Download - Detection of antibody IgG to HIV-1 in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens for diagnosis of HIV-1

Journal of Clinical Laboratory Analysis 7:353-364 (1 993)

Detection of Antibody IgG to HIV-1 in Urine by Sensitive Enzyme lmmunoassay (Immune Complex Transfer Enzyme Immunoassay)

Using Recombinant Proteins as Antigens for Diagnosis of HIV-I Infection

Seiichi Hashida,' Kouichi Hirota,' Katuya Hashinaka,' Atsushi Saitoh,2 Atsuo Nakata,* Hideo Shinagawa,2 Shinichi Oka,3 Kaoru Shirna~ia,~ Jun-ichi Mirna~a,~

Shuzo Mat~ushita,~ 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; 3Department of Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 4Division of Hematology and Oncology, Children's Hospital of Shizuoka Prefecture, Shizuoka, Japan; and The Transfusion

Service Department, Kumamoto UniversiQ Medical School, Kumamoto, Japan

For diagnosis of HIV-1 infection, attempts were made to detect anti-HIV-1 IgG in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) US- ing recombinant reverse transcriptase (RT) and p17 as antigens. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein- enzyme conjugate. The enzymes used as labels were horseradish peroxidase for RT and Escherichia coli p-D-galactosidase for pl7. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with ~N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human

IgG y-chain) IgG. Finally, bound enzyme activity was assayed by fluorometry. Urine samples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positivity was confirmed by preincubation of urine samples with excess of the antigens. The positivity and negativity with one of the two antigens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine samples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme immunoassay using different antigens would make diagnosis of HIV-1 infection possible. C! 1993 Wiley-Liss, Inc

Key words: antibody, human immunodeficiency virus type 1, reverse transcriptase, p17, peroxidase, p-D-galactosidase, ELISA, gelatin particle agglutination

INTRODUCTION

Antibodies to human immunodeficiency virus type 1 (HIV-1) in serum, plasma and whole blood have been detected by various methods such as enzyme-linked imrnunosorbent assay (ELISA), agglutination of latex, red-cells and gelatin parti- cles, dot blotting and Western blotting (1). Antigens used are the whole virus, recombinant proteins and synthetic peptides (1). These tests are sensitive and have been successfully used for the diagnosis of HIV-1 infection (1). However, blood should be collected with due cautions to avoid infections not only with HIV but also with other pathogens. This paper describes the detection of antibody IgG to reverse transcriptase (RT) and p 17 of HIV- 1 in urine, which can be collected more eas- ily with no invasive procedures, less expenses and less pos- sibility of various infections than blood (2). The method used

0 1993 Wiley-Liss, Inc.

was an enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant RT and p17 as antigens, which had advantages in sensitivity and specificity over conventional methods.

MATERIALS AND METHODS

Buffers

The regularly used buffers were 0.1 mol/L sodium phosphate buffer, pH 6.0, containing 5 mmol/L EDTA (buffer A); 10 mmol/L sodium phosphate buffer, pH 7.0, containing 0.1

Received June 17, 1993; accepted June 21, 1993.

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

354 Hashida et al.

DNP-Ag Anti-DNP- solid phase

I

/ / / / ,,

bovine serum albumin was calculated from the absorbance at 280 nm and 360 nm (4). The average number of 2,4-dinitrophenyl groups introduced per albumin molecule was 6.0.

Ab A g - ~ n z Protein-Sepharose 46

2,4-Dinitrophenyl-bovine serum albumin (10 mg) and human IgG (10 mg) were coupled to CNBr-activated Sepharose 4B (1 .0 g, Pharmacia LKB Biotechnology AB, Uppsala, Swe- den) according to the instructions of Pharmacia LKB Biotechnology.

DNP-lysine

Anti-19-solid phase

Fig. 1. Immune complex transfer enzyme inimunoassay for antihody LgG. DNP, 2,4-dinitrophenyl group; Ag, antigen; Ab, antibody; Enz, enzyme; 1g. immunoglobulin.

moliL NaCl (buffer B); I0 mmol/L sodium phosphate buffer, pH 7.0, containing 0.1 g/L bovine serum albumin (fraction V, Intergen Company, Purchase, NY), 1 mmol/L MgCI2 and 1 .0 giL NaN3 (buffer C); and 10 mmoliL sodium phosphate buffer, pH 7.0, containing 1 .0 g/L bovine serum albumin (buffer D) .

Antibodies

Rabbit (anti-2,4-dinitrophenyl-bovine serum albumin) serum was obtained from Shibayagi Co., Ltd., Gumma, Ja- pan. Rabbit (anti-human IgG y-chain) IgG was obtained from Medical and Biological Laboratories Co., Ltd., Nagoya, Ja- pan. IgG was prcpared from serum by fractionation with Na2S04, followed by pacsage through a column of DEAE- cellulose (3). The amount of IgG was calculated from the absorbance at 280 nni ( 3 ) .

2,4-Dinitrophenyl-Bovine Serum Albumin

Thiol groups were introduced into bovine serum albumin molecules using N-succinimidyl-S-acetylniercaptoacetate and were reacted with maleimide groups introduced into EN-2,4- dinitrophenyl-L-lysine molecules using N-succinimidyl- 6-maleimidohexanoate (4). Thc amount of bovine serum albumin, 2,4-dinitrophenyl groups, and 2,4-dinitrophenyl-

Affinity-Purification of Antibodies

(Anti-2,4-dinitrophenyl-bovine serum albumin) IgG and (anti-human IgG y-chain) IgG were affinity-purified by elution at pH 2.5 from columns of 2,4-dinitrophenyl-bovine serum albumin-Sepharose 4B and human IgG-Sepharose 4R, respectively ( 5 ) .

Protein-Coated Polystyrene Balls

Polystyrene balls (3.2 mm in diameter, lmmuno Chemi- cal Inc., Okayama, Japan) were coated by physical adsorp- tion with affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG (0.1 g/L) and affinity-purified (anti-human IgG y-chain) IgG (0.1 giL) (6). Affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG-coated polystyrene balls had been colored pink for discrimination from affinity-purified (anti-human IgG y-chain) IgG-coated polystyrene balls.

Recombinant RT and p l 7

Recombinant RT and p17 were produced in Escherichia coli transformed with expression plasmids carrying the cor- responding cDNA (7,8). Recombinant RT was purified as previously described (7).

For purification of recombinant p17, E . coli BL21 (DE3) cells carrying pTG171 (8) were harvested from the culture medium, resuspended in 15 mmoliL sodium phosphate buffer, pH 6.7, and disrupted by sonication. Thc lysate was clari- fied by centrifugation at 40,000g for 1 h, and the supernatant was fractionated by stepwise ammonium sulfate precipitation. The precipitate obtained by 40-80% saturation of ammonium sulfate was dissolved in, and dialyzed against 15 mmol/L sodium phosphate buffer, pH 6.7, and applied to a column of S-Sepharose (Pharmacia LKB Biotechnology AB). A lin- car gradient from 0 to 0.8 mol/L NaCl was used for elution. The peak fraction of p17 eluted between 0.3 and 0.45 mol/L NaCl was dialyzed against 15 mmol/L sodium phosphate buffer, pH 6.7, and applied to a column of Mono S (Pharmacia LKB Biotechnology AB). The peak fraction of p17 eluted between 0.35 and 0.4 moUL NaCl was dialyzed against 15 mmoliL sodium phosphate buffer, pH 6.7, containing 10 mmoliL 2-mercaptoethanol and applied to a column of hydroxylapatite (Koken Co., Ltd., Tokyo, Japan). A linear gradient from 0.01 5

Enzyme lmmunoassay of Anti-HIV-1 IgG in Urine 355

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Dilution of Urine from Seropositive Subjects with Urine from a Seronegative Subject (-fold)

Fig. 2. Dilution curves of urine samples from seropositive subjects by three different methods. Urine samples from three seropositive subjects were serially diluted with urine from a seronegative subject and were tested by the immune complex transfer enzyme immunoassay using recombinant RT (open circles) and p17 (open triangles) as antigens, the conventional ELISA using five recombinant proteins as antigens (open squares) and gelatin particle agglutination using a lysate of the whole virus as antigcn (open rhombuses).

to 0.7 moliL sodium phosphate buffer, pH 6.7, was used for elution. The peak fraction of p17 eluted between 0.35 and 0.42 moliL sodium phosphate buffer, pH 6.7, was collected. l h e purified recombinant proteins were found to be homoge- neous by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate.

2,4-Dinitrophenyl-Bovine Serum Albumin-Recombinant RT Conjugate

6-Maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin

2.4-Dinitrophenyl-bovine serum albumin prepared as described above (3.7 mg, 56 nmol) in 1 .0 ml of 0. I mol/L sodium phosphate buffer, pH 7.0, was incubated with 30 ~1 of 16.5 mmol/L N-succininiidyl-6-maleimidohexanoate (DO- JINDO Laboratories, Kumamoto. Japan) in N,N-dimethylformamide at 30°C for 30 min. After incubation, the reaction mixture was subjected to gel filtration on a column ( I X 30 cm) of Sephadex G-25 (Pharmacia LKB Biotechnnlogy AB) using buffer A. The average number of maleimide groups introduced per albumin molecule was 5.9 (3).

Mercaptoacetyl-recombinant RT

Recombinant RT (0.8 mg, 13 nmol) in 4.2 ml of 0. I moliL sodium phosphate buffer, pH 7.0, containing 0.01 o/c Triton X-lo0 was incubated with 0.2 ml of 11 mmoYLN-succinimidyl- S-acetylmercaptoacetate (Boehringer Mannheim GmbH, Mann- heim, Germany) in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was incubated with 0.2 ml of 0.1 mol/L EDTA, pH 7.0, 0.4 ml of 1 moliL Tris-HCI buffer, pH 7.0, and 0.6 ml of 1 mol/L hydroxylamine, pH 7.0, at 30°C for 15 min. After incubation, the reaction mixture was subjected to gel filtration on a column (1 X 30 cm) of Sephadex G-25 using buffer A containing 0.01% Triton X-100. The amount of recombinant RT was determined by the estimation of amino groups using fluorescamine (Sigma Chemical Co., St. Louis, MO) (9) and bovine serum albumin as standard. and its molecular weight was taken to be 64,000 (7). The average number of thiol groups introduced per recombinant RT molecule was 2.5 (3).

2,4-Dinitrophenyl-bovine serum albumin-recombinant RT conjugate

Mercaptoacetyl-recombinant RT (0.14 mg, 2.2 nmol) in 0.1 ml of buffer A containing 0.01% Triton X-100 was

356 Hashida et al.

250

x > + .- .- .c.

2

h v) c a, C

u C a, 0 v)

0

LL

c .-

c - a, 100

z 2 3

1 I I I I

' 5 6 7 8

pH for incubation with the Two Conjugates

Fig. 3. Effect of pH in the immune complex transfer enzyme immunoassay. Urine samples were incubated with 2.4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate at pH 5-8. Circles and triangles indicate results with recombinant RT and p17, respectively. Open and closed symbols indicate experiments with urine from a seropositive subject and a seronegative subject, respectively.

incubated with 6-maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin (0.15 mg, 2.2 nmol) in 10 pl o f buffer A at 30°C for 60 min. After incubation, the reaction mixture was incubated with 10 p1 of 10 mmol/L 2-mercaptoethylamine in buffer A at 30°C for 15 min and subsequently with 20 pl of 10 mmoliL N-ethylmaleimide in buffer A at 30°C for 15 min. The reaction mixture was subjected to gel filtration on acolumn (1.5 x 45 cm) of Ultrogel AcA 34 (IBF Biotechnics, Villeneuve-la-Garenne, France) using buffer B , containing 0.01% Triton X-100. The average number of recombinant RT molecules conjugated per 2,4-dinitrophenyl-bovine serum albumin molecule was 1.9, calculated from the concentration of 2,4-dinitrophenyl-bovine serum albumin and the total protein concentration determined by the estimation of amino group as described above. The amount of2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate was calculated as described for 2,4-dinitrophenyl-bovine serum albumin.

2,4-Dinitrophenyl-Bovine Serum Albumin-Recombinant pl7 Conjugate 6-Maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin

2,4-Dinitrophenyl-bovine serum albumin prepared as described above (3.0 mg, 45 nmol) in 0.49 ml of 0.1 mol/L

sodium phosphate buffer, pH 7.0, was incubated with 10 pl of 20 mmoliL N-succinimidyl-6-maleimidohexanoate in N, N- dimethylformamide at 30°C for 30 min. After incubation. the reaction mixture was subjected to gel filtration by the centrifuged column procedure ( 10) using a column (1.1 X 5.3 cm) of Sephadex G-50 fine (Pharrnacia LKB Biotechnology AB). equilibrated with buffer A. The average number of nialeimide groups introduced per albumin molecule was 3.8.

Mercaptoacetyl-recombinant p l 7

Recombinant p17 (0.76 mg, 45 nmol) in 0.48 ml of 0.1 moliL sodium phosphate buffer, pH 7.0, was incubated with 20 p l of 5 mmoliL N-succinimidyl-S-acetylmercaptoacetate in N,N-dimethylformamide at 30°C for 30 min. After incubation, the reaction mixture was incubated with 30 p1 o f 0.1 rnoliL EDTA, pH 7.0,60 pl of 1 moliL Tris-HC1 buffer, pH 7.0, and 70 pl of 1 moliL hydroxylamine, pH 7.0, at 30°C for 15 min and subjected to gel filtration on a column (I .O x 45 cm) of Sephadex G-25, using buffer A containing 0.35 moliL urea. The concentration of recombinant p17 was determined by a commercial protein assay kit (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as standard, and its molecular weight


0 20 40 60 80 100 20 40 60 80 100

Urine Added ( pI / tube )

Fig. 4. Effect of urine volumes in the immune complex transfer enzyme immnunoassay. Increasing volumes of urine samples from three seropositive subjects were tested by the immune complex transfer enzyme immunoassay. Open circles and triangles indicate results with recombinant RT and p17, respectively.

was taken to be 17,000 (8). The average number of thiol groups introduced per recombinant p17 molecule was 1.4.

2,4-Dinitrophenyl-bovine serum albumin-recombinant p l 7 conjugate

Mercaptoacetyl-recombinant p17 (0.5 1 mg, 30 nmol) in 0.3 ml of buffer A containing 0.35 mol/L urea was incubated with 6-maleimidohexanoyl-2,4-dinitrophenyl-bovine serum albumin (0.5 mg, 7.5 nmol) in 90 p1 of buffer A at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 p1 of 0.1 mol/L 2-mercaptoethylamine in buffer A at 30°C for 15 min and subsequently with 20 ~1 of 0. I mol/L N-ethylmaleimide in buffer A at 30°C for 15 min. The reaction mixture was subjected to gel filtration on a column (I .5 x 45 cm) of Ultrogel AcA 44 (IBF Biotechnics), using buffer B. The average number of recombinant p17 molecules conjugated per albumin molecule was 2.8, calculated from the concentration of 2,4-dinitrophenyl-bovine serum albumin and the total protein concentration determined by the commercial protein assay kit as described above. The amount of the conjugate was calculated in the same way as described for 2,4-dinitrophenyl-bovine serum albumin.

Recombinant RT-Peroxidase Conjugate

6-Maleimidohexanoyl-peroxidase

Maleimide groups were introduced into horseradish peroxidase using N-succinimidyl-6-maleimidohexanoate (1 1). The

amount of peroxidase was calculated from the absorbance at 403 nm (3). The average number of maleimide groups introduced per peroxidase molecule was I. 1 (3).

Mercaptoacetyl-recombinant RT

Recombinant RT (0.5 mg, 78 nmol) in 0.6 ml of 0.1 mol/L sodium phosphate buffer, pH 7.0, containing 0.01% Triton X-100 was incubated with 30 pl of 1 1 mmol/LN-succinimidyl- S-acetylmercaptoacetate in N,N-dimethylformamide at 30°C for 30 min. The reaction mixture was processed as described above. The average number of thiol groups introduced per recombinant RT molecule was 4.0 (3).

Recombinant RT-peroxidase conjugate

Mercaptoacetyl-recombinant RT (0.26 mg, 4.0 nmol) in 0.4 ml of buffer A containing 0.01% Triton X-100 was incubated with 6-maleimidohexanoyl-peroxidase (52 pg, 1.3 nmol) in 4.3 ~1 of buffer A at 30°C for 60 min. After incubation. the reaction mixture was incubated with 5 pl of 0.1 moliL 2-mercaptoethylamine in buffer A at 30°C for 15 min and subsequently with 10 p1 of 0.1 mol/L N-ethylmaleimide in buffer A at 30°C for 15 min. The reaction mixture was subjected to gel filtration on a column (1.5 x 45 cm) of Ultrogel AcA 44 using buffer B, containing 0.01% Triton X-100. The average number of recombinant RT molecules conjugated per peroxidase molecule was 1.1, calculated from the concen-

358 Hashida et al.

103

1 0 2 r

6 A A 70

0 '

A 0

0 0 a

f

" 1 10 20 30 40 50 60 70

No. of Urine Samples from Seropositive Subjects

"

Urine SamDles from Seronegative Subjects

Fig. 5. Detection of anti-HIV-1 IgG in unconcentrated urine by the immune complex transfer enzyme immunoassay. Seventy urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tcsted. Open and closed symbols indicate signals for urine samples from seropositive and seronegative subjects. rcspectively. Opcn smaller symbols

TABLE 1. Improved Test Results With Concentrated Urine Samples by the Immune Complex Transfer Enzyme Immunoassav

Fluorescence intensity Urine for bound enzyme activity

Antigen Samulc No. uH lrG Unconcentrated Concentrated

mgiL RT Seronegative

(n = 100) Seropositive

No. 1 6.5 21 No. 2 6.5 15 No. 3 7.0 0.6 No. 4 6.5 3.2 No. 5 7.0 2.4

p17 Seroncgative

Seropositive (n = 100)

No. 19 8.0 11 No. 50 8.0 27 No. 62 6.5 5.1

0.0-3.6

4.0 5.0 5.3 5.3 7.6

0.0-0.8

2.2 1.5 2. I

0.0-4.2

11 80 64 16 45

0.0-0.9

6.6 2.7

18

indicate signals obtained by preincubation of urine samples with excess of antigens. Circles and triangles indicate results with recombinant RT and p17, rcspectively. Small numbers indicate those of urine samples from seropositive subjects. The broken line indicates tentative cut-off values.

tration of peroxidase and recombinant RT. The amount of the conjugate was calculated from peroxidase activity (3).

Recombinant p l 7-p-D-Galactosidase Conjugate

Maleimide-p-D-galactosidase

Maleimide groups were introduced into P-l>-galactosidase from E . coli with hi,"-o-phenylenedimaleimide (3). 'Thc average number of maleimide groups introduced per p-D- galactosidase molecule was 14 (3).

Recombinant pl7-p-D-galactosidase conjugate

Mercaptoacetyl-recombinant p17 (0.1 mg, 6.0 nmol) in 0.5 ml of buffer A containing 0.35 mol/L urea was incubated with maleimide-P-D-galactosidase (1.1 mg, 2.0 nmol) in 0.5 ml of buffer A at 4°C for 20 h. After incubation, the reaction mixture was incubated with 10 pl of 0.1 mol/L 2-mercaptoethylamine in buffer A at 30°C for I5 min and subsequently with 20 pl of 10 mmoliL N-ethylmaleimide in buffer A at


1 00 -

10-

2 0

3 0

6

4 0

I 0

Urine Samples from No. of Urine Samples from Seronegative Subjects Seropositive Subjects

Fig. 6. Confirmation of the positivity and negativity by concentration of urine samples. Urine samples, which gave low signals as shorn in Fig. 5 (Nos. 1-5, 19, S O , and 62), were concentrated approximately 10-fold and tested by the immune complex transfer enzyme immunoassay. See Fig. 5 for symbols.

30°C for 15 min. The reaction mixture was subjected to gel filtration on acolumn (1.5 x 45 cm) of Ultrogel AcA 22 (IBF Biotechnics) using buffer C containing 0.1 moliL NaCl. The average number of recombinant p 17 molecules conjugated per P-D-galactosidase molecule was 2.5, calculated from the de- crease in the number of maleimide groups (3). The amount of the conjugate was calculated from P-D-galactosidase activity.

Immune Complex Transfer Enzyme lmmunoassay Using Recombinant RT-Peroxidase Conjugate

An aliquot (100 pl) of urine samples was incubated with 20 p1 of buffer D containing 0.4 moliL NaCl or 20 pl of buffer D containing 0.4 mol/L NaCl and 1.5 pmol or 15 pmol of recombinant RT at room temperature for 3 h, unless other- wise specified. Recombinant RT was added to confirm the presence of anti-HIV-1 RT IgG in samples. The reaction mixture (120 pl) was incubated with 30 pJ of buffer D containing 0.4 moliL NaCl, 100 fmol of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate and 100 fmol of recombinant RT-peroxidase conjugate at room temperature for 3 h. In experiments designed to examine the effect of urine

volumes, the volume of buffer D containing 0.4 moliL NaCl and the two conjugates was increased to a total volume of 150 pl. In experiments to examine the effect of pH, 5 p1 of urine and 145 p1 of 10mmoliL sodiumphosphate, pH 5.0-8.0, containing 0.4 moliL NaCl, 1 giL bovine serum albumin and the two conjugates were incubated at room temperature for 3 h. To the reaction mixture, two pink polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG were added, and the incubation was continued at room temperature overnight. After removing the reaction mixture, the pink polystyrene balls were washed twice by addition and aspira- tion of 2 ml of buffer B and incubated with 150 pl of buffer D containing 0.1 moliL NaCl and 1 mmol/L EN-2,4-dinitrophenyl-L-lysine and two white polystyrene balls coated with affinity-puiified (anti-human IgG y-chain) IgG at room temperature for 1 h. The pink polystyrene balls were removed, and the incubation was continued at room temperature for 2 h. The white polystyrene balls were washed as described above. Peroxidase activity bound to the white polystyrene balls was assayed by fluorometry at 30°C for 150 min using 3-(4-hy- droxypheny1)propionic acid as hydrogen donor (12). The fluorescence intensity was measured relative to 0.2 mgiL

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AIDS

Urine Samples from Seropositive Subjects

Fig. 7. Level of anti-HIV-l IgG in urine samples from seropositive subjects at different stages of HIV-1 infection measured by the immune complcx transfer enzyme immunoassay. Open circles and triangles indicate levels measured using recombinant RT and p17. respectively. AC, asymptomatic carriers; ARC, paticnts with AIDS- related complex; AIDS. patients with AIDS.

quinine in SO mmol/L H2S04 using 320 nm for excitation and 405 nm for emission in a spectrofluorophotometer (F- 3010, Hitachi, Ltd., Tokyo. Japan).

Immune Complex Transfer Enzyme lmmunoassays Using Recombinant pl7-p-~-Galactosidase Conjugates

An aliquot ( 1 00 pl) of urine samples was incubated with 20 p1 of buffer C containing 0.4 moliL NaCl and SO pg of inactive P-D-galactosidase (P-galactosidase-mutein, Boehringer Mannheim GmbH) or 20 pl of buffer C containing 0.4 moliL NaCl, SO k g of inactive P-D-galactosidase and 15 pmol or 150 pmol of recombinant p17 at room temperature for 3 h. Inactive P-D-galactosidase was used to eliminate interference by anti-P-~-galactosidase antibodies (13). The amount of inactive P-u-galactosidase was calculated from the absorbance at 280 nm, using the extinction coefficient for 6-D-galactosidase ( 3 ) . Recombinant p17 was added to confirm the presence of anti-p17 IgG in samples. The reaction mixture (120 p1) was incubated with 30 p1 of buffer C containing 0.4 mol/L NaCl, 100 fmol of 2,4-dinitrophenyl-bovine serum albumin-recombinant p17 conjugate and 100 fmol of recombinant p 17-P-D-galactosidase conjugate at room temperature for 3 h. In experiments designed to examine the effect of urine volumes, the volume of buffer C containing 0.4 moliL NaCl and the two conjugates was increased to a total volume of

150 pl. In experiments designed to examine the effect of pH, 1 pl of urine and 149 pl of 10 mmoliL sodium phosphate buffer, pH 5.0-8.0, containing 0.4 moliL NaCl, 0.1 g/L bovine serum albumin, 1 mmol/L MgC12, 1 .0 giL NaN3, and the two conjugates were incubated at room temperature for 3 h. To the reaction mixture, two pink polystyrene balls coated with affinity-purified (anti-2.4-dinitrophenyl goup) IgG were added, and the incubation was continued at room temperature overnight. After removing the reaction mixture, the pink polystyrene balls were washed twice by addition and aspira- tion of 2 ml of buffer C containing 0.1 moliL NaCl and incubated with 150 pl of buffer C containing 0. I moliL NaCl and 1 mmol/L cN-2,4-dinitrophenyI-~-lysine and two white polystyrene balls coated with affinity-purified (anti-human IgG y-chain) TgG at room temperature for 1 h. The pink polystyrene balls were removed, and the incubation was continued at room temperature for 2 h. The white polystyrene balls were washed as described above. p-D-Galactosidase activity bound to the white polystyrene balls was assayed at 30°C for 1.50 min by fluommetry using 4-methylumbellifery-~-D-galactoside as a substrate (14). The fluorescence intensity was measured relativeto I x lO-*mol/L4-rnethylumbelliferoneinO. 1 moliL glycine-NaOH buffer, pH 10.3, using 360 nm for excitation and 450 nm for emission analysis with a spectrofluorophotometer (F-3010, Hitachi, Ltd.).


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Urine Samples Urine Samples

Seronegative Seropositive Subjects Seronegative Seropositive Subjects from No. of Urine Samples from from No. of Urine Samples from

Subjects Subjects

Fig. 8. Detection of anti-HIV-1 lgG or anti-I-IIV-I antibodies in unconcentrated urine by conventional methods. Seventy urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested by the conventional enzyme-linked immunosorbent assay (ELISA) using five recombinant proteins of HIV- 1 as antigens and gelatin particle agglutination

Conventional Enzyme-Linked lmmunosorbent Assay (ELISA)

The conventional ELISA was performed by using a commercial kit (ABBOTT HTLV-111 EIA, ABBOTT Laboratories, North Chicago, IL). A polystyrene ball coated with five recombinant proteins of HIV-1 (gp120, gp41, p24. p17, and pl5) was incubated with 100 p1 of urine samples and 3 10 ~1 of the diluent included in the kit at 40°C for 30 min and, after washing, with (anti-human IgG) antibody-peroxidase conjugate at 40°C for 30 min. Bound peroxidase activity was assayed by colorimetry using 2-phenylenediamine as hydrogen donor.

Gelatin Particle Agglutination Gelatin particle agglutination was performed using a com-

mercial kit with a lysate of HIV-I produced by MOLT #4/HTLV-III cell line as antigen (SERODIA-HIV, Fujirebio Inc., Tokyo, Japan) (15). Serum and urine samples were diluted at least 16-fold and 2-hld, respectively. with the diluent included in the kit. An aliquot (25 pl) of diluted samples was mixed with the particle solution (25 pl) in U-shaped wells of microplates and allowed to stand at room temperature €or 2 h.

_j test using a lysate of the whole virus as antigen. Open and closed symbols indicate results for urine samples from seropositive subjects and ceronega- tive subjects. respectively. Squares and rhombuses indicate results by the conventional ELISA and gelatin particle agglutination tcst, respectivcly. The broken line indicates a tentative cut-off value.

Western Blotting

Western blotting for anti-HIV- 1 antibodies was performed using a commercial kit (Ortho HIV Western Blot Kit, Ortho Diagnostic Systems Inc.. Raritan, NJ). Serum samples were diluted 10-fold with the diluent included in the kit. A nitrocellulose membrane preblotted with nine HIV- 1 proteins (gp160. gp120, p66, p55, p51, gp41, p31, p24, and p17) was incubated with 2.02 nil of the diluted samples at room temperature overnight and, after washing, with biotinyl-(anti- human IgG) antibody at room temperature for 1 h. After washing, the nitrocellulose membrane was incubated with avidin-peroxidase conjugate at mum temperature for 1 h. Bound peroxidase activity was detected using 4-chloro- 1-naphthol as hydrogen donor.

Urine Samples Urine samples were collected from 100 seronegative sub-

jects aged 24-68 yr and 70 seropositive subjects aged 10-60 yr. Negative and positive sera were discriminated by gelatin particle agglutination. The seropositivity was confirmed by Western blotting. Urine samples collected were mixed with lil00 volume of 10 giL bovine serum albumin (fraction V,

362 Hashida et al.

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5 6 26 41 a 0 0

13 22 25 49 0 0 0 0

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Urine Samples Urine Samples

Seronegative Seropositive Subjects Seronegative Seropositive Subjects from No. of Urine Samples from from No. of Urine Samples from

Subjects Subjects

Fig. 9. Detection of anti-HIV-1 IgC or anti-HIV-1 antibodies in concentrated urine by conventional methods. Urine samples. which gave low signals or were negative, were concentrated approximately 10-fold and tested by the conventional ELISA and gelatin particle agglutination. See Fig. 8 for symbols.

Intergen Company) and lil00 volume of 5 g/L thimerosal and stored at - 20°C. Before use, urine samples were thawed and centrifuged 1,500g at 4°C for 10 min to remove pre- cipihtes. some urine samples were concentrated approximately 1 ()-fold by centrifugation in a microconcentrator with a molecular sieve (CENTRICON-30, Amicon Division W.R. Grace &Co., Beverly, MA) at 5.000g for 15-20min.

Sensitivity of the Methods Used

Three urine samples from seropositive subjects were serially diluted with urine from a seronegative subject and were tested by the above methods. The immune complex transfer enzyme immunoassay wdS 30- to 10,000-fold more sensitive than the other methods (Fig. 2).

Measurement of Human IgG in Urine

munoassay as described previously ( 16). Human IgG in urine was measured by two-site enzyme im-

RESULTS AND DISCUSSION

Anti-HIV-1 IgG in urine was detected by an enzyme immunoassay (immune complex transfer enzyme immunoasssay) using recombinant RT and p17 as antigens as shown sche- matically in Figure 1. Recombinant RT was labeled with horseradish peroxidase, and recombinant p17 was labeled with P-D-galactosidase from Escherichia coli. The results were com- pared with those by the conventional ELISA using five recombinant proteins as antigens and gelatin particle agglutination using a lysate of the whole virus as antigen.

Interference of the Methods Used by Urine

Effect of pH in the immune complex transfer enzyme immunoassay was examined by incubation of urine samples with 2,4-dinitrophenyl- bovine serum albumin-recombinant protein conjugate and recombinant protein-enzyme conjugate at pH 5 .O-8.0, since pH of urine samples ranged from 5.3 through 8.0. The specific signal at pH 5.5 was 48-79% of the maxi- mal one at pH 7.0-8.0, although the nonspecific signals were not significantly different at pH 5.0-8.0 (Fig. 3). Therefore, neutralization of urine samples before use was recommended.

Increasing volumes of urine samples from seropositive subjects were tested by the immune complex transfer enzyme immunoassay. Up to 100 p1 of urine samples could be uscd with only slight interference (Fig. 4).


positive with RT, were clearly positive with p17, and others, which were weakly positive with p17, were clearly positive with RT. By approximately 10-fold concentration of urine samples, low signals for some seropositive subjects (Nos. 1-5, 19, 50, and 62 in Fig. 5 ) , were enhanced unequivocally, although signals for seronegative subjects were not significantly changed (Table 1 and Fig. 6). Thus, the sensitivity and specificity by the present method were both loo%, and test results by the present method appeared to be fairly reliable, since the positivity and negativity observed with one of the two antigens could be confirmed with the other antigen as well as with approximately 10-fold concentrated urine samples. The level of anti-HIV-1 RT IgG appeared not to be related to the stage of HIV-1 infection, although the level of anti-HIV-1 p17 IgG tended to be low in patients with AIDS (Fig. 7).

The sensitivity and specificity of the conventional ELISA were 91% and 99%, respectively, with unconcentrated urine samples (Fig. 8 ) and 97% and 57%, respectively, with approximately 10-fold concentrated urine samples, since signals for concentrated urine samples from seronegative subjects were much higher than those for unconcentrated ones (Fig. 9). The sensitivity and specificity of the gelatin particle agglutination test were 83% and 97%, respectively, with unconcentrated urine samples (Fig. 8) and 97% and 76%, respectively, with approximately 10-fold concentrated urine samples (Fig. 9).

Assay Variation in the Immune Complex Transfer Enzyme lmmunoassay

The assay variation in the immune complex transfer enzyme immunoassay was examined by using urine samples, which showed three different levels of the fluorescence intensity for bound enzyme activity (8.0. 40, and 318 with RT and 18,517 and 2,260 with p17 for within-assay and 12,64, and 5 10 with RT and 21,27 1, and 3 3 10 with p 17 for between- assay). The number of determinations at each level was 10 for within-assay and 8 for between-assay. The variation coef- ficients for within-assay and between-assay were 6.0-1 1.9% and 7.8-8.3%, respectively with RT and 4.9-8.5% and 6.8-9.6%, respectively, with p17.

Stability of Anti-HIV-1 IgG in Urine

Urine samples immediately after collection were mixed with lil00 volume of 10 giL bovine serum albumin and stored at - 20°C. Underthiscondition, anti-HIV-1 IgGin unnedetected by the immune complex transfer enzyme immunoassay using recombinant RT and p17 as antigens was stable for at least 6 months.

Detection of Anti-HIV-1 Antibodies in Urine

Seventy urine samples were collected from seropositive subjects (49 asymptomatic carriers aged 14-47 yr, 13 patients with acquired immune-deficiency syndrome (AIDS)-related complex aged 10-56 yr, and 8 patients with AIDS aged 21-60 yr), and 100 urine samples were collected from seronegative subjects (65 males aged 24-68 yr and 35 females aged 35-68 yr). The seropositivity was confirmed by Western blotting. The concentrations of IgG in urine samples from seropositive and seronegative subjects were 3.8 i 4.1 (SD) mg/L (range, 0.04-44, 120, 660 nig/L) and 1 .8 2 3.4 (SD) mg/L (range, 0.1-22 mgiL), respectively, calculated by transforming each value to natural logarithm, since they varied to a great extent. The concentrations of IgG in 12 urine samples ( 1 7%j from seropositive subjects and 5 urine samples (5%) from seronegative subjects were higher than 10 mgiL. These samples were tested by the above methods.

Results by the immune complex transfer enzyme immunoassay using recombinant RT and p17 as antigens are shown in Figure 5. All signals for seropositive subjects were higher than those for seronegative subjects and lowered to levels for seronegative subjects by preincubation of urine samples with excess of recombinant RT or p17. Low signals for some urine samples (Nos. 1-5, 19, 50, and 62 in Fig. 5) from seropositive subjects were neither due to low pH of the samples nor due to low concentrations of IgG in the samples (Table 1). When the highest signal for seronegative subjects was taken as a cut-off value, approximately 80% of the samples from seropositive subjects were unequivocally positive not only with RT but also with p17. Some samples, which were weakly

Comparison With Previous Reports

Anti-HIV- 1 antibodies in urine were detected previously by the conventional ELISA (17-19), IgG antibody-capture enzyme-linked immunosorbent assay (GACELISA) (20-22) and IgG antibody-capture particle adherence test (GACPAT) (20,2 I ) . The sensitivity and specificity of the conventional ELISA performed using various commercial kits were 93 - 100% and 92-loo%, respectively (17-19j. In only one report, both the sensitivity and specificity were 100% (18), but the ratio of signal to noise was not improved by concentration of urine samples (18), which is consistent with our finding that nonspecific signals for seronegative subjects by the conventional ELISA markedly enhanced with concentrated urine samples (Fig. 9). The sensitivity and specificity of GACELISA wcre 88.1-99.4%and97.9-100%,respectively(20-22). Thoseof GACPAT were 95.2-100% and 97.9-99.6%. respectivcly (20,21). In the last two methods, the sensitivity would not be improved by using concentrated urine samples, as long as the level of IgG in urine samples reached the low level needed to saturate the assay anti-IgG binding sites (20).

By contrast, the present method had advantages over the previously reported methods. The specificity of test results was confirmed by preincubation with excess of recombinant RT or p17 (see Fig. 5) . The positivity and negativity with one antigen were confirmed with the other antigen as well as

364 Hashida et al.

with concentrated urine samples (Figs. 5, 6). The sensitivity of the present method could be improved with Concentrated urine samples with no loss of the specificity (loo%), sincc the immune complex consisting of 2 ,4-dinitrophenyl-antigen1 antibody IgG to be detected and antigen-enzyme conjugate was transferred from one solid phase to another to eliminate interfering substance(s) and, as a result, nonspecific signals for seronegative subjects did not significantly change by concentration of urine samples (see Fig. 6 and Table 1).

Test results will become more reliable by increasing the number of antigens such as RT and p17, with which the sensitivity and specificity are both 100%.

REFERENCES

1

2

3

4

5

6

7

8

9

10

1 1

Constantine NT: Serologic tests for the retroviruses: approaching a de- cadeofevolution. AIDS7:1-13, 1993. Fricdland GH, Klein RS: Transmission of the human immunodeficiency virus. N En@ J Med 3 17: 1 125-1 135, 1987. Ishikawa E, Imagawa M, Hashida S. Yoshitake S, Hamaguchi Y, Ueno T: Enzyme-labeling of antibodies and their fragments for enzyme iin- munoassay and immunohistochemical staining. J Immunoassay 4: 209-327, 1983. Hashida S, Tanaka K. Yamamoto N, Uno T, Yamaguchi K, Ishikawa E: Detection of one attomole of I Arg’l-vasopressin by novel noncompeti- tive enzyme imrnunoassay (hetero-two-site complex transfer cnzynie immunoassay). J Biochem IlO:486-492, 1991. Kohno T, Ishikawa E: A highly sensitive enzyme immunoassay of anti- insulin antibodies in guinea pig serum. J Biochem 100:1247- 1251. 1986. Ishikawa E, Kato K: Liltrasensitivc enzyme immunoassay. Scand J Immunol X(Supp1.7):43-55. 1978. Saitoh A, lwasaki H. Nakata A, Adachi A, Shinagawa H: Overproduc- tion of human immunodeficiency virus type 1 reverse transcriptase in Escherichiu culi and purification of the enzyme. Microbiol Immunol 34509-521, 1990. Saitoh A . Tanaka N. Nakata A, Ikuta K, Shinagawa H: A unique monoclonal antibody that recognizes inaturc p17 of HIV-I but not its precursor. Microbiol Immunol36:105-1 I I . 1992. Udenfriend S, Stein S. Bijhlen P, Dainnan W. Leimgruber W. Weigcle M: Fluorescamine: a reagent fur assay of amino acids, peptides, proteins, and primary amines in the picomole range. Scierzce 178:87 1-872. 1972. Penefsky HS: A centrifuged-column procedure for the measurement of ligand binding by beef heart F,. Methods EnzynolS6:527-530, 1979. Hashida S. lmagawa M. lnoue S, Kuan K-H, Ishikawa E: More useful

maleiniide compounds for the conjugation of Fab’ to horseradish peroxidase through thiol groups in the hinge. J Appl Biochenl 656-63. 1984.

12. lmagawa M, Hashida S , Ishikawa E, Mori H? Nakai C, Ichioka Y. Nakajima K: A highly sensitive sandwich enzyme immunoassay for insulin in human serum developed using capybara anti-insulin Fab’- horseradish peroxidase conjugate. Anal Leff 16:1509-1523, 1983.

13. Kohno T, Katsumaru H , Nakamoto H, Yasuda T, Mitsukawa T. Matsukura S, Tsunetoshi Y, IshikawaE: Use of inactive P-o-galactosidase for e h - ination of interference by anti-P-n-galactosidase antibodies in immune complex transfer enzyme immunoassay for anti-thyroglobulin IgG in serum using P-o-galactosidasc from Escherichia coli as label. J Clin Lab Anal 5 : 197-205, 199 1 .

14. Imagawa M, Hashida S. Ohta Y, lshikawa E: Evaluation of P-D-galactosidase from EJchrrichiu coli and horseradish peroxidase as labels by sandwich enzyme imrnunoassay technique. Ann Clin Biochem 21 310- 317, 1984.

15. Yoshida T, Matsui T, Kobayashi S, Harada S , Kurimura T, Hinuma Y, Yamamoto N: A novel agglutination test for the human imniunodefi- ciency virus antibody: a comparative study with enzyme-linked immunosorbent assay and immunofluorescence. Jpn J Cancer Res (Gann) 77:1211-1213, 1986.

16. Yogi Y, Hirota K. Kohno T, Toshimori H, Mastukura S, Setoguchi T, Ishikawa E: Measurement of anti-thyroglobulin IgG in urine of patients with autoimmune thyroid diseases by- sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). J Clirz Lab A n d 7:70-79. 1993.

17. Cao Y, Hosein B, Borkowsky W. Mirabile M, Baker L, Baldwin D. Poiesz BJ, Friedman-Kien AE: Antibudies to human immunodeficiency virus type 1 in the urine specimens of HIV-1-Seropositive individuals. AIDSResHumRetrovirusrs5:311-319. 1989.

18. Reagan KJ. Lile CC, Book GW, Devash Y. Winslow DL, Bincsik A: Use of urine for HIV-1 antibody screening. Lancet 335:358-359, 1990.

19. Desai S, Bates H, Michalski FJ: Detection of antibody to HIV-I in urine. Lancet 337:383-184, 1991.

20. Connell JA, Parry JV, Mortimer PP, Duncan KJS, McLean KA, John- son AM, Hambling MH. Barbara I ? Farrington CP: Preliminary report: Accurate assays for anti-HIV in urine. Lancer 335: 1366- 1369, 1990.

21. Klokke AH, Ocheng U. Kalluvya SE. Nicoll AG. Laukamm-Jostcn U, Parry JV, Mortimer PP, Connell JA: Field evaluation of immunoglobulin G antibody capture tests for HIV-1 and HIV-2 antibodies in African serum. salivaandurine.AIDS5:1391-1392, 1991.

22. Thongcharoen P, Wasi C. Louisirirotchanakul S. Parry J , Connell J , Mortimer P Immunoglobulin G antibody capture enzyme-linked imiuno- sorbent assay: a versatile assay for detection of anti-human immunodeficiency virus type 1 and 2 antibodies in body fluids. J Clin Microbiol 30:3288-3289. 1992.

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