ~ ~ ~ & ~ 2 PCI~/US90/06922
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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.
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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)
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Uelta ~eaa (Ta~le 2 - Table 1) .
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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.
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