Abstract
For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.
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 ɛN-2,4-dinitrophenyl-l-lysine and transferred to (anti-human IgG γ-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–β-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–β-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
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 γ-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 γ-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′– β-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 β-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′–β-d-galactosidase conjugate, and 50 μg of inactive β-d-galactosidase (β-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 ɛN-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 β-d-galactosidase activity was assayed by fluorometry using 4-methylumbelliferyl-β-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 10−8 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′–β-d-galactosidase conjugate, 50 μg of inactive β-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 ɛN-2,4-dinitrophenyl-l-lysine. The incubations with polystyrene beads were performed at room temperature with 180 shakings per min throughout (21). Bound β-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–β-d-galactosidase conjugate.
Thiol groups introduced into rp17 molecules were reacted with 6-maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin and maleimide-β-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 β-d-galactosidase, and 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin–rp17 conjugate and rp17–β-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 γ-chain) IgG in 150 μl of buffer A containing 0.1 M NaCl and 1 mM ɛN-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 β-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 β-d-galactosidase, and 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin–rp17 conjugate and rp17–β-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 γ-chain) IgG in the presence of ɛN-2,4-dinitrophenyl-l-lysine. The incubations were performed at room temperature with 180 shakings per min throughout (21). Bound β-d-galactosidase activity was assayed at 30°C for 2 h as described above.
The sensitivity of the immunoassay was expressed as the ratio of the specific signal to the negative (nonspecific) one, which was proportional to the dilution of serum from an HIV-1-seropositive subject with serum from an HIV-1-seronegative subject to provide the positive signal twice as high as the negative signal. The specific signal was calculated by subtraction of the negative signal (N) obtained with serum of an HIV-1-seronegative subject from the positive signal (P) obtained with serum of an HIV-1-seropositive subject and was divided by the negative signal as follows: (P − N)/N. The improved immunoassay of antibody IgG to HIV-1 p17 antigen with shaking using 100-μl serum samples (21) in the present study was approximately 90-fold more sensitive than the previous one without shaking using 10-μl serum samples (13).
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 γ-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 β-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).
TABLE 5.
Test results of HIV-1 seroconversion serum panels by various methods (panels 16 to 19)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
Maximal dilution to cause gelatin particle agglutination | Western blot
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p24 antigen by previous immunoassay | p24 antigen by improved immunoassay | RNA | Anti-p17 IgG by previous immunoassay | Anti-p17 IgG by improved immunoassay | Anti-p17 IgM | Anti-HIV-1 antibodies by conventional EIA | gp160 | gp120 | gp41 | p24 | p17 | |||
| 16 (panel X) | 0 | 0.0 | 0.2 | <0.1 | 0.4 | 12 | 20 | − | − | − | − | − | ||
| 2 | 0.1 | 0.2 | <0.1 | 0.4 | 21 | 23 | − | − | − | − | − | |||
| 8 | 0.5 | 0.3 | <0.1 | 0.4 | 26 | 16 | − | − | − | − | − | |||
| 10 | 0.1 | 0.2 | <0.1 | 0.6 | 20 | 28 | − | − | − | − | − | |||
| 26 | 128 | 2,635 | 336 | 0.2 | 8.9 | 15 | − | − | − | − | − | |||
| 33 | 24 | 592 | 80 | 24 | 9,387 | 4,128 | 8 | − | − | − | − | − | ||
| 35 | 6.0 | 103 | 30 | 91 | 36,165 | 11,197 | 16 | − | − | − | + | − | ||
| 40 | 5.8 | 118 | 13 | 881 | 64,908 | 20,371 | 16 | + | − | − | + | − | ||
| 17 (panel Y) | 0 | 0.3 | 0.4 | <0.1 | 0.1 | 1.4 | 0.2 | − | − | − | − | − | ||
| 10 | 0.2 | 0.2 | <0.1 | 0.1 | 1.3 | 0.2 | − | − | − | − | − | |||
| 18 | 0.0 | 0.2 | <0.1 | 0.2 | 0.8 | 0.2 | − | − | − | − | − | |||
| 22 | 0.3 | 0.2 | <0.1 | 0.1 | 1.2 | 0.1 | − | − | − | − | − | |||
| 44 | 42 | 354 | 393 | 0.3 | 953 | 859 | 2 | − | − | − | + | − | ||
| 49 | 23 | 162 | 427 | 32 | 5,013 | 1350 | 8 | − | − | − | + | − | ||
| 18 (panel AR) | 0 | 0.1 | 0.4 | <0.1 | 0.1 | 6.3 | 0.5 | − | − | − | − | − | ||
| 2 | 0.1 | 0.3 | <0.1 | 0.2 | 5.2 | 0.6 | − | − | − | − | − | |||
| 9 | 0.4 | 9.1 | 25 | 0.0 | 7.3 | 0.6 | − | − | − | − | − | |||
| 14 | 47 | 776 | 2,200 | 0.1 | 11 | 0.5 | − | − | − | + | − | |||
| 19 (panel 6240) | 0 | 0.3 | 0.2 | <0.1 | 0.1 | 27 | 1.7 | − | − | − | − | − | ||
| 2 | 0.1 | 0.2 | <0.1 | 0.1 | 19 | 0.0 | − | − | − | − | − | |||
| 7 | 0.1 | 0.1 | <0.1 | 0.1 | 13 | 0.3 | − | − | − | − | − | |||
| 9 | 0.0 | 0.1 | <0.1 | 0.1 | 12 | 0.1 | − | − | − | − | − | |||
| 14 | 0.3 | 0.1 | <0.1 | 0.6 | 7.2 | 0.1 | − | − | − | − | − | |||
| 16 | 0.3 | 0.3 | <0.1 | 0.1 | 8.1 | 6.5 | − | − | − | − | − | |||
| 21 | 2.8 | 25 | 46 | 0.1 | 12 | 0.2 | − | − | − | − | − | |||
| 23 | 21 | 311 | 377 | 0.3 | 5.1 | 0.2 | − | − | − | − | − | |||
| 28 | 459 | 5,186 | 2,510 | 0.1 | 2.1 | 0.2 | − | − | − | − | − | |||
| 30 | 1,443 | 7,859 | 2,480 | 0.2 | 18 | 9.5 | − | − | − | + | − | |||
| 36 | 102 | 1,071 | 723 | 87 | 2,815 | 1,435 | 16 | + | − | − | + | − | ||
| 45 | 11 | 182 | 64 | 3,483 | 179,104 | 18,909 | 32 | + | − | − | + | − | ||
| 53 | 5.1 | 99 | 47 | 3,920 | 146,818 | 11,962 | 64 | + | − | − | + | − | ||
| Cutoff values | 0.9 | 0.5 | 400 | 1.9 | 0.7 | 1.5 | 0.1 | |||||||
| Signalsa | ||||||||||||||
| Mean | 0.5 | 0.1 | 0.7 | 0.2 | 0.2 | |||||||||
| SD | 0.1 | 0.1 | 0.4 | 0.1 | 0.2 | |||||||||
| Range | 0.1–0.9 | 0.1–0.5 | 0.1–1.9 | 0.1–0.7 | 0.1–1.5 | |||||||||
The signals are the fluorescence intensities of bound β-d-galactosidase activities.
TABLE 1.
Test results of HIV-1 seroconversion serum panels by various methods (panels 1 to 4)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
Maximal dilution to cause gelatin particle agglutination | Western blot
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p24 antigen by previous immunoassay | p24 antigen by improved immunoassay | RNA | Anti-p17 IgG by previous immunoassay | Anti-p17 IgG by improved immunoassay | Anti-p17 IgM | Anti-HIV-1 antibodies by conventional EIA | gp160 | gp120 | gp41 | p24 | p17 | |||
| 1 (panel Z) | 0 | 0.5 | 0.6 | <0.1 | 0.2 | 0.9 | 0.3 | 0.2 | − | − | − | − | − | |
| 2 | 0.6 | 3.1 | 3.5 | 0.1 | 0.9 | 0.2 | 0.2 | − | − | − | − | − | ||
| 7 | 13 | 803 | 1,350 | 0.1 | 0.6 | 0.3 | 0.3 | − | − | − | − | − | ||
| 9 | 103 | 4,965 | 1,560 | 0.5 | 0.6 | 0.3 | 0.3 | − | − | − | − | − | ||
| 27 | 4.9 | 106 | 249 | 968 | 45,107 | 4,009 | 4.7 | 16 | + | − | − | + | − | |
| 32 | 3.7 | 117 | 286 | 1,848 | 55,020 | 3,059 | 3.4 | 16 | + | − | − | + | − | |
| 2 (panel J) | 0 | 0.5 | 0.1 | 0.5 | 1.3 | 0.3 | 0.3 | − | − | − | − | − | ||
| 14 | 163 | 1,033 | 0.4 | 0.1 | 0.3 | 0.4 | − | − | − | − | − | |||
| 26 | 3.8 | 48 | 2,807 | 171,410 | 13,265 | 2.3 | 32 | + | − | − | + | − | ||
| 28 | 2.2 | 20 | 3,589 | 230,589 | 22,440 | 2.3 | 32 | + | − | − | + | − | ||
| 32 | 1.3 | 7.0 | 4,897 | 621,713 | 20,421 | 2.8 | 32 | + | − | − | + | − | ||
| 35 | 1.2 | 8.0 | 5,458 | 784,008 | 17,509 | 2.9 | 32 | + | − | − | + | − | ||
| 40 | 0.9 | 4.4 | 7,415 | 1,286,138 | 13,127 | 3.7 | 32 | + | − | − | + | − | ||
| 3 (panel P) | 0 | 0.4 | 0.2 | <0.1 | 0.0 | 0.1 | 0.3 | 0.2 | − | − | − | − | − | |
| 4 | 0.4 | 0.2 | <0.1 | 0.0 | 0.2 | 0.3 | 0.2 | − | − | − | − | − | ||
| 9 | 0.7 | 12 | 2.4 | 0.0 | 0.1 | 0.3 | 0.3 | − | − | − | − | − | ||
| 15 | 341 | 4,199 | 519 | 0.1 | 0.2 | 0.3 | 0.4 | − | − | − | − | − | ||
| 30 | 4.6 | 57 | 30 | 2,816 | 177,391 | 40,393 | 2.9 | 32 | + | − | − | + | − | |
| 35 | 2.7 | 35 | 28 | 4,142 | 521,757 | 33,542 | 2.9 | 32 | + | − | − | + | − | |
| 4 (panel AF) | 0 | 0.6 | −0.3 | <0.1 | 0.3 | 0.9 | 0.4 | 0.3 | − | − | − | − | − | |
| 2 | 0.2 | −0.2 | <0.1 | 0.4 | 0.2 | 0.3 | 0.2 | − | − | − | − | − | ||
| 7 | 0.3 | 0.0 | <0.1 | 0.3 | 0.5 | 0.4 | 0.2 | − | − | − | − | − | ||
| 9 | 0.4 | 0.1 | <0.1 | 1.4 | 0.1 | 0.4 | 0.2 | − | − | − | − | − | ||
| 15 | 2.0 | 57 | 58 | 0.5 | 0.3 | 0.4 | 0.2 | − | − | − | − | − | ||
| 28 | 9.3 | 658 | 2,520 | 1.1 | 43 | 2.1 | 4.0 | 4 | − | − | − | − | − | |
| 33 | 11 | 399 | 1,050 | 65 | 3,438 | 1,277 | 5.3 | 8 | + | − | − | + | − | |
| 35 | 8.6 | 185 | 666 | 262 | 15,498 | 4,005 | 5.7 | 16 | + | − | − | + | − | |
| 42 | 19 | 115 | 983 | 2,640 | 130,641 | 8,868 | 5.9 | 32 | + | − | − | + | − | |
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
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).
By the improved immunoassay, HIV-1 p24 antigen was detected 4 to 6 days (one sample) earlier in 5 panels than and as early in another 13 panels as by the previous immunoassay. The five panels were panels 1 (Z), 3 (P), 5 (AJ), 10 (SV-0241), and 18 (AR). In panel 12 (K), the antigen was unequivocally detectable in the present study, whereas it was not detectable at all in any samples in the previous study. In 3 panels out of the 13, the cutoff indexes of the earliest positivities increased from 1.2 to 2.8 by the previous immunoassay to 25 to 57 by the improved immunoassay. The three panels were panels 4 (AF), 7 (W), and 19 (6240).
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).
By the improved immunoassay, antibody IgG to p17 antigen was detected 5 to 126 days (1 to 10 samples) earlier in ten panels and as early in seven panels compared to the previous immunoassay. The ten panels were panels 9 (E), 10 (SV-0241), 11 (SV-0161), 12 (K), 14 (SV-0211), 15 (S), 16 (X), 17 (Y), 18 (AR), and 19 (6240). The seven panels were panels 1 (Z), 2 (J), 3 (P), 6 (SV-0051), 7 (W), 8 (SV-0031), and 13 (SV-0111). In three panels out of the seven, the cutoff indexes of the earliest positivities increased from 1.5 to 2.5 by the previous immunoassay to 9.3 to 55 by the improved immunoassay. The three panels were panels 6 (SV-0051), 8 (SV-0031), and 13 (SV-0111). In another two panels, the earliest weak positivities obtained by the previous immunoassay were not confirmed by the improved immunoassay. The two panels were panels 4 (AF) and 5 (AJ). In one of these panels (panel 4 [AF]), the cutoff index of the second-earliest positivity by the previous immunoassay (1.1) increased to 43 by the improved immunoassay. In the other panel, panel 5 (AJ), strong positivities were observed on the same day by the two immunoassays.
By the improved immunoassay, antibody IgG to p17 antigen was detected 5 to 30 days (one to nine samples) earlier in eight panels than and as early in ten panels as and 37 days (one sample) later in one panel (panel 7 [W]) than antibody IgM to p17 antigen. 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). The 10 panels were panels 1 (Z), 2 (J), 3 (P), 4 (AF), 5 (AJ), 6 (SV-0051), 9 (E), 11 (SV-0161), 16 (X), and 17 (Y). The cutoff indexes of the earliest positivities for antibody IgG to p17 antigen in two panels and for antibody IgM to p17 antigen in three panels were small (1.2 to 2.3). The two panels were panels 12 (K) and 14 (SV-0211). The three panels were panels 4 (AF), 7 (W), and 11 (SV-0161). The earliest positivities for antibody IgG to p17 antigen in panel 17 (Y) and for antibody IgM to p17 antigen in panel 19 (6240) did not appear to be unequivocal.
By the improved immunoassay, antibody IgG to p17 antigen was detected 7 to 126 days (one to nine samples) earlier in seven panels than and as early in seven panels as and 2 days (one sample) later in one panel (panel 6 [SV-0051]) than by a conventional ELISA. The former seven panels were panels 9 (E), 10 (SV-0241), 11 (SV-0161), 12 (K), 13 (SV-0111), 14 (SV-0211), and 15 (S). The latter seven panels were panels 1 (Z), 2(J), 3 (P), 4 (AF), 5 (AJ), 7 (W), and 8 (SV-0031). Four panels were not tested by the conventional ELISA: panels 16 (X), 17 (Y), 18 (AR), and 19 (6240).
By the improved immunoassay, antibody IgG to p17 antigen was detected 7 to 126 days (1 to 10 samples) earlier in 10 panels than and as early in 9 panels as by a gelatin particle agglutination test. The 10 panels were panels 9 (E), 10 (SV-0241), 11 (SV-0161), 12 (K), 13 (SV-0111), 14 (SV-0211), 15 (S), 16 (X), 18 (AR), and 19 (6240). The nine panels were panels 1 (Z), 2 (J), 3 (P), 4 (AF), 5 (AJ), 6 (SV-0051), 7 (W), 8 (SV-0031), and 17 (Y).
By the improved immunoassay, antibody IgG to p17 antigen was detected 5 to 126 days (one to nine samples) earlier in 14 panels than any band by Western blotting and as early in 5 panels as gp160, p24, and/or p17 band by Western blotting. The 14 panels were panels 4 (AF), 6 (SV-0051), 8 (SV-0031), 9 (E), 10 (SV-0241), 11 (SV-0161), 12 (K), 13 (SV-0111), 14 (SV-0211), 15 (S), 16 (X), 17 (Y), 18 (AR), and 19 (6240). The five panels were panels 1 (Z), 2 (J), 3 (P), 5 (AJ), and 7 (W). Antibody IgG to p17 antigen was detected by the improved immunoassay 5 to 126 days earlier than p17 band by Western blotting in all of the panels tested except panel 7 (W).
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).
TABLE 3.
Test results of HIV-1 seroconversion serum panels by various methods (panels 8, 9, and 10)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
Maximal dilution to cause gelatin particle agglutination | Western blot
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p24 antigen by previous immunoassay | p24 antigen by improved immunoassay | RNA | Anti-p17 IgG by previous immunoassay | Anti-p17 IgG by improved immunoassay | Anti-p17 IgM | Anti-HIV-1 antibodies by conventional EIA | gp160 | gp120 | gp41 | p24 | p17 | |||
| 8 (SV-0031) | 0 | 0.2 | 0.3 | 0.2 | 0.3 | 0.4 | 0.2 | − | − | − | − | − | ||
| 4 | 0.1 | 0.3 | 0.1 | 0.6 | 0.4 | 0.2 | − | − | − | − | − | |||
| 7 | 0.1 | 0.2 | 0.1 | 0.9 | 0.7 | 0.3 | − | − | − | − | − | |||
| 12 | 0.2 | 0.3 | 0.1 | 0.4 | 0.5 | 0.2 | − | − | − | − | − | |||
| 19 | 0.1 | 0.3 | 0.0 | 0.6 | 0.4 | 0.4 | − | − | − | − | − | |||
| 26 | 0.2 | 0.6 | 0.4 | 1.2 | 1.0 | 0.3 | − | − | − | − | − | |||
| 28 | 0.3 | 0.5 | 0.3 | 0.3 | 0.7 | 0.3 | − | − | − | − | − | |||
| 46 | 201 | 5,374 | 2.0 | 48 | 0.8 | 4.2 | 4 | − | − | − | − | − | ||
| 48 | 255 | 6,807 | 1.4 | 16 | 0.9 | 5.1 | 8 | − | − | − | − | − | ||
| 53 | 324 | 6,580 | 57 | 4,103 | 470 | 2.9 | 32 | − | − | − | − | − | ||
| 55 | 208 | 5,471 | 262 | 1,662 | 1.9 | 32 | − | − | − | − | − | |||
| 60 | 30 | 1,092 | 2,163 | 3,871 | 2.5 | 16 | − | − | − | + | − | |||
| 62 | 18 | 595 | 3,732 | 4,767 | 2.8 | 16 | − | − | − | + | − | |||
| 67 | 16 | 441 | 6,789 | 4,304 | 3.7 | 32 | + | − | − | + | + | |||
| 9 (panel E) | 0 | 0.3 | 0.3 | <0.1 | 0.2 | 4.7 | 3.9 | 0.3 | − | − | − | − | − | |
| 7 | 0.4 | 0.2 | <0.1 | 0.6 | 22 | 30 | 0.3 | − | − | − | − | − | ||
| 21 | 0.4 | 0.2 | <0.1 | 0.3 | 16 | 22 | 0.2 | − | − | − | − | − | ||
| 35 | 0.5 | 0.3 | <0.1 | 0.4 | 3.0 | 2.3 | 0.3 | − | − | − | − | − | ||
| 42 | 0.5 | 0.3 | <0.1 | 0.4 | 2.0 | 1.0 | 0.3 | − | − | − | − | − | ||
| 49 | 0.5 | 0.3 | <0.1 | 0.7 | 1.7 | 0.8 | 0.2 | − | − | − | − | − | ||
| 63 | 0.5 | 0.2 | <0.1 | 0.4 | 1.6 | 0.6 | 0.2 | − | − | − | − | − | ||
| 84 | 25 | 677 | 0.8 | 0.6 | 1.4 | 0.5 | 0.2 | − | − | − | − | − | ||
| 91 | 8,092 | 8,707 | 825 | 0.4 | 1.4 | 0.8 | 0.8 | − | − | − | − | − | ||
| 126 | 14 | 45 | 2.8 | 9,786 | 445,068 | 11,078 | 4.6 | 32 | + | − | − | + | + | |
| 10 (SV-0241) | 0 | 0.7 | 29 | 108 | 0.5 | 21 | 0.4 | 0.2 | − | − | − | − | − | |
| 6 | 77 | 5,754 | 7,500 | 0.8 | 23 | 0.3 | 0.2 | − | − | − | − | − | ||
| 8 | 31 | 2,358 | 2,500 | 0.3 | 5.9 | 0.4 | 0.3 | − | − | − | − | − | ||
| 14 | 3.1 | 154 | 300 | 42 | 2,249 | 164 | 7.4 | 8 | + | − | − | + | − | |
| 16 | 1.6 | 48 | 88 | 433 | 712 | 6.0 | 16 | + | − | − | + | − | ||
| 21 | 1.2 | 1.8 | 9.8 | 3,554 | 2,966 | 5.6 | 16 | + | − | − | + | − | ||
| 23 | 0.7 | 0.7 | 3.8 | 5,454 | 3,062 | 4.2 | 16 | + | − | − | + | − | ||
In four panels, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were clearly detected, although at low levels, 9 to 84 days (two to seven samples) earlier than HIV-1 p24 antigen and RNA, and the antibody levels tended to decline slightly before the usually observed steep rises following the peaks of HIV-1 p24 antigen and RNA. The four panels were panels 9 (E), 16 (X), 18 (AR), and 19 (6240). In two panels, both p24 antigen and antibody IgG to p17 antigen were detected in the earliest serum samples before the peaks of p24 antigen, and the levels of antibody IgG to p17 antigen decreased in the second- or third-earliest serum samples before the usually observed steep rises, suggesting earlier detections of antibody IgG to p17 antigen than p24 antigen in much earlier samples, if available. The two panels were panels 10 (SV-0241) and 11 (SV-0161).
To test the validity of these results, serum samples, in which antibody IgG to p17 antigen was detected earlier than p24 antigen and RNA, were mixed with excess rp17 antigen and subjected to the improved immunoassay of antibody IgG to p17 antigen (Table 6). The cutoff indexes were reduced 7.0- to 63-fold in the presence of excess rp17 antigen, confirming the presence of antibody IgG to p17 antigen in serum samples earlier than the detections of p24 antigen and RNA.
TABLE 6.
Confirmation of the presence of antibody IgG to p17 antigen in serum samples earlier than the detection of p24 antigen and RNAa
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
|
|---|---|---|---|
| No antigen added | rp17 antigen added | ||
| 9 (panel E) | 7 | 19 | 0.7 |
| 21 | 14 | 1.3 | |
| 126 | 379,000 | 87 | |
| 16 (panel X) | 0 | 11 | 0.6 |
| 2 | 18 | 0.9 | |
| 8 | 25 | 0.4 | |
| 33 | 8,450 | 8.4 | |
| 19 (panel 6240) | 0 | 21 | 3.0 |
| 2 | 16 | 1.1 | |
| 7 | 12 | 1.0 | |
| 36 | 2,250 | 2.3 | |
Finally, the possibility that the panel sera, in which antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, might have derived from subjects transfused with HIV-1-infected blood was tested by comparing cutoff indexes for antibodies IgG and IgM to p17 antigen in those panel sera and in sera randomly collected from HIV-1-infected subjects. As shown in Table 7, the ratios of cutoff indexes for antibody IgG to p17 antigen to those for antibody IgM to p17 antigen in those panel sera before the detection of p24 antigen and RNA were much smaller than the corresponding ratios in sera from HIV-1 asymptomatic carriers and patients with ARC and AIDS. Therefore, it was very unlikely that the panel sera were collected from subjects transfused with HIV-1-infected blood.
TABLE 7.
Cutoff indexes for antibodies IgG and IgM to p17 antigen in serum samples of HIV-1-infected subjects at different stages of the infection
| Source of serum sample(s)a | Days after initial blood collection or serum no. | Cutoff indexesb
|
Cutoff index ratio (anti-p17 IgG/anti-p17 IgM) | |
|---|---|---|---|---|
| Anti-p17 IgG | Anti-p17 IgM | |||
| HIV-1 seroconversion serum panel no. | ||||
| 9 (panel E) | 0 | 4.7 | 3.9 | 1.2 |
| 7 | 22 | 30 | 0.7 | |
| 21 | 16 | 22 | 0.7 | |
| 35 | 3.0 | 2.3 | 1.3 | |
| 42 | 2.0 | 1.0 | 2.0 | |
| 49 | 1.7 | 0.8 | ||
| 63 | 1.6 | 0.6 | ||
| 16 (panel X) | 0 | 12 | 20 | 0.6 |
| 2 | 21 | 23 | 0.9 | |
| 8 | 26 | 16 | 1.6 | |
| 10 | 20 | 28 | 0.7 | |
| 19 (panel 6240) | 0 | 27 | 1.7 | 15 |
| 2 | 19 | 0.0 | ||
| 7 | 13 | 0.3 | ||
| 9 | 12 | 0.1 | ||
| 14 | 7.2 | 0.1 | ||
| 16 | 8.1 | 6.5 | ||
| Asymptomatic carriers | T-28 | 6,382,500 | 863 | 7,393 |
| T-64 | 8,205,000 | 211 | 38,866 | |
| T-76 | 47,414,000 | 46 | 1,028,266 | |
| T-17 | 81,177,000 | 111 | 732,791 | |
| T-61 | 109,789,500 | 39 | 2,791,258 | |
| Patients with ARC | T-15 | 243,100 | 207 | 1,176 |
| T-62 | 1,097,600 | 131 | 8,407 | |
| T-79 | 1,410,225 | 410 | 3,436 | |
| T-23 | 1,559,475 | 204 | 7,657 | |
| T-42 | 2,657,100 | 188 | 14,167 | |
| Patients with AIDS | T-74 | 153,350 | 28 | 5,434 |
| T-36 | 184,100 | 33 | 5,579 | |
| T-11 | 197,350 | 63 | 3,116 | |
| T-4 | 333,725 | 84 | 3,994 | |
| T-53 | 1,653,475 | 385 | 4,297 | |
DISCUSSION
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.
TABLE 8.
Concentrations of free HIV-1 p24 antigen measured by the improved immunoassay and HIV-1 virion-associated p24 antigen calculated from HIV-1 RNA copies measured by RT-PCR (panels 1, 3, 4, 5, 7, and 9)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | HIV-1 p24 antigen measured by the improved immunoassay
|
HIV-1 RNA measured by RT-PCR
|
Ratio (F/A) | |||
|---|---|---|---|---|---|---|---|
| Cutoff index | Amt (pg/ml) (F) | Cutoff index | Copies/ml | Virion-associated p24 antigen (pg/ml) (A) | |||
| 1 (panel Z) | 0 | 0.59 | <0.1 | ||||
| 2 | 3.1 | 0.10 | 3.5 | 1,400 | 0.03 | 3.1 | |
| 7 | 800 | 27 | 1,300 | 540,000 | 13 | 2.1 | |
| 9 | 5,000 | 170 | 1,600 | 620,000 | 15 | 11 | |
| 27 | 110 | 3.5 | 250 | 100,000 | 2.4 | 1.5 | |
| 32 | 120 | 3.9 | 290 | 110,000 | 2.8 | 1.4 | |
| 3 (panel P) | 0 | 0.20 | <0.1 | ||||
| 4 | 0.20 | <0.1 | |||||
| 9 | 12 | 0.41 | 2.4 | 970 | 0.02 | 18 | |
| 15 | 4,200 | 140 | 520 | 210,000 | 5.0 | 28 | |
| 30 | 56 | 1.9 | 30 | 12,000 | 0.29 | 6.6 | |
| 35 | 35 | 1.2 | 28 | 11,000 | 0.27 | 4.3 | |
| 4 (panel AF) | 0 | −0.29 | <0.1 | ||||
| 2 | −0.24 | <0.1 | |||||
| 7 | 0.05 | <0.1 | |||||
| 9 | 0.07 | <0.1 | |||||
| 15 | 57 | 1.9 | 58 | 23,000 | 0.55 | 3.5 | |
| 28 | 660 | 22 | 2,500 | 1,000,000 | 24 | 0.91 | |
| 33 | 400 | 13 | 1,000 | 420,000 | 10 | 1.3 | |
| 35 | 190 | 6.2 | 670 | 270,000 | 6.4 | 1.0 | |
| 42 | 120 | 3.8 | 980 | 390,000 | 9.4 | 0.41 | |
| 5 (panel AJ) | 0 | 0.18 | <0.1 | ||||
| 10 | 0.36 | <0.1 | |||||
| 16 | 0.17 | <0.1 | |||||
| 21 | 0.23 | <0.1 | |||||
| 24 | 4.7 | 0.16 | 9.1 | 3,700 | 0.09 | 1.8 | |
| 28 | 1,000 | 33 | 870 | 350,000 | 8.4 | 4.0 | |
| 43 | 750 | 25 | 570 | 230,000 | 5.5 | 4.5 | |
| 7 (panel W) | 0 | 0.92 | <0.1 | ||||
| 7 | 0.97 | <0.1 | |||||
| 12 | 0.75 | <0.1 | |||||
| 14 | 0.93 | <0.1 | |||||
| 28 | 0.89 | <0.1 | |||||
| 30 | 0.92 | <0.1 | |||||
| 35 | 29 | 0.97 | 19 | 7,800 | 0.19 | 5.2 | |
| 37 | 320 | 11 | 200 | 80,000 | 1.9 | 5.6 | |
| 47 | 8,600 | 290 | 3,200 | 1,300,000 | 31 | 9.3 | |
| 84 | 22 | 0.73 | 470 | 190,000 | 4.5 | 0.16 | |
| 86 | 17 | 0.57 | 240 | 95,000 | 2.3 | 0.25 | |
| 145 | 26 | 0.87 | 89 | 35,000 | 0.85 | 1.0 | |
| 161 | 68 | 2.3 | 100 | 40,000 | 0.96 | 2.4 | |
| 9 (panel E) | 0 | 0.28 | <0.1 | ||||
| 7 | 0.23 | <0.1 | |||||
| 21 | 0.24 | <0.1 | |||||
| 35 | 0.30 | <0.1 | |||||
| 42 | 0.31 | <0.1 | |||||
| 49 | 0.30 | <0.1 | |||||
| 63 | 0.23 | <0.1 | |||||
| 84 | 680 | 23 | 0.8 | 320 | 0.01 | 2,900 | |
| 91 | 8,700 | 290 | 830 | 330,000 | 7.9 | 37 | |
| 126 | 45 | 1.5 | 2.8 | 1,100 | 0.03 | 56 | |
TABLE 9.
Concentrations of free HIV-1 p24 antigen measured by the improved immunoassay and HIV-1 virion-associated p24 antigen calculated from HIV-1 RNA copies measured by RT-PCR (panels 10, 14, 16, 17, 18, and 19)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | HIV-1 p24 antigen measured by the improved immunoassay
|
HIV-1 RNA measured by RT-PCR
|
Ratio (F/A) | |||
|---|---|---|---|---|---|---|---|
| Cutoff index | Amt (pg/ml) (F) | Cut-off index | Copies/ml | Virion-associated p24 antigen (pg/ml) (A) | |||
| 10 (SV-0241) | 0 | 29 | 0.96 | 110 | 43,000 | 1.0 | 0.93 |
| 6 | 5,800 | 190 | 7,500 | 3,000,000 | 72 | 2.7 | |
| 8 | 2,400 | 79 | 2,500 | 1,000,000 | 24 | 3.3 | |
| 14 | 150 | 5.1 | 300 | 120,000 | 2.9 | 1.8 | |
| 16 | 48 | 1.6 | 88 | 35,000 | 0.84 | 1.9 | |
| 21 | 1.8 | 0.06 | 9.8 | 3,900 | 0.09 | 0.66 | |
| 23 | 0.70 | 3.8 | 1,500 | 0.04 | |||
| 14 (SV-0211) | 0 | 1,000 | 34 | 375 | 150,000 | 3.6 | 9.4 |
| 2 | 6,600 | 220 | 1,700 | 660,000 | 16 | 14 | |
| 13 | 3,100 | 100 | 3,300 | 1,300,000 | 31 | 3.4 | |
| 15 | 1,200 | 41 | 950 | 380,000 | 9.1 | 4.5 | |
| 22 | 85 | 2.8 | 22 | 8,800 | 0.21 | 13 | |
| 16 (panel X) | 0 | 0.23 | <0.1 | ||||
| 2 | 0.22 | <0.1 | |||||
| 8 | 0.26 | <0.1 | |||||
| 10 | 0.21 | <0.1 | |||||
| 26 | 2,600 | 88 | 340 | 130,000 | 3.2 | 27 | |
| 33 | 590 | 20 | 80 | 32,000 | 0.77 | 26 | |
| 35 | 100 | 3.4 | 30 | 12,000 | 0.28 | 12 | |
| 40 | 120 | 3.9 | 13 | 5,100 | 0.12 | 32 | |
| 17 (panel Y) | 0 | 0.44 | <0.1 | ||||
| 10 | 0.21 | <0.1 | |||||
| 18 | 0.20 | <0.1 | |||||
| 22 | 0.19 | <0.1 | |||||
| 44 | 350 | 12 | 390 | 140,000 | 3.8 | 3.1 | |
| 49 | 160 | 5.4 | 430 | 170,000 | 4.1 | 1.3 | |
| 18 (panel AR) | 0 | 0.40 | <0.1 | ||||
| 2 | 0.29 | <0.1 | |||||
| 9 | 9.1 | 0.30 | 25 | 9,800 | 0.24 | 1.3 | |
| 14 | 780 | 26 | 2,200 | 880,000 | 21 | 1.2 | |
| 19 (panel 6240) | 0 | 0.20 | <0.1 | ||||
| 2 | 0.17 | <0.1 | |||||
| 7 | 0.11 | <0.1 | |||||
| 9 | 0.13 | <0.1 | |||||
| 14 | 0.11 | <0.1 | |||||
| 16 | 0.26 | <0.1 | |||||
| 21 | 25 | 0.82 | 46 | 19,000 | 0.45 | 1.8 | |
| 23 | 310 | 10 | 380 | 150,000 | 3.6 | 2.9 | |
| 28 | 5,200 | 170 | 2,500 | 1,000,000 | 24 | 7.2 | |
| 30 | 7,900 | 260 | 2,500 | 990,000 | 24 | 11 | |
| 36 | 1,100 | 36 | 720 | 290,000 | 6.9 | 5.1 | |
| 45 | 180 | 6.1 | 64 | 25,000 | 0.61 | 10 | |
| 53 | 99 | 3.3 | 47 | 19,000 | 0.45 | 7.3 | |
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–β-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.
TABLE 2.
Test results of HIV-1 seroconversion serum panels by various methods (panels 5, 6, and 7)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
Maximal dilution to cause gelatin particle agglutination | Western blot
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p24 antigen by previous immunoassay | p24 antigen by improved immunoassay | RNA | Anti-p17 IgG by previous immunoassay | Anti-p17 IgG by improved immunoassay | Anti-p17 IgM | Anti-HIV-1 antibodies by conventional EIA | gp160 | gp120 | gp41 | p24 | p17 | |||
| 5 (panel AJ) | 0 | 0.2 | 0.2 | <0.1 | 0.4 | 0.9 | 0.8 | 0.2 | − | − | − | − | − | |
| 10 | 0.3 | 0.4 | <0.1 | 0.6 | 0.8 | 0.9 | 0.3 | − | − | − | − | − | ||
| 16 | 0.3 | 0.2 | <0.1 | 0.5 | 0.9 | 1.0 | 0.3 | − | − | − | − | − | ||
| 21 | 0.3 | 0.2 | <0.1 | 1.2 | 1.0 | 0.9 | 0.2 | − | − | − | − | − | ||
| 24 | 0.9 | 4.7 | 9.1 | 1.2 | 0.8 | 0.8 | 0.2 | − | − | − | − | − | ||
| 28 | 7.3 | 999 | 873 | 0.6 | 0.9 | 0.9 | 0.2 | − | − | − | − | − | ||
| 43 | 20 | 748 | 571 | 291 | 38,235 | 1,705 | 2.4 | 16 | + | − | − | + | − | |
| 6 (SV-0051) | 0 | 17 | 245 | 0.3 | 0.9 | 0.3 | 0.4 | − | − | − | − | − | ||
| 6 | 232 | 3,279 | 0.2 | 0.5 | 0.4 | 1.4 | − | − | − | − | − | |||
| 8 | 97 | 1,871 | 2.5 | 55 | 6.8 | 4.4 | 1 | − | − | − | − | − | ||
| 15 | 14 | 228 | 6,526 | 202,106 | 21,127 | 4.0 | 8 | + | − | − | + | − | ||
| 27 | 1.3 | 9.6 | 8,632 | 5,183 | 3.5 | 8 | + | − | − | + | − | |||
| 7 (panel W) | 0 | 0.4 | 0.9 | <0.1 | 0.1 | 0.4 | 0.4 | 0.2 | − | − | − | − | − | |
| 7 | 0.3 | 1.0 | <0.1 | 0.1 | 0.2 | 0.4 | 0.2 | − | − | − | − | − | ||
| 12 | 0.3 | 0.7 | <0.1 | 0.1 | 0.2 | 0.4 | 0.2 | − | − | − | − | − | ||
| 14 | 0.7 | 0.9 | <0.1 | 0.2 | 0.2 | 0.6 | 0.2 | − | − | − | − | − | ||
| 28 | 0.4 | 0.9 | <0.1 | 0.3 | 0.2 | 0.4 | 0.2 | − | − | − | − | − | ||
| 30 | 0.3 | 0.9 | <0.1 | 0.1 | 0.3 | 0.4 | 0.2 | − | − | − | − | − | ||
| 35 | 1.2 | 29 | 19 | 0.2 | 0.2 | 0.5 | 0.2 | − | − | − | − | − | ||
| 37 | 7.8 | 318 | 199 | 0.1 | 0.2 | 0.4 | 0.2 | − | − | − | − | − | ||
| 47 | 634 | 8,627 | 3,230 | 0.5 | 0.5 | 1.4 | 0.7 | − | − | − | − | − | ||
| 84 | 11 | 22 | 472 | 5,049 | 279,263 | 4,630 | 2.9 | 8 | − | − | − | + | + | |
| 86 | 7.2 | 17 | 237 | 4,940 | 283,406 | 4,177 | 2.7 | 8 | − | − | − | + | + | |
| 145 | 2.4 | 26 | 89 | 1,229 | 58,267 | 272 | 5.6 | 32 | + | + | + | + | + | |
| 161 | 5.1 | 68 | 100 | 569 | 28,769 | 87 | 6.3 | 32 | + | + | + | + | + | |
TABLE 4.
Test results of HIV-1 seroconversion serum panels by various methods (panels 11 to 15)
| HIV-1 seroconversion serum panel no. | Days after initial blood collection | Cutoff indexes
|
Maximal dilution to cause gelatin particle agglutination | Western blot
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| p24 antigen by previous immunoassay | p24 antigen by improved immunoassay | RNA | Anti-p17 IgG by previous immunoassay | Anti-p17 IgG by improved immunoassay | Anti-p17 IgM | Anti-HIV-1 antibodies by conventional EIA | gp160 | gp120 | gp41 | p24 | p17 | |||
| 11 (SV-0161) | 0 | 12 | 358 | 0.7 | 20 | 2.3 | 0.2 | − | − | − | − | − | ||
| 4 | 38 | 1,171 | 0.2 | 4.4 | 0.7 | 0.2 | − | − | − | − | − | |||
| 7 | 67 | 2,217 | 1.3 | 21 | 2.9 | 0.5 | − | − | − | − | − | |||
| 11 | 133 | 3,575 | 89 | 2,446 | 160 | 5.2 | 8 | − | − | − | + | − | ||
| 15 | 4.0 | 101 | 1,537 | 499 | 2.8 | 16 | − | − | − | + | − | |||
| 18 | 1.4 | 34 | 2,516 | 410 | 2.1 | 16 | − | − | − | + | − | |||
| 12 (panel K) | 0 | 0.3 | 2.0 | 0.0 | 0.2 | 0.4 | 0.3 | − | − | − | − | − | ||
| 6 | 0.5 | 3.8 | 0.1 | 0.9 | 0.4 | 0.3 | − | − | − | − | − | |||
| 8 | 0.5 | 7.2 | 0.8 | 1.6 | 0.5 | 0.4 | − | − | − | − | − | |||
| 13 | 0.7 | 11 | 364 | 31,483 | 4,261 | 0.7 | − | − | − | + | − | |||
| 20 | 0.4 | 2.0 | 3,232 | 21,177 | 1.4 | 1 | − | − | − | + | − | |||
| 29 | 0.5 | 0.9 | 4,805 | 6,361 | 1.4 | 2 | + | − | − | + | − | |||
| 33 | 0.3 | 0.5 | 6,316 | 3,857 | 2.1 | 4 | + | − | − | + | + | |||
| 36 | 0.4 | 0.7 | 6,211 | 2,863 | 2.7 | 4 | + | − | − | + | + | |||
| 13 (SV-0111) | 0 | 366 | 7,575 | 1.5 | 9.3 | 0.4 | 0.3 | − | − | − | − | − | ||
| 1 | 559 | 8,083 | 1.8 | 36 | 0.5 | 0.5 | − | − | − | − | − | |||
| 7 | 124 | 4,731 | 3.6 | 228 | 7.1 | 4.8 | 32 | − | − | − | + | − | ||
| 15 | 16 | 480 | 4,716 | 314,671 | 47,440 | 3.7 | 64 | + | − | − | + | − | ||
| 19 | 5.7 | 158 | 5,142 | 31,359 | 3.0 | 64 | + | − | − | + | − | |||
| 21 | 6.1 | 167 | 5,368 | 26,090 | 3.2 | 64 | + | − | − | + | − | |||
| 26 | 3.3 | 75 | 6,316 | 26,709 | 3.3 | 64 | + | − | − | + | − | |||
| 14 (SV-0211) | 0 | 11 | 1,013 | 375 | 0.1 | 1.2 | 0.4 | 0.2 | − | − | − | − | − | |
| 2 | 58 | 6,649 | 1,650 | 0.3 | 4.4 | 0.3 | 0.4 | − | − | − | − | − | ||
| 13 | 107 | 3,147 | 3,250 | 8,367 | 762,671 | 23,121 | 6.3 | 4 | + | − | − | + | − | |
| 15 | 65 | 1,241 | 950 | 10,239 | 26,359 | 6.2 | 16 | + | − | − | + | − | ||
| 22 | 8.5 | 85 | 22 | 10,749 | 18,790 | 3.3 | 32 | + | − | − | + | − | ||
| 15 (panel S) | 0 | 37 | 502 | 0.3 | 2.8 | 0.3 | 0.3 | − | − | − | − | − | ||
| 9 | 8.1 | 89 | 103 | 5,143 | 2,200 | 4.2 | 8 | − | − | − | + | − | ||
| 11 | 3.2 | 29 | 388 | 49,071 | 6,576 | 3.5 | 8 | + | − | − | + | − | ||
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