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 infection
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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-dinitro- phenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein- enzyme conjugate. The enzymes used as la- bels 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-dini- trophenyl group) IgG, eluted with ~N-2,4-dini- trophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human
IgG y-chain) IgG. Finally, bound enzyme ac- tivity was assayed by fluorometry. Urine sam- ples were collected from 100 seronegative subjects and 70 seropositive subjects. The sensitivity and specificity were both 100% with unconcentrated urine samples. The positiv- ity was confirmed by preincubation of urine samples with excess of the antigens. The pos- itivity and negativity with one of the two anti- gens could be confirmed with the other antigen. The positivity with low signals could be confirmed by concentration of urine sam- ples. Detection of anti-HIV-1 IgG in urine by the immune complex transfer enzyme im- munoassay 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, per- oxidase, p-D-galactosidase, ELISA, gelatin particle agglutination
Antibodies to human immunodeficiency virus type 1 (HIV-1) in serum, plasma and whole blood have been detected by var- ious 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 en- zyme immunoassay) using recombinant RT and p17 as anti- gens, which had advantages in sensitivity and specificity over conventional methods.
MATERIALS AND METHODS
The regularly used buffers were 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, 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
/ / / / ,,
bovine serum albumin was calculated from the absorbance at 280 nm and 360 nm (4). The average number of 2,4-dinitro- phenyl 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 hu- man 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.
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) .
Rabbit (anti-2,4-dinitrophenyl-bovine serum albumin) se- rum 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 elu- tion at pH 2.5 from columns of 2,4-dinitrophenyl-bovine se- rum 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-dini- trophenyl-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 pre- viously 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
1 o4 h
4 - 0 ._ 0 s l o 3 8 5 uI l o 2 F m
c a, C
a, 0 C a,
L - -% 10' c
x loo P 0 3 LL -
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 trian- gles) 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 de- scribed above (3.7 mg, 56 nmol) in 1 .0 ml of 0. I mol/L so- dium 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-dimethyl- formamide at 30C 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 in- troduced per albumin molecule was 5.9 (3).
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 30C 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 30C 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 estima- tion of amino groups using fluorescamine (Sigma Chemical Co., St. Louis, MO) (9) and bovine serum albumin as stan- dard. and its molecular weight was taken to be 64,000 (7). The average number of thiol groups introduced per recombi- nant 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.
x > + .- .- .c.
h v) c a, C
u C a, 0 v)
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 conju- gate 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-bo- vine serum albumin (0.15 mg, 2.2 nmol) in 10 pl o f buffer A at 30C for 60 min. After incubation, the reaction mixture was incubated with 10 p1 of 10 mmol/L 2-mercaptoethylamine in buffer A at 30C for 15 min and subsequently with 20 pl of 10 mmoliL N-ethylmaleimide in buffer A at 30C 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 de- scribed 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 30C for 30 min. After incubation. the reaction mixture was subjected to gel filtration by the centri- fuged 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 30C for 30 min. After incu- bation, 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 30C 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 de- termined by a commercial protein assay kit (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bo- vine serum albumin as standard, and its molecular weight
Enzyme lmmunoassay of Anti-HIV-1 IgG in Urine 357
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 immunoas- say. 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 4C for 20 h. After incubation,...