INTRODUCTION

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The positive rates of immunoglobulin G (IgG) antibodies to human immunodeficiency virus type 1 (HIV-1) antigens in serum samples from HIV-1-infected subjects have been reported by the conventional enzyme-linked immunosorbent assay (ELISA) and Western blotting, and it has been generally accepted that the positive rates are high with gp160, gp41 and reverse transcriptase (RT), or p66 (a subunit of RT) as antigens (98 to 100% in asymptomatic carriers, 86 to 100% in patients with AIDS-related complex [ARC], and 77 to 100% in patients with AIDS) but low with gag proteins as antigens (63 to 92% in asymptomatic carriers, 50 to 97% in patients with ARC, and 21 to 77% in patients with AIDS using p24 as antigen and 41% in asymptomatic carriers, 30% in patients with ARC, and 14% in patients with AIDS using p17 as antigen) (2, 4, 6, 8). In seroconversion serum panels, the earliest positive band seen by Western blotting has been observed with p24 in some cases (26, 29, 34) and with gp160 in other cases (30, 32, 35) but not with p17 (13).

Recently, ultrasensitive enzyme immunoassays (EIAs) (immune complex transfer EIAs) of antibody IgGs to HIV-1 antigens have been developed using recombinant RT, p24, and p17 antigens (10, 12, 18). Antibody IgGs were allowed to react simultaneously with 2,4-dinitrophenyl antigens and antigen-enzyme conjugates. The immune complexes of the three components formed were trapped on (anti-2,4-dinitrophenyl group) IgG-coated solid-phase (first solid phase) and, after washing, eluted with N-2,4-dinitrophenyl-L-lysine and transferred to (anti-human IgG -chain) IgG-coated solid phase (second solid phase). The nonspecific signals were markedly reduced by transfer of the immune complexes from the first solid phase to the second one, improving the sensitivities to antibody IgGs to great extents. The sensitivities to antibody IgGs against RT, p24, and p17 achieved by this method were 56,000-, 22-, and 680-fold higher, respectively, than those achieved by Western blotting for the corresponding bands (12). The positive rates of antibody IgGs to RT, p24, and p17 in serum samples of HIV-1-infected subjects by these ultrasensitive EIAs were much higher (100% in asymptomatic carriers and patients with ARC and 90 to 100% in patients with AIDS [12]) than the previously reported ones described above. In seroconversion serum panels, antibody IgGs to RT, p24, and p17 were detected as early as antibodies to HIV-1 by the conventional ELISA and by Western blotting, and the signals and cutoff indexes for antibody IgG to p17 were higher than those for antibody IgGs to RT and p24 in nine seroconversion serum panels out of ten tested (13). The positivity of antibody IgG to p17 antigen, although very weak, was observed earlier than those of antibody IgGs to RT and p24 antigens in three seroconversion serum panels out of ten tested (13).

A notable difference between the results obtained by the immune complex transfer EIAs and the previous reports by the conventional ELISA and Western blotting described above is that the positive rates of antibody IgG to p17 antigen in serum samples from HIV-1-infected subjects was as high as those of antibody IgGs to RT and p24 obtained by the former methods but not by the latter methods. A possible reason for this difference is obviously the high sensitivity achieved by immune complex transfer as described above. In addition, an evidence suggesting another reason has been recently reported (22). Antibody IgG to p17 antigen bound to recombinant p17 antigen (rp17) directly immobilized on polystyrene beads by physical adsorption was much less reactive with rp17--D-galactosidase conjugate than that bound to biotinyl-rp17 indirectly immobilized on polystyrene beads coated successively with biotinyl-bovine serum albumin and streptavidin. This suggested that, in immune complex transfer EIA, antibody IgG to p17 antigen might react with 2,4-dinitrophenyl-bovine serum albumin-rp17 conjugate and rp17--D-galactosidase conjugate in solution as freely as or more freely than that bound to indirectly immobilized rp17, making higher sensitivity possible than that achieved by the conventional ELISA, in which rp17 is directly immobilized on a solid phase.

More recently, immune complex transfer EIA of antibody IgG to p17 antigen has been improved in sensitivity by performing immunoreactions with shaking and increasing the serum sample volume (21), and the conditions for this immunoassay have been optimized (15). Similarly, immune complex transfer EIA of HIV-1 p24 antigen has been developed (11, 14) and improved in sensitivity (16). Simultaneous detection of HIV-1 p24 antigen and antibody IgGs to RT and p17 antigens by immune complex transfer EIAs has shortened the window period after HIV-1 infection, during which diagnosis of the infection is not possible due to the absence of detectable antibodies to HIV-1 in the circulation (13).

This report describes earlier detections of HIV-1 p24 antigen and antibody IgG to HIV-1 p17 antigen by the recently improved immunoassays described above.

	MATERIALS AND METHODS

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Buffer. The regularly used buffer was 10 mM sodium phosphate buffer (pH 7.0) containing 1.0 g of bovine serum albumin (fraction V; Intergen Company, Purchase, N.Y.) per liter, 1.0 mM MgCl₂, and 1.0 g of NaN₃ per liter (buffer A).

Recombinant proteins of HIV-1. Recombinant HIV-1 p17 (rp17) and p24 (rp24) antigens were produced in Escherichia coli transformed with expression plasmids carrying the corresponding cDNAs and were purified as described previously (28, 31). The recombinant proviral clone used was pNL4-3 (1), which contained DNA from HIV-1 isolates NY5 (GenBank accession no. HIVNL43) and LAV (33), and the sequences for rp17 and rp24 derived from NY5.

Antibodies. Rabbit (anti-2,4-dinitrophenyl-bovine serum albumin) serum was obtained from Shibayagi Co., Ltd., Gumma, Japan. Rabbit (anti-human IgG -chain) IgG was obtained from Medical and Biological Laboratories Co., Ltd., Nagoya, Japan. Monoclonal mouse (anti-human IgM) IgG1 (product no. 7408) was obtained from Oy Medix Biochemica Ab, Kauniainen, Finland. Monoclonal mouse anti-HIV-1 p24 IgG1 (24C11) was obtained from Innogenetics N.V., Zwijnaarde, Belgium. Rabbit anti-HIV-1 p24 serum was prepared by immunization with rp24 (11).

Protein-coated polystyrene beads. White and colored polystyrene beads (3.2 mm in diameter; Immuno Chemical, Inc., Okayama, Japan) were coated with proteins by physical adsorption (20). Colored polystyrene beads were coated with affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG (0.05 g/liter), which had been eluted from 2,4-dinitrophenyl-bovine serum albumin-Sepharose 4B (9) with 3.2 mM HCl (pH 2.5) (24). White polystyrene beads were coated with affinity-purified rabbit (anti-human IgG -chain) IgG (0.1 g/liter) (23), monoclonal mouse (anti-human IgM) IgG1 (0.01 g/liter) (17), and biotinyl-bovine serum albumin (0.1 g/liter) (25), respectively. Biotinyl-bovine serum albumin-coated polystyrene beads were coated with streptavidin (0.1 g/liter) (11).

2,4-Dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-HIV-1 p24 Fab' conjugate and monoclonal mouse anti-HIV-1 p24 Fab'- -D-galactosidase conjugate. Affinity-purified rabbit anti-HIV-1 p24 Fab' was reacted with 6-maleimidohexanoyl-2,4-dinitrophenyl-biotinyl-bovine serum albumin (14). Monoclonal mouse anti-HIV-1 p24 Fab' was conjugated with -D-galactosidase from E. coli using o-phenylenedimaleimide (14).

Previous (immune complex transfer enzyme) immunoassay of HIV-1 p24 antigen. The antigen, HIV-1 p24, in serum was measured as described previously (13, 14). An aliquot (10 µl) of serum samples mixed with 90 µl of buffer A containing 0.4 M NaCl was incubated for 4 h with 50 µl of buffer A containing 0.4 M NaCl, 100 fmol of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate, 5 fmol of monoclonal mouse anti-p24 Fab'--D-galactosidase conjugate, and 50 µg of inactive -D-galactosidase (-D-galactosidase-mutein; Boehringer Mannheim GmbH, Mannheim, Germany) and subsequently overnight with two colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The colored polystyrene beads were incubated, after washing, for 3 h with two white polystyrene beads coated with streptavidin in 150 µl of buffer A containing 0.1 M NaCl and 1 mM N-2,4-dinitrophenyl-L-lysine. The incubations were performed at room temperature without shaking throughout. After removal of the colored polystyrene beads with tweezers, the white polystyrene beads were washed, and bound -D-galactosidase activity was assayed by fluorometry using 4-methylumbelliferyl--D-galactoside as substrate (19) at 30°C for 2.5 h. The fluorescence intensity was measured with a spectrofluorophotometer (F-3010; Hitachi, Ltd., Tokyo, Japan) using 360 nm for excitation and 450 nm for emission analysis. The fluorescence intensity of 10⁸ M 4-methylumbelliferone in 0.1 M glycine-NaOH buffer (pH 10.3) was adjusted to 100.

Improved (immune complex transfer enzyme) immunoassay of HIV-1 p24 antigen. The previous immunoassay of HIV-1 p24 antigen in serum was modified as follows (16). An aliquot (50 µl) of serum samples was incubated for 16 h with 100 µl of buffer A containing 0.5 M NaCl, 100 fmol of 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate, 5 fmol of monoclonal mouse anti-p24 Fab'--D-galactosidase conjugate, 50 µg of inactive -D-galactosidase, and 10 µl of nonspecific rabbit serum and subsequently for 2 h with two colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The colored polystyrene beads after washing were incubated for 2 h with two streptavidin-coated white polystyrene beads in the presence of N-2,4-dinitrophenyl-L-lysine. The incubations with polystyrene beads were performed at room temperature with 180 shakings per min throughout (21). Bound -D-galactosidase activity was assayed at 30°C for 2 h as described above. The detection limit of HIV-1 p24 antigen by the improved immunoassay with shaking (16) using 50-µl serum samples in the present study (0.01 pg/ml) was approximately 25-fold smaller than that by the previous one without shaking using 10-µl serum samples (0.24 pg/ml) (13).

2,4-Dinitrophenyl-bovine serum albumin-rp17 conjugate and rp17--D-galactosidase conjugate. Thiol groups introduced into rp17 molecules were reacted with 6-maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin and maleimide--D-galactosidase (EC 3.2.1.23) (10).

Previous (immune complex transfer enzyme) immunoassay of antibody IgG to HIV-1 p17 antigen. Antibody IgG to p17 antigen was measured essentially in the same way as described previously (12, 13). An aliquot (10 µl) of serum samples mixed with 90 µl of buffer A containing 0.4 M NaCl was incubated for 3 h with 50 µl of buffer A containing 0.4 M NaCl, 50 µg of inactive -D-galactosidase, and 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin-rp17 conjugate and rp17--D-galactosidase conjugate, and subsequently overnight with two colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The colored polystyrene beads were washed and incubated for 1 h with two white polystyrene beads coated with affinity-purified (anti-human IgG -chain) IgG in 150 µl of buffer A containing 0.1 M NaCl and 1 mM N-2,4-dinitrophenyl-L-lysine. The colored polystyrene beads were removed, and the incubation was continued for 2 h. The incubations were performed at room temperature without shaking. The white polystyrene beads were washed, and bound -D-galactosidase activity was assayed at 30°C for 2.5 h as described above.

Improved (immune complex transfer enzyme) immunoassay of antibody IgG to HIV-1 p17 antigen. The previous immunoassay of antibody IgG to p17 antigen was modified as follows (15). An aliquot (100 µl) of serum samples was incubated for 0.5 h with 50 µl of buffer A containing 0.9 M NaCl, 50 µg of inactive -D-galactosidase, and 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin-rp17 conjugate and rp17--D-galactosidase conjugate and subsequently for 1 h with two colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. In experiments to confirm the presence of antibodies IgG to p17, 20 pmol of recombinant p17 was added. The colored polystyrene beads after washing were incubated for 1 h with two white polystyrene beads coated with affinity-purified (anti-human IgG -chain) IgG in the presence of N-2,4-dinitrophenyl-L-lysine. The incubations were performed at room temperature with 180 shakings per min throughout (21). Bound -D-galactosidase activity was assayed at 30°C for 2 h as described above.

Immune complex transfer EIA of antibody IgM to HIV-1 p17 antigen. White polystyrene beads coated with monoclonal mouse (anti-human IgM) IgG1 were substituted for those coated with affinity-purified (anti-human IgG -chain) IgG in the improved immunoassay for antibody IgG to HIV-1 p17 antigen described above.

Cutoff values and indexes of immune complex transfer EIAs. The mean fluorescence intensities of bound -D-galactosidase activities (the mean signals) by the immune complex transfer EIAs with 200 serum samples from HIV-1-seronegative subjects are shown in Table 5 with their standard deviations and ranges, which were similar to those achieved by the previous immunoassays, and the highest fluorescence intensities among those values were taken as the cutoff values (see Table 5). The cutoff indexes were calculated by dividing the fluorescence intensities with test samples by the cutoff values (Tables 1 to 5).

Detection of HIV-1 RNA in serum by RT-PCR. HIV-1 RNA in serum was measured by a commercial RT-PCR kit (Amplicor HIV-1 Monitor; Roche Diagnostic Systems, Basel, Switzerland). The cutoff index of HIV-1 RNA detection was expressed as the ratio of the number of HIV-1 RNA copies per milliliter of serum measured to the cutoff value of 400 copies/ml, the detection rate of which was 75% according to the information of the manufacturer (Roche) and 93% by the report of Coste et al. (5).

Other immunological methods. The conventional EIA for antibodies to HIV-1 was performed using a commercial kit with two recombinant proteins of HIV-1 (gp41 and p24) as antigens (Abbott Recombinant HIV-1/HIV-2 3rd Generation EIA; Abbott Laboratories, North Chicago, Ill.). The gelatin particle agglutination test for antibodies to HIV-1 was performed using a commercial kit with a lysate of HIV-1 as antigen (SERODIA-HIV; Fujirebio Inc., Tokyo, Japan). Western blotting for antibody IgG to HIV-1 was performed using a commercial kit preblotted with nine proteins of HIV-1 (gp160, gp120, p66, p55, p51, gp41, p31, p24, and p17) (Ortho HIV Western Blot Kit; Ortho Diagnostic Systems, Inc., Raritan, N.J.).

HIV-1 seroconversion serum panels. One seroconversion serum panel (panel HIV 6240) was obtained from BioClinical Partners, Inc., Franklin, Mass. Six seroconversion serum panels (SV-0031, SV-0051, SV-0111, SV-0161, SV-0211, and SV-0241) were obtained from North American Biologicals, Inc., Miami, Fla. Twelve seroconversion serum panels (panels E, J, K, P, S, W, X, Y, Z, AF, AJ, and AR) were obtained from Boston Biomedica, Inc., West Bridgewater, Mass.

Serum samples randomly collected from HIV-1-seronegative and- seropositive subjects. Serum samples were randomly collected from 200 HIV-1-seronegative subjects (99 males [22 to 62 years old] and 101 females [27 to 73 years old]) and 79 HIV-1-seropositive subjects (42 male asymptomatic carriers [17 to 47 years old], 8 female asymptomatic carriers [18 to 39 years old], 7 male patients [10 to 52 years old] with ARC, 2 female patients [18 and 42 years old] with ARC, and 20 male patients [14 to 61 years old] with AIDS) and were stored at 20°C until use. These serum samples were tested by the gelatin particle agglutination test. The seropositivity was confirmed by Western blotting.

	RESULTS

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Earlier detection of HIV-1 p24 antigen by the improved immunoassay than by the previous one. Nineteen HIV-1 seroconversion serum panels were tested by the improved immunoassay of HIV-1 p24 antigen, and the test results were compared with those obtained by the previous one (Tables 1 to 5).

Comparison of detections of HIV-1 p24 antigen and RNA. Of the 19 panels, 12 panels which had not been thawed for other purposes after purchase were tested by RT-PCR of HIV-1 RNA, and the results were compared with the detections of HIV-1 p24 antigen described above (Tables 1 to 5). The antigen was detected as early as HIV-1 RNA in 11 panels and earlier in 1 panel (panel 9 [E]). In one panel (panel 3 [P]) of the 11, the cutoff index of the earliest positivity was determined to be 12 by the improved immunoassay but 2.4 by RT-PCR.

Earlier detection of antibody IgG to HIV-1 p17 antigen by the improved immunoassay than by other methods for antibodies. Nineteen panels were tested by the improved immunoassay of antibody IgG to p17 antigen, and the test results were compared to those obtained by other methods for antibodies (Tables 1 to 5).

Earlier detection of antibodies IgG and IgM to HIV-1 p17 antigen than HIV-1 p24 antigen and RNA. In some panels, antibodies IgG and IgM to p17 antigen were detected earlier than HIV-1 p24 antigen and RNA (Tables 3 to 5).

	DISCUSSION

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According to the information provided from Boston Biomedica, Inc., and North American Biologicals, Inc., 18 seroconversion serum panels of the 19 used in this study and 11 panels of the 12 used for the detection of HIV-1 RNA were collected in the United States, suggesting that these panels were most probably infected with subtype B, RNA of which can be detected with high sensitivity by the PCR kit (Roche) used in this study, although RNA of subtype E is detected with low sensitivity due to inappropriate sequences of the primers used in the kit (3, 7). Therefore, it is unlikely that HIV-1 p24 antigen was detected as early as HIV-1 RNA due to low sensitivity of the RT-PCR kit used, although subtypes of the seroconversion serum panels used remain to be determined.

According to the information of the manufacturer (Roche), the detection rates of 400 and 800 copies/ml of HIV-1 RNA were 75 and 100%, respectively, and Coste et al. have reported that the detection rate of 400 copies/ml using the commercial kit used in the present study was 93% (5). On the other hand, Layne et al. have reported that the number of HIV-1 p24 molecules per HIV-1 virion was 1,200 (27), which is equal to 2 zmol or 0.048 fg. Since each HIV-1 virion contains two copies of RNA, 400 and 800 copies of HIV-1 RNA per ml must be associated with 2.4 × 10⁵ and 4.8 × 10⁵ molecules, 0.4 and 0.8 amol, or 0.0096 and 0.019 pg, respectively, of HIV-1 p24 antigen per ml, values which were close to the detection limit of HIV-1 p24 antigen (0.01 pg/ml) by the improved immunoassay used. In the present study, HIV-1 p24 antigen was measured without using any method to release the antigen from HIV-1 virions, and the ratios of the free-antigen concentrations measured by the improved immunoassay to the HIV-1 virion-associated p24 antigen concentrations calculated from HIV-1 RNA copies measured by RT-PCR were 0.16 to 2,900 (Tables 8 and 9). Therefore, HIV-1 p24 antigen may be more easily detected by pretreatment of serum samples to release the antigen from the virions in some cases.

In eight seroconversion serum panels, antibody IgG to p17 antigen was detected earlier than antibody IgM to p17 antigen (Tables 1 to 5). The eight panels were panels 8 (SV-0031), 10 (SV-0241), 12 (K), 13 (SV-0111), 14 (SV-0211), 15 (S), 18 (AR), and 19 (6240). This might have been at least partly due to the lower affinities of antibody IgM to p17 antigen than antibody IgG to p17 antigen. This possibility is supported by the fact that longer times were required for the formation of the immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-rp17 conjugate, antibody IgM to p17 antigen, and rp17--D-galactosidase conjugate than for the formation of the corresponding IgG antibody immune complex (15, 17).

In three seroconversion serum panels, antibody IgG to p17 antigen was detected earlier than p24 antigen and RNA. The three panels were panels 9 (E), 16 (X), and 19 (6240). The possibility that these serum samples might have derived from subjects transfused with HIV-1-infected blood appeared to be unlikely due to the fact that the ratios of cutoff indexes for antibody IgG to p17 antigen to those for antibody IgM to p17 antigen in the panel sera before the detection of p24 antigen were much lower than the corresponding ratios in sera randomly collected from HIV-1-infected subjects (Table 7). This was further supported by the fact that the ratios of cutoff indexes for antibody IgG to p17 antigen to those for antibody IgG to RT in the panel sera before the detection of p24 antigen (3.2 to 74) were much larger than the corresponding ratios in most of the sera (50 sera from asymptomatic carriers, 29 sera from patients with ARC and AIDS) randomly collected from HIV-1-infected subjects (0.001 to 0.1 in 56%, 0.11 to 1.0 in 30%, 1.2 to 3.0 in 8.9%, and 3.7 to 11 in 5.1%).

In three seroconversion serum panels, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, and the antibody levels tended to decline before the usually observed steep rises (Tables 3 to 5). The three panels were panels 9 (E), 16 (X), and 19 (6240). Namely, there were two rises of antibodies IgG and IgM to p17 antigen: one was early and small, and the other was late, steep, and large. This suggested the presence of p24 antigen and RNA earlier than the small rises of antibodies IgG and IgM to p17 antigen, although this was not detectable by currently available methods. Namely, there might be two increases in the levels of p24 antigen and RNA. More generally, the combination of HIV-1 antigens, RNA, and anti-HIV-1 antibodies might appear repeatedly in the circulation and finally result in their large increases, which have been detected by conventional methods. Moreover, the finding that the number of days between the small and usually observed steep and large rises of antibodies IgG and IgM to p17 antigen varied in different seroconversion serum panels (Tables 3 to 5) suggested the difference of HIV-1 replication rates in different individuals.

Finally, the improved immunoassays of HIV-1 p24 antigen and antibody IgG and IgM to HIV-1 p17 antigen shortened the window period in the diagnosis of HIV-1 infection, reducing the risk of HIV-1 infection by blood transfusion, compared with previous immunoassays. Further improvement of the sensitivities to HIV antigens and antibodies might further reduce the risk of HIV infection by blood transfusion.