Note: Descriptions are shown in the official language in which they were submitted.
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
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(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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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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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
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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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