diagnosis of hiv-1 infection by detection of antibody igg to hiv-1 in urine with ultrasensitive...

Journal of Clinical Laboratory Analysis 8:237-246 (1994)

Diagnosis of HIV-1 Infection by Detection of Antibody IgG to HIV-1 in Urine with Ultrasensitive Enzyme lmmunoassay

(Immune Complex Transfer Enzyme Immunoassay) Using Recombinant Proteins as Antigens

Sei ic h i Has hida,’ Kazuya Hash i naka,’ Atsus h i Saito h ,* A ki hisa Ta kam i ~ a w a , ~ Hideo Shinagawa,2 Shinichi Oka,4 Kaoru Shimada,4 Kouichi Hirota,‘ Takeyuki Kohno,’

Setsuko Ishikawa,’ and Eiji Ishikawa’ Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki, Japan; *Department of

Experimental Chemotherapy, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 3Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonii, Kagawa, Japan; 4Department of Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan

1

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p i 7 and p24 as antigens, and p-o-galactosidase from Escherichia coli as label. Anti-HIV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant prOtein-P-D-galaCtOSidaSe conjugate. The immune complex formed, con- sisting of the three components, was trapped onto polystyrene balls coated with affinity- purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of &N-2,4-dinitrophenyl-~-lysine and transferred to clean polystyrene balls coated with

aff inity-purified (anti-human IgG ychain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 1 OO%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p l 7 and p24 as antigens. The sensitivity could be improved by a longer assay of bound p-o-galactosidase activity, by using concentrated urine samples and by the combined use of recombinant RT, p l 7, and p24.Thus, reliable diagnosis of HIV- 1 infection was possible for asymptomatic carriers. Q 1994 Wiley-Liss, inc.

Key words: reverse transcriptase, p17, p24, recombinant antigen, P-D-galaCtOSidaSe

INTRODUCTION

Antibodies to HIV- 1 in urine has been detected by various methods including the conventional ELISA (14), gelatin particle agglutination test (GPA) (4), IgG antibody-capture enzyme-linked immunosorbent assay (GACELISA) (5-7), and IgG antibody-capture particle adherence test (GACPAT) (5,6). In some of the above reports, the sensitivity and specificity were fairly high (2). However, none of the methods appeared to be satisfactory for diagnosis of HIV-I infection. Using the conventional ELISA, the ratio of signal to noise was not improved by concentration of urine samples (2) . The specificity of the conventional ELISA and GPA was considerably lowered with concentrated urine samples due to enhanced nonspecific signals (4). As anticipated from the principle of GACELISA and GACPAT, the sensitivity would not be improved with concentrated urine samples, as long as the level of IgG in urine samples reached the low level needed to satu- rate the assay anti-IgG binding sites (5).

0 1994 Wiley-Liss, Inc.

This article reviews the detection of antibody IgG to HIV- 1 in urine by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for reliable diagnosis of HIV- 1 infection.

PRINCIPLE OF IMMUNE COMPLEX TRANSFER ENZYME IMM U NOASSAY

The principle of immune complex transfer enzyme immunoassay has been described in detail elsewhere (8). In brief, the immune complex formed by reacting antibody to be detected with labeled antigen is trapped onto solid phase and,

Received January 12, 1994; accepted January 19, 1994.

Address reprint requests to Dr. Eiji Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

238 Hashida et al.

P + - DNP-Ag Anti-DNP-

solid phase

I no0

nant protein molecules using N-succinimidyl-s-acetyl- mercaptoacetate. Maleimide groups were introduced into 2,4- dinitrophenyl-bovine serum albumin and peroxidase molecules using N-succinimidyl-6-maleimidohexanoate

Ab Ag-Enz and into P-D-galactosidase molecules using N,N'-o-phenyl- enedimaleimide.

Polystyrene balls of 3.2 mm in diameter used as solid phase were coated by physical adsorption with affinity-purified (anti- 2,4-dinitrophenyl group) IgG and affinity-purified (anti-human IgG y-chain) IgG (4,13). Polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG had been colored for discrimination from others.

The immune complex transfer enzyme immunoassay for anti-HIV-1 IgG using recombinant proteins as antigens was performed as shown schematically in Fig. 1 (4,13-15). Urine samples (100 11) were incubated simultaneously with 2,4- dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate in a total volume of 150 p1 for 3 hours. The reaction mixture was fur-

DNP-lysine

Anti-lg-solid phase

Fig. 1. Immune complex transfer enzyme immunoassay forantibody IgG. a $ 4 0 0 ~ ; ; ~ ~ ~ - - ~ - 4 - - ~ - ~

DNP 2,4-dinitrophenyl group. Ag: antigen. Ab: antibody. Enz: enzyme. 4 *.>.;*2 0 v) Ig: immunoglobulin. c 0

- 8 m Q

after washing the solid phase, transferred to another clean solid phase to minimize nonspecific signal for achieving high sensitivity. The most sensitive version among several variations of the immune complex transfer enzyme immunoassay is shown schematically in Fig. 1 (8). Antibody to be detected is reacted simultaneously with 2,4-dinitrophenyl-antigen and enzyme-labeled antigen. The immune complex formed, con- sisting of the three components, is trapped onto solid phase coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex is eluted from the solid phase with excess of ~N-2,4-dinitrophenyl-~-lysine and transferred to clean solid phase coated with anti-immunoglobulin IgG. Finally, the enzyme activity bound to the last solid phase is assayed.

8oo 'D 5 m 2 400

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MATERIALS AND METHODS 400

Recombinant proteins of HIV- 1 (NL4-3 (9)) (reverse transcriptase (RT), p17, and p24) were produced in Escherkhia 0

coli transformed with expression plasmids cawing the corresponding cDNAs, purified (4,1&12) and conjugated to 2,4-

Urine Added ( FI /tube )

dinitrophenyl-bovine serum albumin and enzymes as labels Fig. 2. Effect of urine volumes on the signal by the immune complex (horseradish peroxidase and Escherichia coli P-D-galactosi- transfer enzyme immunoassay using RT p17 (B), and

dase) by the reaction of thiol groups and maleimide groups ~ 2 4 ((3 as antieens and B-o-Ealactosidase as label. Three different urine I ~, .. . -

(413-15). "hi01 groups were introduced into the recombi- samples were tested.

Enzyme lmmunoassay of Anti-HIV-1 IgG in Urine 239

.- 5 > ._ c

450 5501 A

350

170

150 t"

a, 0 t a, 0 m

0 3

2

ii

9 3400

2600

2200i

,.5/ ~ /-y 6 7 a 0.5

pH for Incubation with the Two Conjugates

Fig. 3. Effect of pH for incubation with the two conjugates on the signal by the immune complex transfer enzyme immunoassay using recombinant RT (A), p17 (B), and p24 (C) as antigens and P-o-galactosidase as label. Open and closed symbols indicate results with urine samples from a seropositive and a seronegative, respectively.

ther incubated overnight with two colored polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The colored polystyrene balls were washed and incubated with excess of ~N-2,4-dinitrophenyl-~-lysine and two white polystyrene balls coated with affinity-purified (anti- human JgG y-chain) IgG in a total volume of 150 pl for 1 hour. The colored polystyrene balls were removed, and the incubation was continued for 2 hours.Al1 these processes were performed at room temperature. After washing, the enzyme activity bound to the white polystyrene balls was assayed for 2.5 hours or 25 hours by fluorometry using 3-(4-hydroxy- pheny1)propionic acid as hydrogen donor for peroxidase and 4-methylumbelliferyl-~-~-galactoside as substrate for p-D- galactosidase. The fluorescence intensity was measured rela- tive to 0.2 mg/L quinine for peroxidase and 1 x lo-* mol/L 4-methylumbelliferone for P-o-galactosidase.

For the conventional ELISA and gelatin particle agglutination test, commercial kits using five recombinant proteins

(gp41, gp120, p15, p17, and p24) (ABBOTT HTLV-111 EIA, ABBOTT Laboratories, North Chicago, IL) and a lysate of HIV-1 (SERODIA-HIV, Fujirebio Inc.,Tokyo, Japan), respectively, as antigens were used (4).

INTERFERENCE BY URINE

Only slight interference was observed with up to 100 pl of unconcentrated and approximately 10-fold concentrated urine samples in a total volume of 150 yl for incubation with 2,4- dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate (Fig.

The signal (the fluorescence intensity for enzyme activity bound to polystyrene balls coated with affinity-purified (anti- human IgG y-chain) IgG) was altered by different pH for incubation with 2,4-dinitrophenyl-bovine serum albumin- recombinant protein conjugate and recombinant protein-enzyme conjugate (Fig. 3) (4,13-15). The maximal signal was observed at pH 7.5-8.0 with recombinant RT (15) and p17 (4) as antigens, and at pH 5.0 with recombinant p24 as antigen (13). The signal was lowered by 21-30% at pH 5.0-5.5 with recombinant RT (15) and p17 (4) as compared with the maximal signal, and by 29% at pH 8.0 with recombinant p24 (13). The nonspecific signal for seronegatives was slightly enhanced at lower pH with recombinant RT (15) and p17 (4),

2) (4,13-15).

c

e, I , I T k , I , L m I 3x104 104 103 i oz 101 100

Dilution of Urine from Seroposilives with Urine from a Seronegative (-fold)

Fig. 4. Dilution curves of urine samples from seropositives by the immune complex transfer enzyme immunoassay using recombinant RT as antigen and P-o-gdlactosidase (open circles) and peroxidase (closed circles) as labels. Urine samples from three seropositives were serially diluted with urine from a seronegative. Bound enzyme activities were assayed for 2.5 hours.

240 Hashida et al.

I&L- .. oo lo5 lo3 10' oo 105 lo3 10'

Diluticn of Urine from Seropositives with Urine from a Seronegative (-fold)

Fig. 5. Dilution curves of urine samples from seropositives by the immune complex transfer enzyme immunoassay using recombinant RT (A), p17 (B), and p24 (C) as antigens and P-D-galactosidase as label, the conventional ELISA using five recombinant proteins (gp41, gp120, p15, p17, and p24) as antigens and peroxidase as label (closed circles, D) and gelatin particle agglutination test using a lysate of HIV-1 as antigen (closed rhombuses, D). Urine samples from three seropositives were serially diluted with urine from a seronegative. Bound P-o-galactosidase activity was assayed for 25 hours.

and at higher pH with recombinant p24 (13). From these results, appropriate adjustment of pH of urine samples was rec- ommended for effective detection of antibody IgC to HIV- 1 , since pH of urine samples varied from 5.0 through 8.0.

ASSAY VARIATION

The assay variations with recombinant RT, p17, and p24 as antigens were satisfactorily small (4,13-15). The variation coefficients for within-assay and between-assay were 2.8-8.5% and 4.7-9.7%, respectively.

SENSITIVITY

When urine samples from seropositives serially diluted with urine samples from seronegatives were tested, the immune complex transfer enzyme immunoassay was more sensitive than the conventional ELISA using five recombinant proteins (gp41, gp120, p15, p17, andp24) as antigens and gelatin particle agglutination test using a lysate of HIV-1 as antigen. The sensitivity by using recombinant RT as antigen and peroxidase as label was 30-3,000-fold higher than those of the above conventional methods (4). By substitution of P-o-galactosidase from Escherichia coli for horseradish peroxidase as la-

> I 10 20 30 40 50 601 1 0 1 I (

ARC AIDS Unne Samples AC from

Seronegatives No. of Unne Samples from Seropositives

Fig. 6. Detection of anti-HIV-1 IgG in unconcentrated urine by the immune complex transfer enzyme immunoassay using recombinant RT as antigen and P-D-gdactosidase as label. Eighty-three urine samples from seropositives and 100 urine samples from seronegatives were tested. Bound P-o-galactosidase activity was assayed for 2.5 hours. Open and closed circles indicate signals for seropositives and seronegatives, respectively. Open smaller circles indicate signals obtained by preincubation of urine samples with excess of recombinant RT. 'The broken line indicates a tentative cut- off value. AC: asymptomatic carriers. ARC: patients with AIDS-related complex. AIDS: patients with AIDS.

bel, the sensitivity was improved approximately 30-fold (Fig. 4) (15). Bound P-D-galactosidase activity was assayed for 2.5 hours. In addition, a longer assay of bound P-o-galactosidase activity further improved the sensitivity. When bound P-D-galactosidase activity was assayed for 25 hours, the sensitivities by using recombinant RT, p17, and p24 were 3,000-100,000- fold higher than those of the above conventional methods (Fig. 5 ) (4,13,14).

SPECIFICITY

All signals for seropositives by the immune complex transfer enzyme immunoassay using recombinant RT, p17, and p24 were lowered to levels of those for seronegatives by preincubation of urine samples with excess of recombinant RT, p17, and p24, respectively (Fig. 6 ) (4,13-15). This indi- cated that all signals for seropositives were not due to un- known or nonspecific reaction(s) but due to the presence of anti-HIV-1 IgG. The possibility that antibody IgG(s) to some protein(s) other than HIV- 1 antigens present in the recombinant protein preparations was detected was unlikely, since all the recombinant proteins were purified to homogeneity (13-15).

The reactivity of the immune complex transfer enzyme immunoassay using recombinant RT, p17, and p24 as anti-

Enzyme lmmunoassay of Anti-HIV-1 IgG in Urlne 241

..

0

G 0

0 00

0

Serum Samples from Serum Samples from Noncarriers HTLV-I-Infected Subjects

Fig. 7. No cross-reaction with serum samples from HTLV-I-infected subjects of the immune complex transfer enzyme immunoassay for anti-HIV- 1 IgG using recombinant proteins and P-o-galactosidase as label. Closed and open symbols indicate results with serum samples from noncarriers and HTLV-I-infected subjects, respectively. Circles, triangles, and squares indicate results by the immune complex transfer enzyme immunoassay using recombinant RT, p17, and p24, respectively, as antigens. Rhombuses indicate results by the immune complex transfer enzyme immunoassay for anti- HTLV-I IgG using a synthetic peptide, Cys-env gp46(188-224) of HTLV-I, as antigen and P-o-galactosidase as label.

gens with serum and urine samples from HTLV-I-infected subjects was not significant (Figs. 7 and 8) (13,15). The serum and urine samples tested were strongly positive by the immune complex transfer enzyme immunoassay using a synthetic peptides, Cys-gp46(188-224), as antigen (16) and gelatin particle agglutination test using a lysate of HTLV-I as antigen (1 6) (Figs. 7 and 8).

STABILITY OF ANTI-HIV-1 IGG IN URINE

Urine samples were mixed with 1/100 volume each of 10 g/l bovine serum albumin and 10 g/l NaN3 or 5 gJ thimerosal and stored at-2O0C (4,13-15). Under this condition, anti-HIV-1 IgG detectable by the immune complex transfer enzyme immunoassay using recombinant RT as antigen and horseradish peroxidase as label was stable for at least 6 months (Fig. 9) (14).

DIAGNOSIS OF HIV-1 INFECTION WITH URINE SAMPLES

When 83 urine samples from seropositives (60 asymptomatic carriers, 11 patients with AIDS-related complex (ARC), and 12 patients with AIDS) and 100 urine samples from seronegatives were tested by the immune complex transfer enzyme immunoassay using recombinant RT as antigen and

li A AA u A

AA -

+

0" 8 + + +

Urine Samples from Noncarriers

0

08 Q

0 0

0

0

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n

Urine Samples from HTLV-I-Infected Subjects

Fig. 8. No cross-reaction with urine samples from HTLV-I-infected subjects of the immune complex transfer enzyme immunoassay for anti-HIV- 1 IgG using recombinant proteins and P-o-galactosidase as label. Closed and open symbols indicate results with urine samples from noncaniers and HTLV-I-infected subjects, respectively. Circles. triangles, and squares indicate results by the immune complex transfer enzyme immunoassay using recombinant RT, p17, and p24, respectively, as antigens. Rhombuses indicate results by the immune complex transfer enzyme irnmunoassay for anti-HTLV-I IgG using a synthetic peptide, Cys-em gp46(188-224) of HTLV-I, as antigen and P-o-gdactosidase as label.

t 0 %

0

io'l 00 10' 1 o2 1 o3

Fluorescence Intensity for Bound Peroxidase Activity Obtained Shortly after Collection of Urine

Fig. 9. Stability of anti-HIV-1 IgG in urine. Anti-HIV-1 IgG in urine was measured by the immune complex transfer enzyme imrnunoassay using recombinant RT as antigen and peroxidase as label shortly after collection (abscissa) and approximately 6 months after storage at -20°C (ordinate).

242 Hashida et al.

~~~ * ='=.. . . . ji$P'::: -."+"""

TABLE 1. Lowest Cut-Off Index (Ratio of the Lowest Signal for Seropositives to the Highest Nonspecific Signal for Seronegatives) for 60 Asymptomatic Carriers (AC), 11 Patients With AIDS-Related Complex (ARC), and 12 Patients With AIDS

Assay time for bound P-D-gdactosidase Lowest cut-off index

Antigen activity (hour) AC ARC AIDS

RT 2.5 8.2 11.0 7.2 25.0 14.0 29.0 10.0

P17 25.0 1.1 1.8 4.1 P24 25.0 1.7 1.9 1.7

0

. . . . . . . . . . . -. . . . . . . . . .

P-o-galactosidase from Escherichiu coli as label, all signals for seropositives were higher than those for seronegatives (Fig. 6) (15). The lowest signals for the asymptomatic carriers and the patients with ARC and AIDS were 8.2, 11, and 7.2-fold,

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101 10 ARC AIDS

Seronegatives No of Urine Samples from Seropositives

Fig. 10. Detection of anti-HIV-1 IgG in unconcentrated urine by the immune complex transfer enzyme immunoassay using recombinant RT (A), p17 (B), and p24 (C), as antigens and P-D-galactosidase as label. Eighty- three urine samples from seropositives and 100 urine samples from seronegatives were tested. Bound 0-o-galactosidase activity was assayed for 25 hours. Open and closed symbols indicate signals for seropositives and seronegatives, respectively. The broken lines indicate tentative cut-off values. See Fig. 6 for AC, ARC, and AIDS.

Unne Samples from No. of Urine Samples from Seronegatives Seropositives

Fig. 11. Confirmation of the positivity and negativity by concentration of urine samples. Urine samples, which gave low signals by the immune complex transfer enzyme imrnunoassay using recombinant RT (circles) and p17 (triangles) as labels and horseradish peroxidase and Escherichia coli P-o-galactosidase, respectively, as labels were concentrated approximately 10-fold and tested in the same way. Open and closed symbols indicate signals for seropositives and seronegatives, respectively. Arrows indicate changes by the concentration. Open small circles and triangles indicate signals obtained by preincubation of the concentrated urine samples with excess of recombinant RT and p17, respectively. The broken line indicates tentative cut-off values for unconcentrated urine samples.

respectively, higher than the highest signal for the seronegatives (Fig. 6 and Table 1). Bound fl-D-galactosidase activity was assayed for 2.5 hours.

The positivity described above could be made more reliable or confirmed in various ways. By a longer assay (25 hours) of bound P-o-galactosidase activity, the ratios of the lowest signals for the asymptomatic camers and the patients with ARC and AIDS, to the highest signal for the seronegatives,

TABLE 2. Combined Use of Recombinant RT, p17, and p24 for Detection of Anti-HIV-1 IgG in Urine Samples

Fluorescence intensity for bound P-D-galactosidase activity

Urine Mixture of sample RT P'7 p24 RT, p17, and p24

Seronegative 1.1-1.2 0.2-1.0 0.7-0.8 0.9-1.0 (n = 3)

Seropositive 1 30 35 255 232 2 54 34 12 71 3 106 293 11 379 4 1000 315 67 1263 5 4701 1465 1727 7722

Enzyme lmmunoassay of Anti-HIV-1 IgG in Urine

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Fig. 12. Detection of anti-HIV-1 IgG or anti-HIV-I antibodies in unconcentrated urine by conventional methods. Eighty-three urine samples from seropositives and 100 urine samples from seronegatives were tested by the conventional ELISA using five recombinant proteins (gp41, gp120, p15, p17, and p24) of HIV-1 as antigens (A) and gelatin particle agglutination test using a lysate of HIV-1 as antigen (B). Open and closed symbols indicate results for urine samples from the seropositives and the seronegatives, respectively. The broken line indicates a tentative cut-off value.

were enhanced to 14,29, and 10, respectively (Fig. 10A and Table I ) . The positivity with recombinant RT as antigen could be confirmed by demonstrating antibody IgG to p17 and p24 (Fig. 10B and C) (4,13,14). The signal was enhanced by using approximately 10-fold concentrated urine samples with little enhancement of signals for the seronegatives (Fig. 11) (4). Low signals for some seropositives with recombinant RT as antigen could be enhanced by the combined use of recombinant RT, p17, and p24 with no significant enhancement of signals for the seronegatives (Table 2) ( 1 5).

Thus, diagnosis of HIV-1 infection was possible by detecting anti-HIV-1 IgG in 100 p1 of unconcentrated urine samples with the immune complex transfer enzyme immunoassay using recombinant RT as antigen or the three recombinant proteins (RT, p 17, and p24) as antigens, and p-o-galactosidase from Escherichia coli as label.

In contrast with the above results, the sensitivity and specificity for the seropositives and the seronegatives by the conventional ELISA and gelatin particle agglutination test were 7749% and 97-99%, respectively (Fig. 12) (4). With approximately 10-fold concentrated urine samdes. the sensi-

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Fig. 13. Detection of anti-HIV-1 IgG or anti-HIV-1 antibodies in concentrated urine by conventional methods. Urine samples, which gave low signals or were negative as shown in Fig. 12, were concentrated approximately 10-fold and tested by the conventional ELISA (A) and gelatin particle agglutination test (B). See Fig. 12 for symbols.

1 o4

f " u = 10' c f Io3l

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8 t

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Fig. 14. Concentration of IgG in serum, urine, and oral fluids (whole saliva) from healthy and seronegative subjects. open and closed circles indi-

I --- ------ cates values for males aged 7-85 yr and females aged 8-87 YT, respectively.

244 Hashida et al.

tivity was slightly improved, but the specificity was lowered to 57-76% due to enhanced nonspecific signals (Fig. 13) (4).

POSSIBILITIES SUGGESTED AND PROBLEMS TO BE SOLVED

From the above results, the following possibilities were suggested. Diagnosis of HIV-1 infection may be possible with a smaller volume of oral fluids (whole saliva) and a much smaller volume of serum or blood than that of urine samples used above, since the concentrations of IgG in oral fluids and serum are approximately 10-fold and 4,000-fold, respectively, higher than that inurine (Fig. 14 andTable 3). In fact, diagnosis of HIV-1 infection has been possible with only 1 pl of oral fluids using recombinant RT as antigen and Escherichia coli P-D-galactosidase as label, which will be described in detail elsewhere. The volume of serum or blood required for diagnosis of HIV- 1 infection may be 1-1 0 nl after seroconversion by conventional methods. By using 10-20 yl of serum samples, anti-HIV- 1 IgG may be detected earlier than at the time of seroconversion by conventional methods, and more rapidly after seroconversion than described above.

The processes of the immune complex transfer enzyme immunoassay remain to be simplified especially for transfer of the immune complex from solid phase to solid phase. A more sensitive assay method for label enzymes or an alterna- tive label which can be detected more sensitively and more rapidly remains to be developed.

Amino acid substitutions by mutations have been found in RT molecules of HIV-1 isolated from seropositives after pro- longed treatment with 3 '-azido-3 '-deoxythymidine (1 7-22),

TABLE 3. Concentration of IgG in Serum, Urine, and Oral Fluids (Whole Saliva) from Healthy and Seronegative Subjects.

Body fluids Sex

Serum Male

Female

Male + Female

Urine Male

Female

Male + Female

Oral fluids Male

Female

Male + Female

Number Concentration Age of of IgG (yr) samples (md)

7-85 49 14,900 f 6,200

16-87 56 15,400 k 6,400

7-87 105 15,200 f 6,300

7-85 148 3.3 f 3.5

8-87 131 4.4 f 5.8

7-87 279 3.8 f 4.7

19-57 35 42.4 k 32.8

19-25 20 37.2 f 51.2

19-57 55 40.5 f 40.1 (0.7 - 1871

(3,800 - 36,000)

(6,100 - 32,000)

(3,800 - 36,000)

(0.15 - 21.7)

(0.05 - 41.4)

(0.05 - 41.4)

(5.6 - 135)

(0.7 - 187)

0

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Fig. 15. Concentration of IgG in urine samples from seronegatives (closed circles) and seropositives at different stages of HTV-1 infection (open circles) shown in Figs. 6 and 10. See Fig. 6 for AC, ARC, and AIDS.

2',3'-dideoxyinosine and 2',3'-dideoxycytidine (19,23). Therefore, it remains to be investigated how long the production of antibodies to RT before mutations lasts after the ap- pearance of HIV-1 carrying RT with amino acid substitutions by mutations, and whether RT before mutations reacts with antibodies to RT after mutations. It also remains to be tested to what extent antigens of HIV-1 including RT, p17, and p24 react with antibodies in body fluids of HIV-2 seropositives and whether hagnosis of HIV-2 infection is possible by detecting anti-HIV-2 1gG in urine with immune complex transfer enzyme immunoassay using RT of HIV-2 as antigen.

For improving the specificity by detecting antibodies to increasing numbers of antigens or epitopes of HIV-1, antigens other than RT, p17, and p24 (e.g. e m proteins) remain to be prepared. By this, the sensitivity may also be improved.

Signals for patients withARC andAIDS tended to be lower than those for the asymptomatic carriers (Fig. 10) (4,13-15). Therefore, levels of anti-HIV-1 IgG measured with RT, p17, and p24 as antigens, may be used as a marker for progress of the disease. However, this makes less reliable the diagnosis of HIV-1 infection at later stages of HIV-1 infection. Low levels of anti-HIV-1 IgG in urine samples from the patients withARC andAIDS than from the asymptomatic carriers were not due to lower concentrations of IgG (Figs. 15 and 16). However, it remains to be investigated whether the level of detectable anti-HIV-1 IgG is low due to high concentrations of the corresponding antigens (RT, p17, and p24) in patients with ARC and AIDS.

Anti-HIV-1 antibodies have been detected in urine samples

Enzyme lmmunoassay of Anti-HIV-1 IgG in Urine 245

AC ARC

0

A 0

d o 0

0

Al DS

Urine Samples from Seropositives

Fig. 16. Level of anti-HIV-1 IgG in urine samples from seropositives at different stages of HIV-1 infection. Levels of anti-HIV-1 TgG in urine samples from seropositives at different stages of HIV-I infection were measured by the immune complex transfer enzyme immunoassay using recombinant RT (circles), p17 (triangles), and p24 (squares), as antigens and P-o-galactosidase as label, and fluorescence intensities for bound $-.- galactosidase activity were divided by the urinary concentration of IgG in terms of mg/l. See Fig. 6 for AC, ARC, and AIDS.

from seronegative subjects by conventional methods (24). The above sensitive immunoassay may more frequently detect anti- HIV-1 IgG in urine samples from seronegative high-risk groups than so far reported.

Finally, it remains to be tested whether anti-HIV-1 IgG can be detected in urine at the time of seroconversion in an early stage of HIV-1 infection.

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