anti-htlv-i igg in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme...

Journal of Clinical Laboratory Analysis 8:149-156 (1994)

Anti-HTLV-l IgG in Urine Detected by Sensitive Enzyme lmmunoassay (Immune Complex Transfer Enzyme Immunoassay) Using a Synthetic Peptide, Cys-Env

gp46(188-224), as Antigen Seiichi Hashida, Kouichi Hirota, Takeyuki Kohno, and Eiji Ishikawa Deparfment of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki, Japan

Antibody IgG to human T-cell leukemia virus type I (HTLV-I) in urine was detected by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys-env gp46(18&224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples. Anti- HTLV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-envgp46(188-224) conjugate and Cys-env gp46( 188-224)-P-~-galactosidase (fscherichia coli) conjugate. The complex formed, consisting of the three corn- ponents, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dini-

Key words: antibody, adultT-cell leukemia, P-D-galactosidase, Western blotting, gelatin particle agglutination, ELISA

trophenyl 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 ~-D-galactosidase activity was assayed by fluorometry. Thirty-one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested.The sensitivity and specificity were 87 and I OO%, respectively, with unconcentrated urine samples and 94 and 1 OO%, respectively, with approximately 10-fold concentrated urine samples. These results were superior to those by the conventional ELISA and gelatin particle agglutination test. o 1994 Wiley-Liss, inc.

INTRODUCTION

HumanT-cell leukemia virus type I (HTLV-I), isolated from a patient with cutaneousT-cell lymphoma (l), is etiologically associated with adultT-cell leukemia (2) and adult T-cell cancers (3). The virus is transmitted by blood transfusion (4) and breast-feeding (5). For prevention of the virus transmissions, plasma or serum of blood donors and pregnant women have been tested for anti-HTLV-I antibodies by gelatin particle agglutination, enzyme-linked immunosorbent assay, fluoroimmunoassay, and/or Western blotting (6). These tests are sensitive and have been successfully used for the diagno- sis of HTLV-I infection (6). However, blood should be collected with due cautions to avoid infections not only with HTLV-I but also with other pathogens. This paper describes the detection of antibody IgG to HTLV-I in urine, which can be collected more easily with no invasive procedures, less expenses, and less possibility of various infections than blood (7). The method used was an enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide. Cys-env gp46( 188-224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples (8).

0 1994 Wiley-Liss, Inc.

MATERIALS AND METHODS Buffer

The regularly used buffer was 10 mmolfiiter sodium phosphate buffer, pH 7.0, containing 1 .O mmolhiter MgC12, 1 .O g/ liter NaN3, and 0.1 g/liter bovine serum albumin (fraction V; Intergen Company, Purchase, NY) (buffer A).

Cys-em gp46(188-224) of HTLV-I

Cys-env gp46( 188-224) of HTLV-I (H2N-Cys-Pro'88-Pro- Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-~u-Glu-Pro-Ser- Ile-Pro-Trp-Lys-Ser-Lys-Leu-Leu-Thr-Leu-Val-Gln- Leu-Thr-Leu-Gln-Ser-Thr-Asn-Tyr-~2x-COOH) was obtained from Peptide Institute, Inc., Osaka, Japan.

Antibodies

Rabbit (anti-2,4-dinitrophenyl-bovine serum albumin) serum was obtained from Shibayagi Co., Ltd., Gumma, Japan.

Received October 7, 1993; accepted October 12, 1993

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

150 Hashida et al.

Rabbit (anti-human IgG y-chain) IgG was obtained from Medical and Biological Laboratories Co., Ltd., Nagoya, Ja- pan. IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of DEAE- cellulose (9). The amount of IgG was calculated from the absorbance at 280 nm (9).

2,4-Dinitrophenyl-Bovine Serum Albumin

Thiol groups were introduced into bovine serum albumin molecules using N-succinimidyl-S-acetylmer- captoacetate and were reacted with maleimide groups introduced into &N-2,4-dinitrophenyl-~-lysine molecules using N-succinimidyl-6-maleimidohexanoate (10). The amount of bovine serum albumin, 2,4-dinitrophenyl groups, and 2,4-dinitrophenyl-bovine serum albumin was calculated from the absorbance at 280 and 360 nm (10). The average number of 2,4-dinitrophenyl groups introduced per albumin molecule was 6.0.

Protein-Sepharose 4B

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

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 serum albumin-Sepharose 4B and human IgG-Sepharose 4B, respectively (1 1).

Protein-Coated Polystyrene Balls

Polystyrene balls (3.2 mm in diameter; Immuno 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/liter) and affinity-purified (anti- human IgG y-chain) IgG (0.1 g/liter) (12). Affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG-coated polystyrene balls had been colored pink for discrimination from other polystyrene balls.

2,4-Dinitrophenyl-Bovine Serum Albumin-Cys-env gp46(188-224) Conjugate and Cys-env gp46(188-224)-j3-~-Galactosidase Conjugate

Maleimide groups were introduced into 2,4-dinitrophenyl bovine serum albumin molecules using N-succinimidyl-6- maleimidohexanoate and P-D-gdactosidase molecules from

E. coEi using NSJ'-o-phenylenedimaleimide, and reacted with Cys-env gp46(188-224) (8).

Immune Complex Transfer Enzyme lmmunoassay

Serum samples (20 p1) were incubated with 50 pg of inactive P-D-galactosidase (P-Calactosidase-Mutein; Roehringer Mannheim GmbH, Mannheim, Germany) in 30 p1 of buffer A containing 0.1 mol/liter NaCl at 20°C for 3 hr. After incubation, the reaction mixture was incubated simultaneously with 100 fmol of 2,4-dinitrophenyl-bovine serum albumin- Cys-env gp46(188-224) conjugate and 100 fmol of Cys-env gp46( 188-224)-P-~-galactosidase conjugate in 0.1 ml of buffer A containing 0.55 mol/liter XaC1 at 20°C for 3 hr (8). Inactive P-~-galactosidase was used to eliminate interference by anti-P-D-galactosidase antibodies (13). The amount of inactive P-D-galactosidase was calculated from the absorbance at 280 nm using the extinction coefficient for P-D-galactosidase (9).

Urine samples (1 00 pl) were incubated with 20 1.11 of nonspecific rabbit serum and 1 0 pl of buffer A containing 1 mol/ liter NaCl and 50 pg of inactive P-D-galactosidase at room temperature for 3 hr, unless otherwise specified. In experiments to confirm the presence of anti-HTLV-I env gp46(188-224) IgG, 150 pmol (0.61 pg) of Cys-env gp46( 188-224) was added. In experiments to examine the effect of urine volumes, the total volume of buffer Acontain- ing 0.4 mol/liter NaCl and 50 pg of inactive P-D-galactosidase was increased from 10 to 11 0 pl depending on the volume of urine used. In experiments to examine the effect of pH, 1 p1 of urine and 109 p1 of 10 mmolfiiter sodium phosphate buffer, pH 5.0-8.0, containing 0.4 molfliter NaCl, 0.1 g/liter bovine serum albumin, 1 .O mmolfiter MgC12, 1 .O g/liter NaN3, and 50 pg of inactive P-D-galactosidase were used. The reaction mixture (130 pl) was incubated with 20 pl of buffer A containing 1 mol/liter NaC1, 100 fmol of 2,4-dinitrophenyl- bovine serum albumin-Cys-env gp46( 1 88-224) conjugate, and 100 fmol of Cys-env gp46( 188-224)-P-~-galactosidase conjugate at room temperature for 3 hr.

To the reaction mixture (150 pl) with either serum or urine samples, two colored 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 colored polystyrene balls were washed twice by addition and aspiration of 2 ml of buffer A containing 0.1 mol/liter NaCl and incubated with 150 pl of bufferAcontaining 0.1 mol/liter NaCl and 1 mmol/ liter EN-2,4-dinitrophenyl-~-lysine and two white polystyrene balls coated with affinity-purified (anti-human IgG y-chain) IgG at room temperature for 1 hr. The colored polystyrene balls were removed, and the incubation was continued at room temperature for 2 hr. 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 2.5 hr for

Enzyme lmmunoassay of Anti-HTLV-l IgG in Urine 151

serum samples and for 25 hr for urine samples by fluorometry using 4-methylumbelliferyl-~-~-galactoside as a substrate (9). The fluorescence intensity was measured relative to 1 x lo-* molbiter 4-methylumbelliferone in 0.1 mol/liter glycine- NaOH buffer, pH 10.3, using 360 nm for excitation and 450 nm for emission analysis with a spectrofluorophotometer (RF- 5 10, Shimadzu Seisakusho, Ltd., Kyoto, Japan).

Conventional Enzyme-Lin ked lmmunosorbent Assay (ELISA)

The conventional ELISA was performed with a commercial kit (Eitest-ATL; Eisai Co., Ltd.,Tokyo, Japan). Microtiterplates coated with HTLV-I produced by MT-2 cell line ( 1 4) were incubated with 120 pl of urine samples and, after washing, with monoclonal (anti-human IgG Fc) antibody-alkaline phosphatase conjugate. Bound alkaline phosphatase activity was assayed by colorimetry using 4-nitrophenyl-phosphate as substrate.

Gelatin Particle Agglutination Test

Gelatin particle agglutination test was performed using a commercial kit with HTLV-I produced by TCL-Kan cell line (15) as antigen (SERODIA-ATLA; Fujirebio, Inc., Tokyo, Japan). Serum and urine samples were diluted 8-fold or more and 2-fold or more, respectively, with the diluent included in the kit. The diluted samples (25 pl) were mixed with the particle solution (25 pl) in “U”-shaped wells of microplates and allowed to stand at room temperature for 3 hr.

Serum and Urine Samples

Paired serum and urine samples were collected from 600 healthy subjects (382 males aged 19-77 years and 218 females aged 20-69 years). Serum samples were stored at -20°C. Urine samples collected were mixed with 1/100 volume of 10 g/liter bovine serum albumin (fractionv, Intergen Company) and 1/100 volume of 100 gbiter NaN3 and stored at -20°C. Before use, urine samples were thawed and centri- fuged at 1,500g at 4°C for 10 min to remove precipitates. Some urine samples were concentrated approximately 10- fold by centrifugation in a microconcentrator with a molecular sieve (CENTRTCON-30,Amicon Division W.R. Grace & Co., Beverly, MA) at 5,OOOg for 15-20 min.

Measurement of Human IgG in Urine

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

RESULTS

Anti-HTLV-I IgG in urine was detected by an enzyme immunoassay (immune complex transfer enzyme immunoassay)

DNP-Ag Anti-DNP- solid phase

I

I 0 0 0 DNP-lysine

Ab Ag-Enz

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

using a synthetic peptide, Cys-env gp46( 188-224), as antigen as shown schematically in Figure 1, and the results were compared with those by the conventional ELISA and gelatin particle agglutination test using a lysate of the whole virus as antigen.

Sensitivity of the Methods Used

Five urine samples from seropositive subjects were serially diluted with urine from a seronegative subject and were tested by the above three methods. The immune complex transfer enzyme immunoassay was to various degrees up to more than 1,000-fold as sensitive as the conventional ELISA (Fig. 2). The gelatin particle agglutination test was less sensitive than the other two methods, since none of the five urine samples tested was positive by the gelatin particle agglutination test.

Interference of the Immune Complex Transfer Enzyme lmmunoassay 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-Cys-env gp46( 188-224) conjugate and Cys-env gp46( 188-224)-p-~-

152 Hashida et at.

I

L+l loo0 ' 100 ' 10 1

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

Fig. 2. Dilution curves of urine samples from seropositive subjects by two different methods. Unconcentrated urine samples from five seropositive subjects were serially diluted with unconcentrated urine from a seronegative subject and were tested by the immune complex transfer enzyme imrnu- noassay using a synthetic peptide, Cys-env gp46(188-224), as antigen (open symbols) and the conventional ELISA using a lysate of HTLV-I as antigen (closed symbols).

galactosidase conjugate at pH 5.0-8.0, since pH of urine samples ranged from 5.3 through 8.0. The specific signal at pH 5.0 was 34% of the maximal one at pH 7.0, and the nonspecific signals were slightly higher at lower pH (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 used with only slight interference (Fig. 4).

Urine samples from seronegative subjects were concentrated approximately 10-fold, and 100 p1 of the concentrated urine samples were tested by the immune complex transfer enzyme immunoassay. By the concentration, the volume of urine samples was reduced 7.6- to 15-fold, and the concentration of IgG was enhanced 6.1- to 15-fold. Nonspecific signals observed with the concentrated urine samples were not significantly different from those observed with unconcentrated ones (Fig. 5).

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, with which three different levels of the fluorescence intensity for bound P-D-galactosidase activity (16, 183, and 2,190 for

l-'- 5 6 7 8

pH for Incubation with the Two Conjugate

Fig. 3. Effect of pH in the immune complex transfer enzyme immunoassay. Open and closed circles indicate experiments with urine samples from a seropositive subject and a seronegative subject. respectively.

within assay and 20, 152, and 1,900 for between assay) were observed. The number of determinations at each level was 10 for both within assay and between assay. The variation coef- ficients for within assay and between assay were 3.9-6.3 and 6.1-8.7%, respectively.

400 /- 4 4000

1 - 2 .-. 2

3000 m

0 " m m

E - - x

2000 : 0 m

1 0 20 40 60 80 100

Volume of Urine Used (ul /tube)

Fig. 4. Effect of urine volumes in the immune complex transfer enzyme immunoassay. Urine samples from three seropositive subjects were tested without concentration.


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Urine Samples from Seronegative Subjects

Fig. 5. Detection of anti-HTLV-I IgG in urine by the immune complex transfer enzyme immunoassay using a synthetic peptide, Cys-env gp46(188-224), as antigen. Thirty-one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested. Open and closed symbols indicate signals for urine samples from seropositive and seronegative subjects, respectively. Triangles and circles indicate signals

Detection of Anti-HTLV-l Antibodies in Urine

Paired serum and urine samples were collected from 600 healthy subjects (382 males aged 19-77 years and 218 females aged 20-69 years). Thirty-one sera (5.2%) out of the 600 were positive by both the immune complex transfer enzyme immunoassay and gelatin particle agglutination test. Thirty-one urine samples from the seropositive subjects (16 males aged 38-64 years and 15 females aged 38-69 years) and 100 urine samples from the seronegative subjects were tested by the above three methods before and after approximately 1 0-fold concentration. The concentrations of IgG in urine samples from the seropositive and seronegative subjects were 1.0 f 4.5 (SD) mgfliter (range, 0.05-15 mg/liter) and 1.8 f 3.4 (SD) mgiter (range, 0.09-22 mditer), respectively, and were enhanced 6.1- to 15-fold by the concentra-

1 10 20 30

No. of Urine Samples from Seropositive Subjects

observed before and after approximately 10-fold concentration, respectively. Smaller triangles and circles indicate signals obtained by preincubation of unconcentrated and approximately 10-fold concentrated urine samples, respectively, with excess of the synthetic peptide, Cys-env gP46(188-2U). The broken line indicates a tentative cut-off value.

tion as described above. These values were calculated by transforming each value to natural logarithm, since they var- ied to a great extent. The concentrations of one urine sample (3%) from the seropositive subjects and 5 urine samples (5%) from the seronegative subjects were higher than 10 mgfliter.

By the immune complex transfer enzyme immunoassay, anti-HTLV-I IgG was detected in 27 unconcentrated urine samples (87%) from the seropositive subjects and in none from the seronegative subjects. The lowest signals for 10 urine samples from the seropositive subjects (Nos. 1-10 in Fig. 5 ) were neither due to low pH of the samples nor due to low concentrations of IgG in the samples, except for two samples (Nos. 2 and 10 in Fig. 5) with low concentrations of IgG (0.05-0.06 mg/liter). The low signals for 8 urine samples out of the 10 were unequivocally enhanced by approximately 10-

154 Hashida et al.

TABLE 1. Test Results of Urine Samples by the Immune Complex Transfer Enzyme Immunoassay, Conventional ELISA and Gelatin Particle Agglutination Test

Sensitivity (%) Specificity (%)

Before After Before After concentration concentration concentration concentration Method

Immune complex 87 (27/31) 94 ('29131) loo (lOO/lOO) 100 (lOO/lOO) transfer enzyme immunoassay

Conventional ELISA 13 (4/31) 26 (8/31) 91 (67/74) 41 (30/74)

Gelatin particle 0 (OD11 23 (7/31) 100 (100/100) 100 (71/71) agglutination test

fold concentration, although those for the rest two samples were not. Nonspecific signals for approximately 10-fold concentrated urine samples from the seronegative subjects were as low as before concentration. Namely. the sensitivity and specificity were 87 and loo%, respectively, before concen-

tration and were improved to 94 and loo%, respectively, after the concentration (Fig. 5 and Table 1).

The sensitivity and specificity of the conventional ELISA and gelatin particle agglutination test were much lower (Figs. 6 and 7 and Table 1). The sensitivity and specificity of the

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Urine Samples No. of Urine Samples from Urine Samples No. of Urine Samples from

Seronegative Seronegative Subjects Subjects

from Seropositive Subjects from Seropositive Subjects

Fig. 6. Detection of anti-HTLV-I IgG or anti-HTLV-I antibodies in unconcentrated urine by conventional methods. Thirty-one unconcentrated urine samples from seropositive subjects and 100 unconcentrated urine samples from seronegative subjects were tested by the conventional ELISA and gelatin particle agglutination test. Open and closed symbols indicate

results for unconcentrated urine samples from seropositive subjects and seronegative subjects, respectively. Squares and rhombuses indicate results by the conventional ELISA and gelatin particle agglutination test, respectively. The broken line indicates a tentative cut-off value.


7 I7

17 0

- n o r n o

3 0%

s 0.2 Q y w , 0

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' 1 10 20 30 0.0

17 0

1 5 6 000

11 0

25

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Unne Samples No. of Urine Samples from Urine Samples No of Urine Samples from

Seronegative Seronegative Subjects Subjects

from Seropositive Subjects from Seropositive Subjects

Fig. 7. Detection of anti-HTLV-I IgG or anti-HTLV-I antibodies in concentrated urine by conventional methods. Urine samples, which gave low signals or were negative, were concentrated approximately 10-fold and tested

conventional ELISA were 13 and 91%, respectively, with unconcentrated urine samples (Fig. 6) and 26 and 41%, respectively, with approximately 10-fold concentrated urine samples, since signals for concentrated urine samples from the seronegative subjects were much higher than those for unconcentrated ones (Fig. 7). The sensitivity and specificity of the gelatin particle agglutination test were 0 and loo%, respectively, with unconcentrated urine samples (Fig. 6 ) and 23 and loo%, respectively, with approximately 10-fold concentrated urine samples (Fig. 7).

DISCUSSION

The immune complex transfer enzyme immunoassay was superior to the conventional ELISA and gelatin particle agglutination test. The immune complex transfer enzyme immunoassay was more sensitive than the conventional methods as described above (Fig. 2). The sensitivity and specificity of the immune complex transfer enzyme immunoassay with unconcentrated urine samples were much higher than those of the conventional methods (Figs. 5-7 andTable 1). In addi-

by the conventional ELISA and gelatin particle agglutination test. See Fig- ure 6 for symbols.

tion, the sensitivity of the immune complex transfer enzyme immunoassay could be improved by using concentrated urine samples with no loss of the specificity ( 1 OO%), since the immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46( 188-224) conjugate, anti-HTLV-I IgG to be detected and Cys-env gp46( 188-224)-P-~-galactosidase conjugate was transferred from one solid phase to another to eliminate interfering substance(s) (Fig. 1) and, as a result, nonspecific signals for seronegative subjects did not significantly change by concentration of urine samples (Fig. 5). By contrast, the sensitivity of the conventional ELISA was improved to some extent using concentrated urine samples only with loss of the specificity (Figs. 6 and 7 and Table l), since nonspecific signals for seronegative subjects were enhanced by concentration of urine samples (Figs. 6 and 7).

However, the sensitivity of the immune complex transfer enzyme immunoassay was lower than 100% even with concentrated urine samples (Fig. 5 andTable 1) . This might have been due at least partly to the fact that Cys-env gp46( 188-224) used as antigen in this study is only a small part of HTLV-1, which has a number of epitopes with various conformations.

156 Hashida et al.

Therefore, the sensitivity would be improved by using a larger synthetic peptide than Cys-env gp46( 188-224) or a recombi- nant protein corresponding to a part or parts of HTLV-I.

ACKNOWLEDGMENTS

This work was supported in part by Grants-in-Aid for Sci- entific Research (No. 05557015) from the Ministry of Edu- cation, Science and Culture of Japan.

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anti-htlv-i igg in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme...

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