© 1998 Wiley-Liss, Inc.

Journal of Clinical Laboratory Analysis 12:197–204 (1998)

Immune Complex Transfer Enzyme Immunoassay for AntibodyIgG to HIV-1 gp41 Antigen Using Synthetic Peptides as Antigens

Seiichi Hashida, 1 Setsuko Ishikawa, 1 Kazuya Hashinaka, 1 Ichiro Nishikata, 1

Shinichi Oka, 2 and Eiji Ishikawa 1*1Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki, Japan

2AIDS Clinical Center, International Medical Center of Japan, Toyama, Shinjuku, Tokyo, Japan

*Correspondence to: Eiji Ishikawa, M.D., Ph.D., Professor of Biochemistry,Department of Biochemistry, Miyazaki Medical College, Kiyotake, Miyazaki889-1692, Japan.

Received 8 December 1997; Accepted 16 December 1997

The immune complex transfer enzymeimmunoassay for antibody IgG to HIV-1 gp41antigen was developed using two syntheticpeptides. An aliquot (10 µl) of serum samplesfrom HIV-1 seropositive subjects was incu-bated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41peptide conjugates and synthetic HIV-1 gp41peptide-β-D-galactosidase conjugates andsubsequently with colored polystyrene beadscoated with affinity-purified (anti-2,4-dinitrophenyl group) IgG to trap the immunecomplexes formed comprising the three com-ponents. After washing, the colored polysty-rene beads were incubated with whitepolystyrene beads coated with affinity-puri-fied (anti-human IgG γ-chain) IgG in the pres-ence of eN-2,4-dinitrophenyl-L-lysine totransfer the immune complexes to the whitepolystyrene beads. β-D-Galactosidase activ-ity bound to the white polystyrene beads was

assayed by fluorometry. The formation,trapping and transferring of the immunecomplexes were completed within 0.5, 0.5and 1.5 hr, respectively. Since each pep-tide appeared to react with its own spe-cific antibody IgG, serum samples weretested by the equimolar combination of thetwo peptides. The lowest signals (fluores-cence intensities for bound β-D-galactosi-dase activity) for serum samples fromHIV-1 asymptomatic carriers, patients withAIDS-related complex and patients withAIDS were 1490-, 2210- and 1460-fold, re-spectively, higher than the highest signalfor serum samples from HIV-1 seronega-tive subjects. In five seroconversion se-rum panels, antibody IgG to HIV-1 gp41antigen was detected as early as antibodiesto HIV-1 detected by three currently commer-cially available methods. J. Clin. Lab. Anal.12:197–204, 1998. © 1998 Wiley-Liss, Inc.

Key words: HIV-1; gp41; antibody IgG; enzyme immunoassay; β-D-galactosidase

INTRODUCTION

Ultrasensitive enzyme immunoassays (immune complextransfer enzyme immunoassays) for antibody IgGs to p17,p24, and reverse transcriptase (RT) of HIV-1 have been de-veloped using recombinant p17, p24, and RT (rp17, rp24, andrRT) as antigens (1-7). The immune complexes, comprising2,4-dinitrophenyl-recombinant protein conjugates, antibodyIgGs to HIV-1, and recombinant protein-β-D-galactosidaseconjugates, were formed by 3-hr incubation and were trappedonto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG by overnight incubation. After wash-ing, the immune complexes were eluted from the polystyrenebeads with eN-2,4-dinitrophenyl-L-lysine and were transferredto polystyrene beads coated with affinity-purified (antihumanIgG γ-chain) IgG by 3-hr incubation. Bound β-D-galactosi-dase activity was assayed by fluorometry for 2.5 hr. The vol-ume of serum samples used was 10 µl. These immunoassayshave made possible the diagnosis of HIV-1 infection withurine (1-4) and whole saliva samples (5,6), and have been

shown to be more useful than Western blotting (7) as a con-firmatory test since they reflect higher sensitivities and speci-ficities. In particular, antibody IgG to p17 of HIV-1 could bedetected as early as, or even earlier than, antibodies to HIV-1by conventional methods (7,8). In addition, an ultrasensitiveenzyme immunoassay based on a similar principle for p24antigen of HIV-1 has been developed (9,10). On the basis ofthese results, the period of time immediately following HIV-1 infection during which actual diagnosis of HIV-1 infectionis not possible due to the absence of detectable antibodies toHIV-1 has been shortened by simultaneous detection of bothp24 antigen and antibody IgGs to p17 and RT (8). In theseimmunoassays, however, incubation time for immuno-reactions was lengthy as described above. Recently, it has

198 Hashida et al.

become possible to perform more sensitive immune complextransfer enzyme immunoassays for antibody IgG to p17 withinshorter periods of time (15 to 60 min each for the formation,trapping, and transferring of the immune complex) by utili-zation of shaking for the immunoreactions, using a larger vol-ume of serum samples (100 µl), and use of solid phases withlarger surface areas (11).

This report describes the time courses of the immuno-reactions in the immune complex transfer enzyme immu-noassays for antibody IgGs to HIV-1 gp41 antigen usingtwo synthetic peptides as antigens and the detection ofantibody IgGs to HIV-1 gp41 antigen in serum samplesrandomly collected from HIV-1-infected subjects and HIV-1 seroconversion serum panels.

MATERIALS AND METHODS

Buffers

The following were used regularly: 0.1 mol/L sodium phos-phate buffer, pH 6.0, containing 5 mmol/L EDTA (buffer A);10 mmol/L sodium phosphate buffer, pH 7.0, containing1 g/L bovine serum albumin (fraction V, Intergen Company,Purchase, NY) with 1.0 mmol/L MgCl2 and 1.0 g/L NaN3(buffer B).

Two Synthetic HIV-1 gp41 Peptides

Synthetic HIV-1 gp41 peptides, p32 and p34, consisting of20 amino acids including one cysteine residue per moleculewere obtained from Innogenetics NV, Zwijnaarde, Belgium.

2,4-Dinitrophenyl-Bovine Serum Albumin-Synthetic HIV-1 gp41 Peptide Conjugates

Maleimide groups were introduced into 2,4-dinitrophenyl-bovine serum albumin molecules using N-succinimidyl-6-maleimidohexanoate (12).

6-Maleimidohexanoyl-2,4-dinitrophenyl-bovine serum al-bumin (0.86 mg, 13 nmol) in 0.45 ml of buffer A was incu-bated with synthetic HIV-1 gp41 peptide, p32, (175 µg, 75nmol) in 0.05 ml of deionized water at 30°C for 1 hr. Subse-quently, the reaction mixture was incubated with 5 µl of 0.1mol/L 2-mercaptoethylamine in buffer A at 30°C for 10 minand was subjected to gel filtration by the centrifuged columnprocedure (13) using a column (1.1 × 5.3 cm) of SephadexG-50 fine (Pharmacia Biotech, Uppsala, Sweden), equili-brated with 10 mmol/L sodium phosphate buffer, pH 7.0,containing 0.1 mol/L NaCl. The average number of syntheticHIV-1 gp41 peptide molecules conjugated per albumin mol-ecule was 4.0, calculated from the decrease in the number ofmaleimide groups (12). The amount of the conjugate was cal-culated from that of 2,4-dinitrophenyl-bovine serum albumin.

2,4-Dinitrophenyl-bovine serum albumin-syntheticHIV-1 gp41 peptide, p34, conjugate was also prepared asdescribed above.

Synthetic HIV-1 gp41 Peptide- b-D-GalactosidaseConjugates

Maleimide groups were introduced into molecules of β-D-galactosidase (EC 3.2.1.23) from E. coli using N,N´-1,2-phenylenedimaleimide (12). Maleimide-β-D-galactosidase(0.54 mg, 1.0 nmol) in 0.45 ml of buffer A was incubatedwith synthetic HIV-1 gp41 peptide, p32, (14 µg, 6.0 nmol) in0.05 ml of deionized water at 30°C for 1 hr. Subsequently,the reaction mixture was incubated with 5 µl of 0.1 mol/L2-mercaptoethylamine in buffer A at 30°C for 10 min andwas subjected to gel filtration by the centrifuged column pro-cedure (13) using a column (1.1 × 5.3 cm) of Sephadex G-50fine (Pharmacia Biotech), equilibrated with 10 mmol/L so-dium phosphate buffer, pH 7.0, containing 0.1 mol/L NaCl.The average number of synthetic HIV-1 gp41 peptide, p32,molecules conjugated per β-D-galactosidase molecule was 5.0,which was calculated from the decrease in the number ofmaleimide groups (12). The amount of the conjugate was cal-culated from β-D-galactosidase activity (12).

Synthetic HIV-1 gp41 peptide, p34, -β-D-galactosidaseconjugate was also prepared as described above.

Polystyrene Beads Coated With Antibody IgGs

Colored and white polystyrene beads (3.2 mm in diam-eter, Immuno Chemical, Okayama, Japan) were coated withaffinity-purified (anti-2,4-dinitrophenyl group) IgG and(antihuman IgG γ-chain) IgG, respectively, by physicaladsorption (12).

Immune Complex Transfer Enzyme Immunoassayfor Antibody IgG to HIV-1 gp41

The immune complex transfer enzyme immunoassay wasperformed essentially as described previously (1-7). An ali-quot (10 µl) of serum was mixed with 90 µl of buffer B con-taining 0.4 mol/L NaCl and was incubated for up to 2 hr with100 fmol each of 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41 peptide conjugates and synthetic HIV-1 gp41 peptide-β-D-galactosidase conjugates in 50 µl of bufferB containing 0.4 mol/L NaCl and 50 µg of inactive β-D-ga-lactosidase (Mutein, Boehringer Mannheim, Mannheim, Ger-many). The reaction mixture was incubated with twocolored polystyrene beads (3.2 mm in diameter) coatedwith affinity-purified (anti-2,4-dinitrophenyl group) IgGfor up to 2 hr. The colored polystyrene beads were washedtwice with 2 ml of buffer B containing 0.1 mol/L NaCland were incubated for up to 2 hr with two white polysty-rene beads (3.2 mm in diameter) coated with affinity-pu-rified (anti-human IgG γ-chain) IgG in 150 µl of buffer Bcontaining 0.1 mol/L NaCl and 1 mmol/L eN-2,4-dini-trophenyl-L-lysine. The incubations were performed atroom temperature with 180 shakings per min throughout.The white polystyrene beads were washed as described

Enzyme Immunoassay of Anti-HIV-1 gp41 IgG 199

above, and β-D-galactosidase activity bound to the whitepolystyrene beads was assayed by fluorometry at 30°C for 1or 20 hr. The fluorescence intensity was measured relative to1 × 10–8 mol/L 4-methylumbelliferone.

Other Immunological Methods

The conventional EIA for antibodies to HIV-1 was per-formed using a commercial kit with two recombinant pro-teins of HIV-1 (gp41 and p24) as antigens (AbbottRecombinant HIV-1/HIV-2 3rd Generation EIA, Abbott Labo-ratories, North Chicago, IL). The gelatin particle agglutina-tion test for antibodies to HIV-1 was performed using acommercial kit with a lysate of HIV-1 as antigen (Serodia-HIV, Fujirebio Inc., Tokyo, Japan). Western blotting for anti-body IgGs to HIV-1 was performed using a commercial kitpreblotted with nine proteins of HIV-1 (gp160, gp120, p66,p55, p51, gp41, p31, p24, and p17) (Ortho HIV Western BlotKit, Ortho Diagnostic Systems Inc., Raritan, NJ).

Seroconversion Serum Panels

Two seroconversion serum panels (SV-0211 and SV-0241)were obtained from North American Biologicals, Inc., Mi-ami, FL. Three seroconversion serum panels (Panels J, W,and AJ) were obtained from Boston Biomedica, Inc., WestBridgewater, MA.

Fig. 1. Immune complex transfer enzyme immunoassay for antibody IgG.DNP: 2,4-dinitrophenyl group. Ag: antigen. Enz: enzyme.

Fig. 2. Time courses for the formation, trapping, and transferring of theimmune complexes comprising 2,4-dinitrophenyl-bovine serum albumin-syn-thetic HIV-1 gp41 peptide conjugates, antibodies to HIV-1 gp41 antigen andsynthetic HIV-1 gp41 peptide-β-D-galactosidase conjugates. A: Formation ofthe immune complex. An aliquot (10 µl) of serum samples was incubated atroom temperature with 100 fmol each of 2,4-dinitrophenyl-bovine serum al-bumin-synthetic HIV-1 gp41 peptide conjugates and synthetic HIV-1 gp41peptide-β-D-galactosidase conjugates for up to 2 hr and subsequently withtwo colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 20 min with shaking.B: Trapping of the immunecomplex. An aliquot (10 µl) of serum samples was incubated at room tem-perature with 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41 peptide conjugates and synthetic HIV-1 gp41 peptide-β-D-galactosidase conjugates for 0.5 hr and subsequently with colored poly-styrene beads coated with (anti-2,4-dinitrophenyl group) IgG at roomtemperature for up to 2 hr. C: Transferring of the immune complex. An aliquot(10 µl) of serum samples was incubated at room temperature with 100 fmoleach of 2,4-dinitrophenyl-bovine serum albumin-synthetic HIV-1 gp41 pep-tide conjugates and synthetic HIV-1 gp41 peptide-β-D-galactosidase conju-gates for 0.5 hr and subsequently with colored polystyrene beads coated with(anti-2,4-dinitrophenyl group) IgG for 1 hr. The colored polystyrene beadsafter washing were incubated with white polystyrene beads coated with (anti-human IgG γ-chain) IgG in the presence of eN-2,4-dinitrophenyl-L-lysine atroom temperature for up to 2 hr.β-D-Galactosidase activity bound to the colored and white polystyrene beadswas assayed at 30°C for 1 hr throughout. Open and closed symbols indicateresults with serum samples from HIV-1 seropositive and seronegative sub-jects, respectively. Circles, triangles, and squares indicate results with syn-thetic HIV-1 gp41 peptides, p32, p34, and p32 plus p34, respectively. Solidand broken lines indicate results with different HIV-1 seropositive serumsamples.

200 Hashida et al.

TABLE 1. Reactivity of Two Synthetic HIV-1 gp41 Peptides, p32 and p34, With Serum Samples

Signal (fluorescence intensity for boundβ-D-galactosidase activity)

Conjugate usedp32-Gal

DNP-BSA-p32+

Serum p32-Gal p34-Gal p34-Galsample No. DNP-BSA-p32 DNP-BSA-p34 DNP-BSA-p34

Mean ± SD (range)Negative 1-100 0.4 ± 0.2 0.2 ± 0.0 0.6 ± 0.2

(0.1 – 1.1) (0.1 – 0.3) (0.1 – 1.4)Positive 101 53.6 13200 14000

102 783 1150 2040103 29700 122000 152000104 1200 1250 2410105 4870 958 6610106 1250 841 2080107 35600 36600 68500108 614 976 2050

Serum samples were collected from 100 HIV-1 seronegative subjects (serum sample Nos. 1 to 100) and 8 HIV-1 seropositive subjects (serum samples Nos.101 to 108) and were tested by the immune complex transfer enzyme immunoassay using synthetic HIV-1 gp41 peptide (p32 and p34) conjugates. Serumsamples (10 µl) were incubated with the two conjugates for 0.5 hr, and the immune complexes formed were trapped for 1 hr and were transferred for 1 hr.Bound β-D-galactosidase activities with the positive and negative serum samples were assayed for 1 hr and 20 hr, respectively, and the signals by 20 hr assayare shown as those by 1 hr assay after correction. Gal, β-D-galactosidase; DNP, 2,4-dinitrophenyl; BSA, bovine serum albumin.

Serum Samples Randomly Collected From HIV-1Seronegative and Seropositive Subjects

Serum samples were randomly collected from 100 HIV-1seronegative subjects (42 males aged 23-67 yr and 58 femalesaged 21–68 yr) and 79 HIV-1 seropositive subjects (42 maleasymptomatic carriers aged 17-47 yr, 8 female asymptomaticcarriers aged 18–39 yr, 7 male patients aged 10–52 yr withAIDS-related complex, 2 female patients aged 18–42 yr withAIDS-related complex, and 20 male patients aged 14–61 yrwith AIDS) and were stored at –20°C until use. The serumsamples were tested by the gelatin particle agglutination test,and the seropositivity was confirmed by Western blotting.

RESULTS AND DISCUSSION

The immune complex transfer enzyme immunoassay forantibody IgG to HIV-1 gp41 antigen using two syntheticHIV-1 gp41 peptides (p32 and p34) as antigens was per-formed as follows. Antibody IgG to HIV-1 gp41 antigenwas reacted simultaneously with 2,4-dinitrophenyl-bovineserum albumin-synthetic HIV-1 gp41 peptide conjugatesand synthetic HIV-1 gp41 peptide-β-D-galactosidase con-jugates, and the immune complexes formed comprisingthe three components was trapped onto colored polysty-rene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The immune complexes wereeluted from the colored polystyrene beads with excessof eN-2,4-dinitrophenyl-L-lysine and were transferred towhite polystyrene beads coated with affinity-purified (anti-human IgG γ-chain) IgG (Fig. 1). β-D-Galactosidase ac-

tivity bound to the white polystyrene beads was assayedby fluorometry.

Time Courses of the Immunoreactions in theImmune Complex Transfer Enzyme Immunoassay

In order to perform the immune complex transfer enzymeimmunoassay for antibody IgG to HIV-1 gp41 antigen usingthe synthetic HIV-1 gp41 peptides (p32 and p34) as antigenswithin as short a time as possible, the time courses of theformation, trapping, and transferring of the immune com-plexes were examined (Fig. 2).

First, serum samples (10 µl) from two HIV-1 seropositivesubjects were incubated with the two conjugates for up to 2hr, and the immune complexes formed were trapped onto thecolored polystyrene beads for 20 min. The signals (fluores-cence intensities for bound β-D-galactosidase activity) reachedthe maximum within 0.5 hr and subsequently declined slowly.

Second, the two serum samples were incubated with thetwo conjugates for 0.5 hr, and the immune complexes formedwere trapped onto the colored polystyrene beads for up to 2hr. The signals reached the maximum within 0.5 hr and sub-sequently declined slowly.

Third, the two serum samples were incubated with the twoconjugates for 0.5 hr, and the immune complexes were trappedfor 1 hr and was transferred for up to 2 hr. The signals reachedthe maximum within 0.5 to 1.5 hr. Transferrence of the im-mune complexes was almost completed within 1.5 hr.

The basis of these results were used in the following experi-ments in which serum samples (10 µl) were incubated with the

Enzyme Immunoassay of Anti-HIV-1 gp41 IgG 201

two conjugates for 0.5 hr, and the immune complexes formedwere trapped onto the colored polystyrene beads for 1 hr andwere transferred to the white polystyrene beads for 1 hr.

Reactivity of Two Synthetic HIV-1 gp41 Peptides,p32 and p34, With Serum Samples From HIV-1Seropositive Subjects

Eight serum samples from HIV-1 seropositive subjects (7males aged 24–42 yr and 1 female aged 28 yr) and 100 serum

Fig. 3. Detection of antibody IgG to HIV-1 gp41 antigen in serum samplesrandomly collected from HIV-1 seropositive subjects. Serum samples fromthe 100 HIV-1 seronegative subjects described in Table 1, 50 asymptomaticcarriers (AC), 9 patients with AIDS-related complex (ARC), and 20 patientswith AIDS were tested by the immune complex transfer enzyme immunoas-say for antibody IgG to HIV-1 gp41 antigen using conjugates with syn-thetic HIV-1 gp41 peptides p32 (A), p34 (B), and p32 plus p34 (C). An

aliquot (10 µl) of serum samples was incubated with the two conju-gates for 0.5 hr, and the immune complexes formed were trapped for 1hr and were transferred for 1 hr. Bound β-D-galactosidase activities withthe positive and negative serum samples were assayed for 1 hr and 20 hr,respectively, and the signals by 20 hr assay were corrected for 1 hr assay.The broken lines indicate the highest signals among 100 HIV-1 seronega-tive subjects.

samples from HIV-1 seronegative subjects (42 males aged23–67 yr, and 58 females aged 21–68 yr) were tested by theimmune complex transfer enzyme immunoassay for antibodyIgG to HIV-1 gp41 using the two synthetic HIV-1 gp41 pep-tides (p32 and p34) (Table 1). For accuracy, bound β-D-ga-lactosidase activities with the negative serum samples wereassayed for 20 hr and the signals were corrected for 1-hrassay, although those with the positive serum samples wereassayed for 1 hr. In two of the eight serum samples, thesignals with p32 and p34 were similar. However, they were

TABLE 2. Test Results of Seroconversion Serum Panels by Various Methods

Cut-off index by theimmune complex Cut-off Maximaltransfer enzyme index by the dilutionimmunoassay for conventional to causeantibody IgG to EIA gelatin

HIV-1 gp41 using (HIV-1/HIV-2 particleSeroconversion Day of the equimolar 3rd aggluti-serum panel blood combination of p32 generation, nation Western blot (Ortho)

No. collection and p34 as antigens Abbott) (Fujirebio) gp160 gp120 p66 p51 gp41 p31 p24 p17

1 (SV-0241) 0 0.5 0.2 – – – – – – – – –6 0.7 0.2 – – – – – – – – –8 0.5 0.3 – – – – – – – – –

14 216 7.4 8 + – – – – – + –16 291 6.0 16 + – – – – – + –21 298 5.6 16 + – – – – – + –23 281 4.2 16 + – – – – – + –

2 (SV-0211) 0 2.3 0.2 – – – – – – – – –2 2.2 0.4 – – – – – – – – –

13 1084 6.3 4 + – – – – – + –15 1595 6.2 16 + – – – – – + –20 2582 3.7 32 + – – – – – + –22 2373 3.3 32 + – – – – – + –

3 (Panel J) 0 0.8 0.3 – – – – – – – – –14 0.6 0.4 – – – – – – – – –26 492 2.3 32 + – + + – – + –28 491 2.3 32 + – + + – – + –32 423 2.8 32 + – + + – – + –35 299 2.9 32 + – + + – – + –40 253 3.7 32 + – + + – – + –

For test by the immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 using the equimolar combination of p32 and p34 as antigens, an aliquot (10 µl) of serum samples wasincubated with the two conjugates for 1 hr, and the immune complexes formed were trapped for 1 hr and were transferred for 1 hr. Bound β-D-galactosidase activity was assayed for 1 hr. The cut-off indexwas calculated by dividing each signal with the highest signal of 1.4 for 100 HIV-1 seronegative serum samples.NT: not tested. The p55 band was not detected.

TABLE 3. Test Results of Seroconversion Serum Panels by Various Methods

Cut-off index by theimmune complex Cut-off Maximaltransfer enzyme index by the dilutionimmunoassay for conventional to causeantibody IgG to EIA gelatin

HIV-1 gp41 using (HIV-1/HIV-2 particleSeroconversion Day of the equimolar 3rd aggluti-serum panel blood combination of p32 generation, nation Western blot (Ortho)

No. collection and p34 as antigens Abbott) (Fujirebio) gp160 gp120 p66 p51 gp41 p31 p24 p17

4 (Panel AJ) 0 NT 0.2 – – – – – – – – –10 NT 0.3 – – – – – – – – –16 NT 0.3 – – – – – – – – –21 NT 0.2 – – – – – – – – –24 0.9 0.2 – – – – – – – – –28 0.8 0.2 – – – – – – – – –43 126 2.4 512 + – – – – – + –

5 (Panel W) 0 1.0 0.2 – – – – – – – – –7 0.7 0.2 – – – – – – – – –

12 0.7 0.2 – – – – – – – – –14 0.6 0.2 – – – – – – – – –28 0.8 0.2 – – – – – – – – –30 0.7 0.2 – – – – – – – – –35 0.7 0.2 – – – – – – – – –37 0.7 0.2 – – – – – – – – –47 1.8 0.7 – – – – – – – – –84 44 2.9 256 – – + + – + + +86 52 2.7 256 – – + + – + + +

145 70 5.6 1024 + + + + + + + +161 73 6.3 1024 + + + + + + + +

For test by the immune complex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 using the combination of p32 and p34 as antigens, an aliquot (10 µl) of serum samples was incubated withthe two conjugates for 1 hr, and the immune complexes formed were trapped for 1 hr and were transferred for 1 hr. Bound β-D-galactosidase activity was assayed for 1 hr. The cut-off index was calculatedby dividing each signal with the highest signal of 1.4 for 100 HIV-1 seronegative serum samples.NT: not tested. The p55 band was not detected.

204 Hashida et al.

higher with p32 than with p34 in two serum samples, andwere higher with p34 than with p32 in four serum samples. Inother words, two serum samples out of the eight were morereactive with p32, and four serum samples were more reac-tive with p34. The signals by the combined use of p32 andp34 were almost equal to the sums of those with p32 andp34. These results suggest that each of the two peptidesreacted with its own specific antibody molecules.

Detection of Antibody IgG to HIV-1 gp41 Antigenin Serum Samples Randomly Collected From HIV-1-Infected Subjects

Serum samples were collected from the 100 HIV-1 serone-gative subjects described above and 79 HIV-1 seropositivesubjects (42 male and 8 female asymptomatic carriers aged17–47 yr, 7 male and 2 female patients with ARC (AIDS-related complex) aged 10–52 yr, and 20 male patients withAIDS aged 14–61 yr) and were tested by the immune com-plex transfer enzyme immunoassay for antibody IgG to HIV-1 gp41 antigen using the two HIV-1 gp41 synthetic peptides(p32 and p34) (Fig. 3). As described above, bound β-D-galac-tosidase activities with the positive and negative serumsamples were assayed for 1 hr and 20 hr, respectively, and allthe signals are shown as those by 1-hr assay. The lowest sig-nals for the asymptomatic carriers and patients with ARCand AIDS were, respectively, 505-, 1459-, and 49-foldhigher than the highest signal for the seronegative sub-jects with p32, 2800, 3850- and 3250-fold higher with p34and 1490-, 2210-, and 1460-fold higher with the equimo-lar combination of the two peptides.

Detection of Antibody IgG to HIV-1 gp41 Antigenin Seroconversion Serum Panels

Five seroconversion serum panels were tested by the im-mune complex transfer enzyme immunoassay for antibodyIgG to HIV-1 gp41 antigen using the equimolar combina-tion of two synthetic HIV-1 gp41 peptides (p32 and p34)(Tables 2 and 3). The seropositivity was detected as earlyas by three commercially available methods (Abbott 3rdgeneration EIA, Fujirebio gelatin particle agglutination test,and Ortho Western blotting).

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