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% NaN ₃ ).

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 10 ⁷ 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.