Journal of Clinical Laboratory Analysis 6:302-310 (1992)
Sensitive Enzyme lmmunoassay (Immune Complex Transfer Enzyme Immunoassay) for (Anti-Human T-cell Leukemia Virus Type I) IgG in Serum Using Recombinant gag p24(14-214) as Antigen Takeyuki Kohno,’ Kouichi Hirota,’ lwane Sakoda,* Motoo Yarna~aki,~ Yoshiharu Yok00,~ and Eiji Ishikawa’
‘
Department of Biochemistry, Medical College of Miyazaki, Miyazaki, Miyazaki Blood Center, The Japanese Red Cross, Miyazaki,2 and Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-humanT-cell leukemia virus type I) IgG (anti-HTLV-lIgG) in serum using recombinant gag p24 (14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination,enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positiveor false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for Key words:
antibody, adult T-cell leukemia, p-D-galactosidase,Western blotting, gelatin particle agglutination, ELISA
INTRODUCTION Human T-cell leukemia virus type I (HTLV-I), isolated from a patient with cutaneous T-cell lymphoma (l), is etiologically associated with adult T-cell leukemia (2) and adult T-cell cancers (3). For prevention of the virus transmissions by blood transfusion (4) and breast-feeding ( 5 ) , plasma or serum of blood donors and pregnant women has been tested for antiHTLV-I antibodies by gelatin particle agglutination (6), enzyme-linked immunosorbent assay (ELISA) (7), fluoroimmunoassay (2), and/or Western blotting (8). HTLV-I used as antigen in these methods has been produced using particular cell lines such as TCL-Kan (9) and MT-2 (10) and purified by density gradient centrifugation (6) or by 0 1992 Wiley-Liss, Inc.
improving the reliability of serodiagnosis by separately demonstrating antibodiesagainst as many different epitopes of HTLV-I as possible. Anti-HTLV-l IgG in test serum, which had been incubated with excess of inactive p-0galactosidase to eliminate interference by antip-D-galactosidaseantibodies, was reacted simultaneouslywith2,4-dinitrophenyl-bovine serum albumin-recombinantgag p24(14-214) conjugate and recombinant gag p24(14-214)p-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyreneballs coatedwith aff inity-purified(anti-2,4-dinitrophenylgroup) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the p-D-galactosidaseconjugate,the complex was eluted from the polystyrene balls with rN-2,4dinitrophenyl-L-lysineand transferred to polystyrene balls coated with affinity-purified (anti-human IgG y-chain) IgG. p-D-Galactosidase activity bound to the (anti-human IgG y-chain) IgG-coated polystyrene balls was s, assayedby fluorometry. o i 9 9 2 ~ i 1 e y - ~ i stnc.
ultracentrifugation (7). This is hazardous and inefficient. In addition, the specificity of these methods has not been tested by preincubation of serum with excess of HTLV-I as antigen. Recently, a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG has been developed using recombinant gag p24( 14-139)-env
Received March 2, 1992; accepted April 9, 1992. Address reprint requests to Dr. Eiji Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.
Enzyme lmmunoassay for Anti-HTLV-l IgG
gp46(217-315) hybrid protein as antigen ( I 1) which was safely and efficiently produced in Escherichia coli (12). This assay was 30-to 300-fold more sensitive than other methods including Western blotting using HTLV-I, gelatin particle agglutination using HTLV-I, and ELISA using the gag-env hybrid protein. Most of positive and negative samples were more clearly discriminated than by the other methods. Anti-HTLV-I IgG was demonstrated in all samples, which were positive by Western blotting. Low levels of anti-HTLV-I IgG below those detected by Western blotting were suggested to be detectable. However, the gag-env hybrid protein was soluble only in the presence of detergents. This hampered the use of enzymes other than horseradish peroxidase as label, and the specificity could not be tested by preincubation of serum with excess of the gag-env hybrid protein. More recently, the recombinant protein has been replaced by synthetic peptides including Cys-Arg-env gp46( 188-209) (13), Cys-gag p19(100-130) (14), Cys-env gp46(188-224) (15), and Ala-Cys-env gp46(237-262) (16). The specificity could be tested by preincubation of serum with excess of the synthetic peptides, which are readily soluble in the absence of detergents. The sensitivities were 30- to 30,OClo-fold higher than those of other methods such as Western blotting using four core proteins of HTLV-I, gelatin particle agglutination using HTLV-I, ELISA using the synthetic peptides, and HTLV-I. Most of negative and positive sera were discriminated. However, some results appeared to be false-negative or false-positive, and the use of as many synthetic peptides and recombinant proteins as possible was suggested to improve the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. This paper describes a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antiHTLV-I IgG using recombinant gag p24(14-214) which can be safely and efficiently produced in E . coEi and is soluble in the absence of detergents.
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 mmoYl sodium phosphate buffer, pH 7.0, containing 1.O mmol/l MgC12, 1.O g/1 NaN3, and 1.O g/l bovine serum albumin (fraction V, Amour Pharmaceutical Co., Kankakee, IL) (buffer B), and 10 mmol/l sodium phosphate buffer, pH 7.0, containing 1.O g/l bovine serum albumin (buffer C).
Recombinantgag p24(14-214) Recombinant gag p24(14-214) was produced in E . coli, which has been transformed with expression plasmid carry-
303
ing cDNA cloned for gag p24( 14-214) of HTLV-I (17), and was purified by centrifugal precipitation, solubilization with urea, and cation exchange chromatography (12). The purity of this preparation was 97% as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ( 18). The concentration of recombinant gag p24( 14-214) was determined by a commercial protein assay kit (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as standard.
Antibodies Rabbit (anti-2,4-dinitrophenyl-bovineserum albumin) serum was obtained from Shibayagi Co., Ltd. (Gumma, Japan). Rabbit (anti-human IgG y-chain) IgG was obtained from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of diethylaminoethyl cellulose, and the amount of IgG was calculated from the absorbance at 280 nm (19).
2,4-Dinitrophenyl-Bovine Serum Albumin Thiol groups were introduced into bovine serum albumin molecules using N-succinimidyl-S-acetylmercaptoacetate(20) and were reacted with maleimide groups introduced into EN-2,4-dinitrophenyl-L-lysine molecules using N-succinimidyl-6-maleimidohexanoate(2 1). The amount of 2 ,Cdinitrophenyl-bovine serum albumin was calculated from the absorbance at 280 nm and 360 nm (13). The average number of 2,4-dinitrophenyl groups introduced per albumin molecule was 6.8.
Affinity Purification of Antibodies 2,4-Dinitrophenyl-bovineserum albumin (10 mg) and nonspecific human IgG (10 mg) were coupled to CNBr-activated Sepharose 4B (1.0 g, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) according to the instructions of the manufacturer. (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 nonspecific human IgGSepharose 4B, respectively (22). Protein-Coated Polystyrene Balls Polystyrene balls (3.2 mm in diameter, Immuno Chemical, Inc., Okayama, Japan) were coated by physical adsorption with affinity-purified (anti-2,4-dinitrophenyl-bovineserum albumin) IgG (0.1 g/l), affinity-purified (anti-human IgG y-chain) IgG (0.1 g/l), and recombinant gag p24(14-214) (0.1 g/l) (23). Affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG-coated polystyrene balls had been colored pink for discrimination from other polystyrene balls.
304
Kohno et al.
2,4-Dinitrophenyl-Bovine Serum AlbuminRecombinantgag p24(14-214) Conjugate Maleimide-2,4-dinitrophenyl-bovineserum albumin
Mercaptoacetyl-recombinantgag p24(14-214) Mercaptoacetyl-recombinantgag p24( 14-214) was prepared as described above.
Maleimide groups were introduced into molecules of Recombinantgag p24(14-214)-P-D-galactosidase 2,4-dinitrophenyl-bovineserum albumin using N-succinim- conjugate idyl-6-maleimidohexanoate( 13). The average number of Maleimide-P-D-galactosidase (0.4 mg, 0.74 nmol) in 0.5 maleimide groups introduced per 2,4-dinitrophenyl-bovine ml of buffer A containing 0.35 mol/l urea was incubated with serum albumin molecule was 2.4 (19). mercaptoacetyl-recombinant gag p24( 14-214) (53 pg, 2.2 nmol) in 0.24 ml of the same buffer at 4°C overnight. SubseMercaptoacetyl-recombinantgag p24(14-214) quently, the reaction mixture was incubated with 10 yl of 0.1 Recombinant gag p24(14-214) (1.0 mg) in 0.7 ml of 0.1 mol/l 2-mercaptoethylamine in buffer A at 30°C for 15 min mol/l sodium phosphate buffer, pH 7.0, containing 5 mmol/l and 20 p1 of 0.1 mol/l N-ethylmaleimide in buffer A, and EDTA and 0.35 moll1 urea was incubated with 70 p1 of 11 subjected to gel filtration on a column ( 1.5 X 45 cm) of Ultrogel mmol/l N-succinimidyl-S-acetylmercaptoacetatein N,N-di- AcA 22 (IBF Biotechnics) using 10 mmol/l sodium phosmethylformamide at 30°C for 30 min. The reaction mixtures phate buffer, pH 7.0, containing 0.1 mol/l NaC1, 1.O mmoYl were further incubated with 70 pl of 1.O mol/l Tris-HCI buffer, MgC12, 1.0 g/l NaN3, 0.35 mol/l urea, and 0.1 g/l bovine pH 7.0, and 0.1 ml of 1.0 mol/l hydroxylamineHC1, pH serum albumin. The average number of recombinant gag 7.0, at 30°C for 15 min and subjected to gel filtration on a p24( 14-214) molecules conjugated per P-D-galactosidase molcolumn (1 .O X 30 cm) of Sephadex G-25 (Pharmacia LKB ecule was 2.8, which was calculated from the decrease in the Biotechnology AB) using buffer A containing 0.35 moVl urea. number of maleimide groups ( 19). The amount of the conjuThe average number of thiol groups introduced per recombi- gate was calculated from P-D-galactosidase activity (24). nant gag p24( 14-214) molecule was 3.0 (19). Immune Complex Transfer Enzyme lmmunoassay 2,4-Dinitrophenyl-bovineserum albumin-recombinantgag Using Recombinantgag p24(14-214) p24(14-214) conjugate Test serum (20 yl) was incubated with 50 pg of inactive
Maleimide-2,4-dinitrophenyl-bovine serum albumin (0.16 mg, 2.4 nmol) in 0.1 ml of buffer A containing 0.35 mol/l urea was incubated with mercaptoacetyl-recombinant gag p24(14-214) (0.29 mg, 12 nmol) in 0.38 ml of the same buffer at 4°C overnight. After incubation, the reaction mixture was incubated with 5 y1 of 0.1 mol/l 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 and subjected to gel filtration on a column (1.5 X 100 cm) of Ultrogel AcA 34 (IBF Biotechnics, Villeneuve-la-Garenne, France) using 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl, 1.O mmol/l MgC12, 1.O g/l NaN3, 0.35 moYl urea, and 0.1 g/l bovine serum albumin. The average number of recombinant gag p24( 14-214) molecules conjugated per albumin molecule was 2.4, which was calculated from the decrease in the number of maleimide groups (19). The amount of the conjugate was calculated from that of 2,4-dinitrophenyl-bovineserum albumin.
Recombinantgag p24(14-214)-p-D-Galactosidase Conjugate Maleimide-p-D-galactosidase Maleimide groups were introduced into molecules of P-D-galactosidase (EC 3.2.1.23) from E . coli using N,N’-l,2phenylenedimaleimide (19).
P-D-galactosidase(P-GalactosidaseMutein, Boehringer Mannheim GmbH, Mannheim, Germany) in 30 y1 of buffer B containing 0.1 moYl NaCl at 20°C for 3 hr to eliminate interference by anti-P-D-galactosidase antibodies (13). In some experiments, recombinant gag p24(14-214) (36 pg, 15 pmol) was added to test the specificity. After incubation, the reaction mixture was incubated simultaneously with 2,4-dinitrophenylbovine serum albumin-recombinant gag p24( 14-214) conjugate (100 fmol) and recombinant gag p24(14-214)-P-Dgalactosidase conjugate (100 fmol) in 0.1 ml of buffer B containing 0.55 mol/l NaCl at 20°C for 3 hr. Two affinitypurified (anti-2,4-dinitrophenyl-bovine serum albumin) IgGcoated polystyrene balls, which had been colored pink, were added to the reaction mixture, and the incubation was continued at 20°C overnight. After incubation, the colored polystyrene balls were washed twice with 2 ml of buffer B containing 0.1 mol/l NaCl and incubated with 0.15 ml of 1.O mmol/l ~N-2,4-dinitrophenyl-Llysine (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in the same buffer and two affinitypurified (anti-human IgG y-chain) IgG-coated polystyrene balls at 20°C for 1 hr. After incubation, the colored polystyrene balls were discarded, and the eluate and the (anti-human IgG y-chain) IgG-coated polystyrene balls were further incubated at 20°C for 2 hr. After washing as described above, P-D-galactosidase activity bound to the polystyrene balls was assayed at 30°C for 150 min by fluorometry using 4-methyl-
Enzyme lmmunoassay for Anti-HTLV-l IgG
umbelliferyl-P-D-galactoside as substrate (25). The fluorescence intensity was measured relative to moll1 4methylumbelliferone in 0.1 moYl glycine-NaOH buffer, pH 10.3 (25).
Immune Complex Transfer Enzyme lmmunoassay Using Synthetic Peptides The immune complex transfer enzyme immunoassays using the synthetic peptides Cys-gag p19( l00-130), Cys-env gp46(1 88-224), and Ala-Cys-env gp46(237-262) were performed as described previously (14- 16).
ELISA Rabbit (anti-human IgG y-chain) IgG was converted to the corresponding Fab' and conjugated to horseradish peroxidase using N-succinimidyl-6-maleimidohexanoate(26). The amount of the conjugate was calculated from peroxidase activity (19). In ELISA using recombinant gag p24( 14-214), test serum (20 p1) was mixed with 0.13 ml of buffer C containing 0.46 moYl NaCl and 1.O s/l NaN3, and was incubated with a recombinant gag p24( 14-214)-coated polystyrene ball at 37°C for 3 hr and at 4°C overnight. After incubation, the polystyrene ball was washed twice with 2 ml of 10 mmoVl sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl, and was incubated with 50 ng of (anti-human IgG y-chain) Fab'peroxidase conjugate in 0.15 ml of buffer C containing 0.1 mol/l NaCl at 37°C for 3 hr. The polystyrene ball was washed as described above, and bound peroxidase activity was assayed at 30°C for 10 min by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor (27). The fluorescenceintensity was measured relative to 1.O mg/l quinine in 50 mmol/l HZS04 (27). ELISA using HTLV-I as antigen was performed with a commercial kit (Eitest-ATL, Eisai Co., Ltd., Tokyo, Japan). Microtiter plates coated with HTLV-I produced by MT-2 cell line (10) were incubated with 20 pl of test sera and, after washing, with monoclonal (anti-human IgG Fc) antibodyalkaline phosphatase conjugate. Bound alkaline phosphatase activity was assayed by colorimetry using 4-nitrophenylphosphate as substrate.
305
Gelatin Particle Agglutination Gelatin particle agglutination was performed using a commercial kit with HTLV-I produced by TCL-Kan cell line (9) as antigen (SERODIA-ATLA, Fujirebio, Inc., Tokyo, Japan). Test serum was diluted eightfold or more with the diluent included in the kit. The diluted serum (25 pl) was mixed with the particle solution (25 pl) in U-shaped wells of microplates and allowed to stand at room temperature for 3 hr.
Western Blotting Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I produced by TCLKan cell line (9) was generously performed by Fujirebio, Inc.
Serum Samples Serum samples were obtained from healthy subjects aged 16-79 years in Miyazaki, Japan. Approximately 7% of healthy subjects were HTLV-I carriers.
RESULTS Recombinant gag p24( 14-214) was conjugated to 2,4-dinitrophenyl-bovine serum albumin and P-D-galactosidase and tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) as shown schematically in Figure 1 (the present assay).
p+-
+ @ + e
Anti-DNPsolid phase
Ag-Enz
U0 0
DNP-lysine
Anti-lgG-solid phase
Expressionof the Detection Limit of Anti-HTLV-l IgG in Serum The detection limit of anti-HTLV-I IgG in serum by enzyme immunoassay was expressed as the maximal dilution of serum containing anti-HTLV-I IgG with pooled normal serum which gave a bound enzyme activity significantly in excess of that in the presence of normal serum (background). A significant difference from the background was confirmed by the t-test (n = 5, P < 0.001).
Fig. 1. Immune complex transfer enzyme immunoassay for antibody IgG. DNP, 2,4-dinitrophenyl group; Ag, antigen; Ab, antibody; Enz, enzyme.
Kohno et al.
306
The present assay was 10- to 10,000-foldmore sensitive than the other methods.
Assay Variation The assay variation in the present assay was examined using serum samples, with which three different levels of the fluorescence intensity for bound P-D-galactosidase activity (16, 61, and 800 for within-assay and 9.6, 97, and 1,200 for between-assay) were obtained. The coefficients of variation for within-assay and between-assay were 5.6-9.0% (n = 12) and 8.6-12% (n = 6), respectively.
Usefulness of the Present Assay Using Recombinant gag p24(14-214) 0.1
40.1
104
w
103
lo2
10'
0"
10'
Dilution of Serum Containing Anti-HTLV-l IgG with Normal Serum ( -fold )
Fig. 2. Sensitivity of various methods. Four sera from HTLV-I carriers were serially diluted with normal serum and subjected to the immune complex transfer enzyme immunoassay using recombinant gag p24( 14-214) (the present assay) (open symbols with solid lines), ELISA using recombinant gag p24( 14-214) (closed symbols with solid lines), and ELISA using HTLV-I (closed symbols with broken lines).
Sensitivity Four sera from HTLV-I carriers were serially diluted with normal serum and were subjected to the present assay using recombinant gag p24( 14-214),ELISA using recombinant gag p24(14-214), ELISA using HTLV-I (Fig. 2), gelatin particle agglutination using HTLV-I, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I (Table 1).
The usefulness of the present assay using recombinant gag p24(14-214) was examined using 305 sera, which had been tested by other methods including gelatin particle agglutination, ELISA using HTLV-I, and Western blotting. Sera, for which the fluorescenceintensity for bound P-D-galactosidase activity by the present assay was over 40, were tentatively taken as positive, and other sera were taken as negative. The results were shown in Figure 3 and Tables 2 and 3. The 305 sera consisted of three groups tested by gelatin particle agglutination, which has been most widely used for screening of HTLV-I carriers in Japan: 90 strongly positive sera (group 1, serum nos. 1-90), in which the maximal dilution with the diluent included in the kit to cause gelatin particle agglutination was 128- to 16,384-fold,99 weakly positive sera (group 2, serum nos. 91-189), in which the maximal dilution was 8- to 64-fold, and 116 negative sera (group 3, serum nos. 190-305). In group 1 (90 sera, serum nos. 1-90), 87 sera (serum nos. 1-87) were consistently positive by gelatin particle agglutination, ELISA using HTLV-I, and the present assay. How-
TABLE l. Sensitivity of Various Methodsa Detection of anti-HTLV-I antibodies or IgG Method Present assay using recombinant gag p24( 14-214)
Gelatin particle agglutination using HTLV-I
Westem blotting using four core proteins of HTLV-I
Serum no. 1 2 3 4 1 2 3 4 1 2 3 4
Dilution of serum containing anti-HTLV-I antibodies with normal serum (-fold) 1 x 104 3 x 103 1 x lo2 3 x 10 1 x 10
+ + +
+ + +
+ + + +
-
-
-
-
-
f f 2
-
+-
''
-
-
-
-
-
-
-
f
-
-
-
-
5 -
"Four sera from HTLV-I carriers were diluted with normal serum and subjected to the three methods indicated.
+ + + + + + +
+ + + + + + + + + f +
-
f
3
+ + + + + + + + + + + +
Enzyme lmmunoassay for Anti-HTLV-l IgG
0 0 0 0
A
0 0
A
0
t
U
1
O
O
l
'
Negative
I
' ' " o m a dL 10 lo2 lo3 104 Maximal Dilution of Serum with Buffer to Cause Gelatin Particle Agglutination ( -fold) '"11111'
1
1
~
1
~
~
~
'
Fig. 3. Comparison of test results for anti-HTLV-I IgC or antibodies by the immune complex transfer enzyme immunoassay using recombinant gag p24(14-214) (the present assay), gelatin particle agglutination using HTLV-I, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I. A tentative cutoff value of 40 in the present assay is indicated by broken line. Open circles indicate groups 1 and 3, and closed triangles, open squares, and open triangles indicate groups 2-1, 2-2-1, and 2-2-2, respectively, as shown in Tables 2 and 3. Group 2-1 was positive and groups 2-2-1 and 2-2-2 were negativehdetenninate by Western blotting.
307
ever, one serum (serum no. 88) appeared to be false-negative by the present assay, and two sera (serum nos. 89 and 90), which were negative by ELISA using HTLV-I, Western blotting, and the present assay, appeared to be false-positive by gelatin particle agglutination as described in the Discussion. Group 2 (99 sera, serum nos. 91-189) was divided into two groups by Western blotting: 29 positive sera (group 2- 1, serum nos. 91- 119)and 70 negativehndeterminatesera (group 2-2, serum nos. 120- 189). In group 2-1 (29 sera, serum nos. 91-1 19), 27 sera (serum nos. 91-1 17) were consistently positive by gelatin particle agglutination, ELISA using HTLV-I, Western blotting, and the present assay. However, two sera (serum nos. 118 and 119), which were positive by gelatin particle agglutination, ELISA using HTLV-I, and Western blotting, appeared to be false-negative by the present assay as described in the Discussion. Group 2-2 (70 sera, serum nos. 120- 189) was divided into two groups by the present assay: 5 positive sera (group 2-2- 1, serum nos. 120- 124)and 65 negative sera (group 2-2-2, serum nos. 125-189). In group 2-2-1 (five sera, serum nos. 120-124), all the sera were positive by the present assay, but were positive, negative, or indeterminate by ELISA using HTLV-I or Western blotting. Notably, high fluorescence intensities for bound P-D-galactosidase activity were observed with some sera (serum nos. 120-122), which were indeterminate by Western blotting. In group 2-2-2 (65 sera, serum nos. 125-189), all the sera were negative by Western blotting and the present assay. The
TABLE 2. Test Results by Various Methods for Groups 1,2-1, and 2-2-1
Group Group 1 (90sera)
Serum no. 1-87 88 89
90 Group 2 OrOup2-1 (29 sera) Group 2-2-1
(5sera)
91-117 118; 119 120 121 122 123 124
Gelatin particle agglutination using HTLV-I +++a
+++ +++ +++ + + + + + + +
Optical density at 405 nm by ELISA using HTLV-I with cutoff valueof0.62 1.9-14 3.1 0.21
Western blotting using four 'Ore proteins Of HTLv-l p19 p24 p28 p53
NTb NT
NT NT
NT NT
0.16
+
-
-
-
2.7; 1.1-15 1.2; 1.5
+ +
+ +
+ +
+ +
+
*
*
2
*
5.5 1.3 0.94 0.17 0.13
2
+
-
NT NT
+
-
-
-
-
-
?
-
-
*
4
-
Fluorescence intensity for bound P-D-galactosidase activity by the present assay using recombinant gag p24( 14-214) with a cutoff value of 40
Inhibition by excess of recombinant gag p24( 14-214)with a cutoff value of 73(%)
230-2,300,OOO 25 7.1 14
81-100 NT
150; 49-46,OOO
74; 84-100
22; 31 38,000 140 2,100 64 51
NT
NT NT
100 89 99 93 40
"The maximal dilution of serum samples with the diluent, included in the kit to cause gelatin particle agglutination was 128- to 16,384-fold for serum nos. 1-90and 16-to 64-fold for serumnos. 91-124. bNT, not tested.
308
Kohno et el.
TABLE 3. Test Results by Various Methods for Groups 2-2-2, and 3
Group Group 2 Group 2-2-2 (65 sera) Group 3 ( 1 16 sera)
Serum no. 125- 186 187; 188 189 190-299 300; 301 302-304 305
Gelatin particle agglutination using HTLV-I
+= +
+
-
-
Optical density at 405 nm by ELlSA using HTLV-I with cutoff valueof0.62
Westem blotting using four core proteins Of HTLv-l p19 p24 p28 p53
0.10-0.58
-
-
1.1;0.82 I .3 0.12-0.43 0.69; 0.97 0.19-0.46 0.14
-
-
+ NT NT NT NT
-
-
-
-
-
NT NT NT NT
NT NT NT NT
NT NT NT NT
-
Fluorescence intensity for bound P-D-galactosidase activity by the present assay using recombinant gag p24( 14-214) with a cutoff value of 40
Inhibition by excess of recombinant gag p24(14-214) with a cutoff value of 73(%)
5.7-35 12; 19 33 3.0-38 22; 11 64-99 1.ooo
NT~ NT NT NT NT 34-70 95
"The maximal dilution of serum samples with the diluent included in the kit to cause gelatin particle agglutination was 8- to 64-fold for serum nos. 125-189. bNT, not tested
results by gelatin particle agglutination for most of these sera might have been false-positive, which was consistent with a previous report that the gelatin particle agglutination kit used in this study often gave false-positiveresults (28). However, some of these sera should be examined more carefully for reliable results as described in the Discussion. In group 3 (1 16 sera, serum nos. 190-305), 110 sera (serum nos. 190-299) were consistently negative by gelatin particle agglutination, ELISA using HTLV-I, and the present assay. However, six sera (serum nos. 300-305) appeared to be falsepositive by ELISA using HTLV-I or the present assay.
Specificity of the Present Assay Using Recombinantgag p24(14-214) In order to test the specificity of the present assay using recombinant gag p24( 14-214), sera, which showed fluorescence intensities for bound P-D-galactosidase activity over the tentative cutoff value of 40, were preincubated with excess of recombinant gag p24( 14-214) and subjected to the present assay (Tables 2 and 3). In most of sera, which were positive by both gelatin particle agglutination and ELISA using HTLV-I (group 1) or by all of gelatin particle agglutination, ELISA using HTLV-I, and Western blotting (group 2-1), the degree of inhibition by the preincubation was more than 80%. When the cutoff value was tentatively taken to be 73%, one serum in group 2-2-1 (serum no. 124) and three sera in group 3 (serum nos. 302-304) were negative, although these sera were positive on the basis of the fluorescence intensity for bound P-D-galactosidase activity by the present assay as described above.
DISCUSSION The results obtained by the present assay using recombinant gag p24( 14-214) may be summarized as follows:
1. The present assay was more sensitive than other methods. 2. Anti-p24 IgG was detected in most of positive sera by ELISA using HTLV-I and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I. Notably, anti-p24 IgG was detected in four sera (serum nos. 120-123) which were indeterminateor negative by Western blotting. 3. However, anti-p24 IgG was not detected in one serum in group 1 (serum no. 88) which was strongly positive by gelatin particle agglutination and ELISA using HTLV-I and in two sera in group 2- 1 (serum nos. 118 and 119) which were positive by gelatin particle agglutination, ELISA using HTLV-I, and Western blotting. 4. False-positiveresults for some sera in group 3 (serum nos. 302-304) which were negative by gelatin particle agglutination and ELISA using HTLV-I may be corrected by the inhibition degree with excess of recombinant gag p24( 14-214). When test results by various methods for serodiagnosis are inconsistent, it is difficult to decide which results are more reliable. This difficulty may be overcome at least in part by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. From such consideration, sensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) using three peptides, Cys-gag p 19(100130), Cys-env gp46( 188-224), and Ala-Cys-env gp46(237262), have been developed ( 14- 16). Since test results by these three assays were mostly consistent with those by other methods, the 305 sera used in this study were tested by these three assays. Eighty-eight sera in group 1 (serum nos. 1-88), 29 sera in group 2- 1 (serum nos. 9 1- 119), and 4 sera in group 2-2-1 (serum nos. 120-123) were positive by at least three of the four assays using the three peptides and recombinant gag p24( 14-214) and could be taken as positive with high probability, although some of these sera (serum nos. 88, 118, 119, and 120- 123) were negativehdeterminate by ELISA using HTLV-I, Western blotting, and/or the present assay. Two sera
Enzyme lrnrnunoassayfor Anti-HTLV-l IgG
in group 1 (serum nos. 89 and 90), 1 serum in group 2-2-1 (serum no. 124), 61 serain group 2-2-2 (serumnos. 125-183, 187, and 188), and 115 sera in group 3 (serum nos. 190-303 and 305) were negative by at least three of the four assays and could be taken as negative with high probability, although some of these sera (serum nos. 89,90, 125-183, 187, 188, 300, 301, and 305) were positive by gelatin particle agglutination, ELISA using HTLV-I, and/or the present assay. Two sera in group 2-2-2 (serum nos. 186 and 189) were positive by at least three of the four assays and could be taken as positive with high probability, although the two sera were negative by the present assay, negative/positive by ELISA using HTLV-I, and indeterminate by Western blotting. However, two sera in group 2-2-2 (serum nos. 184 and 185) and one serum in group 3 (serum no. 3 0 4 ) , which were positive by two of the four assays, required the use of many other different epitopes of HTLV-I as possible to improve the reliability of serodiagnosis. Thus, recombinant gag p24( 14-214) was suggested to be useful, when used with other peptides and/or recombinant proteins, for improving the reliability of serodiagnosis of HTLV-I infection by separately demonstratingantibodies against different epitopes of HTLV-I. Finally, recombinant gag p24( 14-214) remains to be replaced by an appropriate synthetic peptide(s), since it is not easy to completely separate recombinant proteins from proteins of E. coli, against which antibodies are often present in serum.
REFERENCES 1. Poiesz BJ, Ruscetti FW,Gazdar AF, Bunn PA, Minna JD, Gallo RC: Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. P roc NarlAcadSci USA 77:7415-7419, 1980. 2 . Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita K, Shirakawa S, Miyoshi I: Adult T-cell leukemia: Antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc NarlAcad Sci USA 786476-6480, 1981. 3. Gallo RC, Kalyanaraman VS, Sarngadharan MG, Sliski A, Vonderheid EC, Maeda M, Nakao Y, Yamada K, Ito Y, Gutensohn N, Murphy S, Bunn Jr PA, Catovsky D, Greaves MF, Blayney DW, Blattner W, Jarrett WFH, zur Hausen H, Seligmann M, Brouet JC, Haynes BF, Jegasothy BV, Jaffe E, Cossman J, Broder S, Fisher RI,Golde DW, Robert-Guroff M: Association of the human type C retrovirus with a subset of adult T-cell cancers. Cancer Res 43:3892-3899, 1983. 4. Okochi K, Sat0 H, Hinuma Y: A retrospective study on transmission of adult T cell leukemia virus by blood transfusion: Seroconversion in recipients. Vox Sang 46:245-253, 1984. 5 . Ando Y, Saito K, Nakano S, Kakimoto K, Furuki K, Tanigawa T, H a s h o t o H, Moriyama 1, Ichijo M, Toyama T Bottle-feedig can prevent transmission of HTLV-I from mothers to their babies. J Infect 19:25-29, 1989. 6. Ikeda M, Fujino R, Matsui T, Yoshida T, Komoda H, Imai J: A new agglutination test for serum antibodies to adult T-cell leukemia virus. Gann 75:845-848, 1984. I . Taguchi H, Sawada T, Fujishita M, Morimoto T, Niiya K, Miyoshi I: Enzyme-linked immunosorbent assay of antibodies to adult T-cell leukemia-associated antigens. Gann 74:185-187, 1983.
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8. Gallo D, Diggs JL, Hanson CV: Comparison of Western immunoblot antigens and interpretive criteria for detection of antibody to human T-lymphotmpic virus types I and II.J Clin Microbiol28:2045-2050, 1990. 9. Kannagi M, Sugamura K, Sat0 H, Okochi K, Uchino H, Hinuma Y: Establishment of human cytotoxic T cell lines specific for human adult T cell leukemia virus-bearing cells. J Immunoll302942-2946, 1983. 10. Miyoshi I, Kubonishi I, Yoshimoto S, Akagi T, Ohtsuki Y, Shiraishi Y, Nagata K, Hinuma Y: Type C virus particles in a cord T-cell line derived by co-cultivating normal human cord leukocytes and human leukaemic T cells. Nature 294:770-771, 1981. 1 1 . Kohno H, Kohno T, Sakoda I, Ishikawa E: Novel and sensitive enzyme immunoassay (immune-complex-transferenzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG in human serum using recombinant gag-env hybrid protein as antigen. JClin LabAnai4:355-362,19!33. 12. KugaT, Yamasaki M, Sekine S, Fukui M, Yokoo Y,Itoh S, YoshidaM, Hattori T, Takatsuki K: A gag-env hybrid protein of human T-cell leukemia virus type I and its application to serum diagnosis. Jpn J Cancer Res79:1168-1173,1988. 13. Kohno T, Sakoda I, Ishikawa E Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type 1) IgG in serum using a synthetic peptide, env gp46(188-209), as antigen. J Clin Lab Anal 5:25-37,1991. 14. Kohno T, Sakoda I, Suzuki M, Izumi A, Ishikawa E:Immune complex transfer enzyme immunoassay for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-gag p19(100-130), as antigen. J Clin Lab Anal 5:307-316, 1991. 15. Kohno T, Sakoda I, Ishikawa E: Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Cys-env gp46(188-224), asantigen. JClinLabAnal6:105-112, 1992. 16. Kohno T, Sakoda I, Ishikawa E Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-human T-cell leukemia virus type I) IgG in serum using a synthetic peptide, Ala-Cys-env gp46(237-262),as antigen. JClinLabAnal6:162-169, 1992. 17. Sekine S, Amoh T, Kuga T, Itoh S, Hattori S, Taniguchi T, Yoshida M: Expression of HTLV-I gag protein in Escherichia coli.AgricEiol Chem 52:1267-1271, 1988. 18. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage Ts. Nature 227:680-685, 1970. 19. Ishikawa E, Imagawa M, Hashida S, Yoshitake S, Hamaguchi Y, Ueno T Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J Immunoassq 4: 209-327, 1983. 20. Hashida S, Ishikawa E: Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay. J Biochem 108: 960-964,1990. 21. Kohno T, Kitamura T, Tsuda K, Ishikawa E: Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for alpha-fetoprotein from hepatocellular carcinoma. J Clin Lab Anal 4:183-188, 1990. 22. Kohno T, Ishikawa E: A highly sensitive enzyme immunoassay of antiinsulinantibodiesinguineapig serum. JEiochem 100:1247-1251, 1986. 23, lshikawa E, Kato K: Ultrasensitive enzyme immunoassay. Scand J Imrntinot 8(Suppl7):43-55, 1978. 24. Inoue S , Hashida S, Kohno T, Tanaka K, Ishikawa E: A micro-scale method for the conjugation of affinity-purifiedFab’ to P-D-galactosidase from Escherichia coli.JEiochern 98:1387-1394, 1985. 25. Imagawa M, Hashida S, Ohta Y, Ishikawa E: Evaluation of P-D-galactosidase from Escherichia coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique. Ann Clin Biochem 21:310317, 1984. 26. Hashida S, Imagawa M, Inoue S, Ruan K-h, Ishikawa E More useful maleimide compounds for the conjugation of Fab’ to horseradish
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27. Imagawa 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 Lett 16:1509-1523, 1983.
28. Maeda Y, Imai J, Kiyokawa H, Kanamura M, Hino S: Performance certification of gelatin particle agglutination assay for anti-HTLV-1 antibody: Inconclusive positive results. JpnJ CancerRes 80:915-919, 1989.
Sensitive Enzyme lmmunoassay (Immune Complex Transfer Enzyme Immunoassay) for (Anti-Human T-cell Leukemia Virus Type I) IgG in Serum Using Recombinant gag p24(14-214) as Antigen Takeyuki Kohno,’ Kouichi Hirota,’ lwane Sakoda,* Motoo Yarna~aki,~ Yoshiharu Yok00,~ and Eiji Ishikawa’
‘
Department of Biochemistry, Medical College of Miyazaki, Miyazaki, Miyazaki Blood Center, The Japanese Red Cross, Miyazaki,2 and Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Japan A sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (anti-humanT-cell leukemia virus type I) IgG (anti-HTLV-lIgG) in serum using recombinant gag p24 (14-214) of HTLV-I is described. The recombinant gag p24(14-214) is soluble in the absence of detergents and allows the use of enzymes other than horseradish peroxidase as a label in the assays. The usefulness of recombinant gag p24(14-214) was examined with 305 sera characterized by other methods including gelatin particle agglutination,enzyme-linked immunosorbent assay (ELISA) using HTLV-I, and Western blotting. This assay was more sensitive than other methods using HTLV-I as antigen. The specificity could be tested by preincubation of test serum with excess of the recombinant protein. Most of negative and positive sera were discriminated. However, some results appeared to be false-positiveor false-negative, and recombinant gag p24(14-214) was suggested to be useful, when used with other recombinant proteins and/or peptides, for Key words:
antibody, adult T-cell leukemia, p-D-galactosidase,Western blotting, gelatin particle agglutination, ELISA
INTRODUCTION Human T-cell leukemia virus type I (HTLV-I), isolated from a patient with cutaneous T-cell lymphoma (l), is etiologically associated with adult T-cell leukemia (2) and adult T-cell cancers (3). For prevention of the virus transmissions by blood transfusion (4) and breast-feeding ( 5 ) , plasma or serum of blood donors and pregnant women has been tested for antiHTLV-I antibodies by gelatin particle agglutination (6), enzyme-linked immunosorbent assay (ELISA) (7), fluoroimmunoassay (2), and/or Western blotting (8). HTLV-I used as antigen in these methods has been produced using particular cell lines such as TCL-Kan (9) and MT-2 (10) and purified by density gradient centrifugation (6) or by 0 1992 Wiley-Liss, Inc.
improving the reliability of serodiagnosis by separately demonstrating antibodiesagainst as many different epitopes of HTLV-I as possible. Anti-HTLV-l IgG in test serum, which had been incubated with excess of inactive p-0galactosidase to eliminate interference by antip-D-galactosidaseantibodies, was reacted simultaneouslywith2,4-dinitrophenyl-bovine serum albumin-recombinantgag p24(14-214) conjugate and recombinant gag p24(14-214)p-D-galactosidase conjugate. The complex formed consisting of the three components was trapped onto polystyreneballs coatedwith aff inity-purified(anti-2,4-dinitrophenylgroup) IgG. After washing to eliminate nonspecific IgG in the test serum and excess of the p-D-galactosidaseconjugate,the complex was eluted from the polystyrene balls with rN-2,4dinitrophenyl-L-lysineand transferred to polystyrene balls coated with affinity-purified (anti-human IgG y-chain) IgG. p-D-Galactosidase activity bound to the (anti-human IgG y-chain) IgG-coated polystyrene balls was s, assayedby fluorometry. o i 9 9 2 ~ i 1 e y - ~ i stnc.
ultracentrifugation (7). This is hazardous and inefficient. In addition, the specificity of these methods has not been tested by preincubation of serum with excess of HTLV-I as antigen. Recently, a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG has been developed using recombinant gag p24( 14-139)-env
Received March 2, 1992; accepted April 9, 1992. Address reprint requests to Dr. Eiji Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.
Enzyme lmmunoassay for Anti-HTLV-l IgG
gp46(217-315) hybrid protein as antigen ( I 1) which was safely and efficiently produced in Escherichia coli (12). This assay was 30-to 300-fold more sensitive than other methods including Western blotting using HTLV-I, gelatin particle agglutination using HTLV-I, and ELISA using the gag-env hybrid protein. Most of positive and negative samples were more clearly discriminated than by the other methods. Anti-HTLV-I IgG was demonstrated in all samples, which were positive by Western blotting. Low levels of anti-HTLV-I IgG below those detected by Western blotting were suggested to be detectable. However, the gag-env hybrid protein was soluble only in the presence of detergents. This hampered the use of enzymes other than horseradish peroxidase as label, and the specificity could not be tested by preincubation of serum with excess of the gag-env hybrid protein. More recently, the recombinant protein has been replaced by synthetic peptides including Cys-Arg-env gp46( 188-209) (13), Cys-gag p19(100-130) (14), Cys-env gp46(188-224) (15), and Ala-Cys-env gp46(237-262) (16). The specificity could be tested by preincubation of serum with excess of the synthetic peptides, which are readily soluble in the absence of detergents. The sensitivities were 30- to 30,OClo-fold higher than those of other methods such as Western blotting using four core proteins of HTLV-I, gelatin particle agglutination using HTLV-I, ELISA using the synthetic peptides, and HTLV-I. Most of negative and positive sera were discriminated. However, some results appeared to be false-negative or false-positive, and the use of as many synthetic peptides and recombinant proteins as possible was suggested to improve the reliability of serodiagnosis by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. This paper describes a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antiHTLV-I IgG using recombinant gag p24(14-214) which can be safely and efficiently produced in E . coEi and is soluble in the absence of detergents.
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 mmoYl sodium phosphate buffer, pH 7.0, containing 1.O mmol/l MgC12, 1.O g/1 NaN3, and 1.O g/l bovine serum albumin (fraction V, Amour Pharmaceutical Co., Kankakee, IL) (buffer B), and 10 mmol/l sodium phosphate buffer, pH 7.0, containing 1.O g/l bovine serum albumin (buffer C).
Recombinantgag p24(14-214) Recombinant gag p24(14-214) was produced in E . coli, which has been transformed with expression plasmid carry-
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ing cDNA cloned for gag p24( 14-214) of HTLV-I (17), and was purified by centrifugal precipitation, solubilization with urea, and cation exchange chromatography (12). The purity of this preparation was 97% as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate ( 18). The concentration of recombinant gag p24( 14-214) was determined by a commercial protein assay kit (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Richmond, CA) using bovine serum albumin as standard.
Antibodies Rabbit (anti-2,4-dinitrophenyl-bovineserum albumin) serum was obtained from Shibayagi Co., Ltd. (Gumma, Japan). Rabbit (anti-human IgG y-chain) IgG was obtained from Medical and Biological Laboratories Co., Ltd. (Nagoya, Japan). IgG was prepared from serum by fractionation with Na2S04 followed by passage through a column of diethylaminoethyl cellulose, and the amount of IgG was calculated from the absorbance at 280 nm (19).
2,4-Dinitrophenyl-Bovine Serum Albumin Thiol groups were introduced into bovine serum albumin molecules using N-succinimidyl-S-acetylmercaptoacetate(20) and were reacted with maleimide groups introduced into EN-2,4-dinitrophenyl-L-lysine molecules using N-succinimidyl-6-maleimidohexanoate(2 1). The amount of 2 ,Cdinitrophenyl-bovine serum albumin was calculated from the absorbance at 280 nm and 360 nm (13). The average number of 2,4-dinitrophenyl groups introduced per albumin molecule was 6.8.
Affinity Purification of Antibodies 2,4-Dinitrophenyl-bovineserum albumin (10 mg) and nonspecific human IgG (10 mg) were coupled to CNBr-activated Sepharose 4B (1.0 g, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) according to the instructions of the manufacturer. (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 nonspecific human IgGSepharose 4B, respectively (22). Protein-Coated Polystyrene Balls Polystyrene balls (3.2 mm in diameter, Immuno Chemical, Inc., Okayama, Japan) were coated by physical adsorption with affinity-purified (anti-2,4-dinitrophenyl-bovineserum albumin) IgG (0.1 g/l), affinity-purified (anti-human IgG y-chain) IgG (0.1 g/l), and recombinant gag p24(14-214) (0.1 g/l) (23). Affinity-purified (anti-2,4-dinitrophenyl-bovine serum albumin) IgG-coated polystyrene balls had been colored pink for discrimination from other polystyrene balls.
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2,4-Dinitrophenyl-Bovine Serum AlbuminRecombinantgag p24(14-214) Conjugate Maleimide-2,4-dinitrophenyl-bovineserum albumin
Mercaptoacetyl-recombinantgag p24(14-214) Mercaptoacetyl-recombinantgag p24( 14-214) was prepared as described above.
Maleimide groups were introduced into molecules of Recombinantgag p24(14-214)-P-D-galactosidase 2,4-dinitrophenyl-bovineserum albumin using N-succinim- conjugate idyl-6-maleimidohexanoate( 13). The average number of Maleimide-P-D-galactosidase (0.4 mg, 0.74 nmol) in 0.5 maleimide groups introduced per 2,4-dinitrophenyl-bovine ml of buffer A containing 0.35 mol/l urea was incubated with serum albumin molecule was 2.4 (19). mercaptoacetyl-recombinant gag p24( 14-214) (53 pg, 2.2 nmol) in 0.24 ml of the same buffer at 4°C overnight. SubseMercaptoacetyl-recombinantgag p24(14-214) quently, the reaction mixture was incubated with 10 yl of 0.1 Recombinant gag p24(14-214) (1.0 mg) in 0.7 ml of 0.1 mol/l 2-mercaptoethylamine in buffer A at 30°C for 15 min mol/l sodium phosphate buffer, pH 7.0, containing 5 mmol/l and 20 p1 of 0.1 mol/l N-ethylmaleimide in buffer A, and EDTA and 0.35 moll1 urea was incubated with 70 p1 of 11 subjected to gel filtration on a column ( 1.5 X 45 cm) of Ultrogel mmol/l N-succinimidyl-S-acetylmercaptoacetatein N,N-di- AcA 22 (IBF Biotechnics) using 10 mmol/l sodium phosmethylformamide at 30°C for 30 min. The reaction mixtures phate buffer, pH 7.0, containing 0.1 mol/l NaC1, 1.O mmoYl were further incubated with 70 pl of 1.O mol/l Tris-HCI buffer, MgC12, 1.0 g/l NaN3, 0.35 mol/l urea, and 0.1 g/l bovine pH 7.0, and 0.1 ml of 1.0 mol/l hydroxylamineHC1, pH serum albumin. The average number of recombinant gag 7.0, at 30°C for 15 min and subjected to gel filtration on a p24( 14-214) molecules conjugated per P-D-galactosidase molcolumn (1 .O X 30 cm) of Sephadex G-25 (Pharmacia LKB ecule was 2.8, which was calculated from the decrease in the Biotechnology AB) using buffer A containing 0.35 moVl urea. number of maleimide groups ( 19). The amount of the conjuThe average number of thiol groups introduced per recombi- gate was calculated from P-D-galactosidase activity (24). nant gag p24( 14-214) molecule was 3.0 (19). Immune Complex Transfer Enzyme lmmunoassay 2,4-Dinitrophenyl-bovineserum albumin-recombinantgag Using Recombinantgag p24(14-214) p24(14-214) conjugate Test serum (20 yl) was incubated with 50 pg of inactive
Maleimide-2,4-dinitrophenyl-bovine serum albumin (0.16 mg, 2.4 nmol) in 0.1 ml of buffer A containing 0.35 mol/l urea was incubated with mercaptoacetyl-recombinant gag p24(14-214) (0.29 mg, 12 nmol) in 0.38 ml of the same buffer at 4°C overnight. After incubation, the reaction mixture was incubated with 5 y1 of 0.1 mol/l 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 and subjected to gel filtration on a column (1.5 X 100 cm) of Ultrogel AcA 34 (IBF Biotechnics, Villeneuve-la-Garenne, France) using 10 mmol/l sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl, 1.O mmol/l MgC12, 1.O g/l NaN3, 0.35 moYl urea, and 0.1 g/l bovine serum albumin. The average number of recombinant gag p24( 14-214) molecules conjugated per albumin molecule was 2.4, which was calculated from the decrease in the number of maleimide groups (19). The amount of the conjugate was calculated from that of 2,4-dinitrophenyl-bovineserum albumin.
Recombinantgag p24(14-214)-p-D-Galactosidase Conjugate Maleimide-p-D-galactosidase Maleimide groups were introduced into molecules of P-D-galactosidase (EC 3.2.1.23) from E . coli using N,N’-l,2phenylenedimaleimide (19).
P-D-galactosidase(P-GalactosidaseMutein, Boehringer Mannheim GmbH, Mannheim, Germany) in 30 y1 of buffer B containing 0.1 moYl NaCl at 20°C for 3 hr to eliminate interference by anti-P-D-galactosidase antibodies (13). In some experiments, recombinant gag p24(14-214) (36 pg, 15 pmol) was added to test the specificity. After incubation, the reaction mixture was incubated simultaneously with 2,4-dinitrophenylbovine serum albumin-recombinant gag p24( 14-214) conjugate (100 fmol) and recombinant gag p24(14-214)-P-Dgalactosidase conjugate (100 fmol) in 0.1 ml of buffer B containing 0.55 mol/l NaCl at 20°C for 3 hr. Two affinitypurified (anti-2,4-dinitrophenyl-bovine serum albumin) IgGcoated polystyrene balls, which had been colored pink, were added to the reaction mixture, and the incubation was continued at 20°C overnight. After incubation, the colored polystyrene balls were washed twice with 2 ml of buffer B containing 0.1 mol/l NaCl and incubated with 0.15 ml of 1.O mmol/l ~N-2,4-dinitrophenyl-Llysine (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in the same buffer and two affinitypurified (anti-human IgG y-chain) IgG-coated polystyrene balls at 20°C for 1 hr. After incubation, the colored polystyrene balls were discarded, and the eluate and the (anti-human IgG y-chain) IgG-coated polystyrene balls were further incubated at 20°C for 2 hr. After washing as described above, P-D-galactosidase activity bound to the polystyrene balls was assayed at 30°C for 150 min by fluorometry using 4-methyl-
Enzyme lmmunoassay for Anti-HTLV-l IgG
umbelliferyl-P-D-galactoside as substrate (25). The fluorescence intensity was measured relative to moll1 4methylumbelliferone in 0.1 moYl glycine-NaOH buffer, pH 10.3 (25).
Immune Complex Transfer Enzyme lmmunoassay Using Synthetic Peptides The immune complex transfer enzyme immunoassays using the synthetic peptides Cys-gag p19( l00-130), Cys-env gp46(1 88-224), and Ala-Cys-env gp46(237-262) were performed as described previously (14- 16).
ELISA Rabbit (anti-human IgG y-chain) IgG was converted to the corresponding Fab' and conjugated to horseradish peroxidase using N-succinimidyl-6-maleimidohexanoate(26). The amount of the conjugate was calculated from peroxidase activity (19). In ELISA using recombinant gag p24( 14-214), test serum (20 p1) was mixed with 0.13 ml of buffer C containing 0.46 moYl NaCl and 1.O s/l NaN3, and was incubated with a recombinant gag p24( 14-214)-coated polystyrene ball at 37°C for 3 hr and at 4°C overnight. After incubation, the polystyrene ball was washed twice with 2 ml of 10 mmoVl sodium phosphate buffer, pH 7.0, containing 0.1 mol/l NaCl, and was incubated with 50 ng of (anti-human IgG y-chain) Fab'peroxidase conjugate in 0.15 ml of buffer C containing 0.1 mol/l NaCl at 37°C for 3 hr. The polystyrene ball was washed as described above, and bound peroxidase activity was assayed at 30°C for 10 min by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor (27). The fluorescenceintensity was measured relative to 1.O mg/l quinine in 50 mmol/l HZS04 (27). ELISA using HTLV-I as antigen was performed with a commercial kit (Eitest-ATL, Eisai Co., Ltd., Tokyo, Japan). Microtiter plates coated with HTLV-I produced by MT-2 cell line (10) were incubated with 20 pl of test sera and, after washing, with monoclonal (anti-human IgG Fc) antibodyalkaline phosphatase conjugate. Bound alkaline phosphatase activity was assayed by colorimetry using 4-nitrophenylphosphate as substrate.
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Gelatin Particle Agglutination Gelatin particle agglutination was performed using a commercial kit with HTLV-I produced by TCL-Kan cell line (9) as antigen (SERODIA-ATLA, Fujirebio, Inc., Tokyo, Japan). Test serum was diluted eightfold or more with the diluent included in the kit. The diluted serum (25 pl) was mixed with the particle solution (25 pl) in U-shaped wells of microplates and allowed to stand at room temperature for 3 hr.
Western Blotting Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I produced by TCLKan cell line (9) was generously performed by Fujirebio, Inc.
Serum Samples Serum samples were obtained from healthy subjects aged 16-79 years in Miyazaki, Japan. Approximately 7% of healthy subjects were HTLV-I carriers.
RESULTS Recombinant gag p24( 14-214) was conjugated to 2,4-dinitrophenyl-bovine serum albumin and P-D-galactosidase and tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) as shown schematically in Figure 1 (the present assay).
p+-
+ @ + e
Anti-DNPsolid phase
Ag-Enz
U0 0
DNP-lysine
Anti-lgG-solid phase
Expressionof the Detection Limit of Anti-HTLV-l IgG in Serum The detection limit of anti-HTLV-I IgG in serum by enzyme immunoassay was expressed as the maximal dilution of serum containing anti-HTLV-I IgG with pooled normal serum which gave a bound enzyme activity significantly in excess of that in the presence of normal serum (background). A significant difference from the background was confirmed by the t-test (n = 5, P < 0.001).
Fig. 1. Immune complex transfer enzyme immunoassay for antibody IgG. DNP, 2,4-dinitrophenyl group; Ag, antigen; Ab, antibody; Enz, enzyme.
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The present assay was 10- to 10,000-foldmore sensitive than the other methods.
Assay Variation The assay variation in the present assay was examined using serum samples, with which three different levels of the fluorescence intensity for bound P-D-galactosidase activity (16, 61, and 800 for within-assay and 9.6, 97, and 1,200 for between-assay) were obtained. The coefficients of variation for within-assay and between-assay were 5.6-9.0% (n = 12) and 8.6-12% (n = 6), respectively.
Usefulness of the Present Assay Using Recombinant gag p24(14-214) 0.1
40.1
104
w
103
lo2
10'
0"
10'
Dilution of Serum Containing Anti-HTLV-l IgG with Normal Serum ( -fold )
Fig. 2. Sensitivity of various methods. Four sera from HTLV-I carriers were serially diluted with normal serum and subjected to the immune complex transfer enzyme immunoassay using recombinant gag p24( 14-214) (the present assay) (open symbols with solid lines), ELISA using recombinant gag p24( 14-214) (closed symbols with solid lines), and ELISA using HTLV-I (closed symbols with broken lines).
Sensitivity Four sera from HTLV-I carriers were serially diluted with normal serum and were subjected to the present assay using recombinant gag p24( 14-214),ELISA using recombinant gag p24(14-214), ELISA using HTLV-I (Fig. 2), gelatin particle agglutination using HTLV-I, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I (Table 1).
The usefulness of the present assay using recombinant gag p24(14-214) was examined using 305 sera, which had been tested by other methods including gelatin particle agglutination, ELISA using HTLV-I, and Western blotting. Sera, for which the fluorescenceintensity for bound P-D-galactosidase activity by the present assay was over 40, were tentatively taken as positive, and other sera were taken as negative. The results were shown in Figure 3 and Tables 2 and 3. The 305 sera consisted of three groups tested by gelatin particle agglutination, which has been most widely used for screening of HTLV-I carriers in Japan: 90 strongly positive sera (group 1, serum nos. 1-90), in which the maximal dilution with the diluent included in the kit to cause gelatin particle agglutination was 128- to 16,384-fold,99 weakly positive sera (group 2, serum nos. 91-189), in which the maximal dilution was 8- to 64-fold, and 116 negative sera (group 3, serum nos. 190-305). In group 1 (90 sera, serum nos. 1-90), 87 sera (serum nos. 1-87) were consistently positive by gelatin particle agglutination, ELISA using HTLV-I, and the present assay. How-
TABLE l. Sensitivity of Various Methodsa Detection of anti-HTLV-I antibodies or IgG Method Present assay using recombinant gag p24( 14-214)
Gelatin particle agglutination using HTLV-I
Westem blotting using four core proteins of HTLV-I
Serum no. 1 2 3 4 1 2 3 4 1 2 3 4
Dilution of serum containing anti-HTLV-I antibodies with normal serum (-fold) 1 x 104 3 x 103 1 x lo2 3 x 10 1 x 10
+ + +
+ + +
+ + + +
-
-
-
-
-
f f 2
-
+-
''
-
-
-
-
-
-
-
f
-
-
-
-
5 -
"Four sera from HTLV-I carriers were diluted with normal serum and subjected to the three methods indicated.
+ + + + + + +
+ + + + + + + + + f +
-
f
3
+ + + + + + + + + + + +
Enzyme lmmunoassay for Anti-HTLV-l IgG
0 0 0 0
A
0 0
A
0
t
U
1
O
O
l
'
Negative
I
' ' " o m a dL 10 lo2 lo3 104 Maximal Dilution of Serum with Buffer to Cause Gelatin Particle Agglutination ( -fold) '"11111'
1
1
~
1
~
~
~
'
Fig. 3. Comparison of test results for anti-HTLV-I IgC or antibodies by the immune complex transfer enzyme immunoassay using recombinant gag p24(14-214) (the present assay), gelatin particle agglutination using HTLV-I, and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I. A tentative cutoff value of 40 in the present assay is indicated by broken line. Open circles indicate groups 1 and 3, and closed triangles, open squares, and open triangles indicate groups 2-1, 2-2-1, and 2-2-2, respectively, as shown in Tables 2 and 3. Group 2-1 was positive and groups 2-2-1 and 2-2-2 were negativehdetenninate by Western blotting.
307
ever, one serum (serum no. 88) appeared to be false-negative by the present assay, and two sera (serum nos. 89 and 90), which were negative by ELISA using HTLV-I, Western blotting, and the present assay, appeared to be false-positive by gelatin particle agglutination as described in the Discussion. Group 2 (99 sera, serum nos. 91-189) was divided into two groups by Western blotting: 29 positive sera (group 2- 1, serum nos. 91- 119)and 70 negativehndeterminatesera (group 2-2, serum nos. 120- 189). In group 2-1 (29 sera, serum nos. 91-1 19), 27 sera (serum nos. 91-1 17) were consistently positive by gelatin particle agglutination, ELISA using HTLV-I, Western blotting, and the present assay. However, two sera (serum nos. 118 and 119), which were positive by gelatin particle agglutination, ELISA using HTLV-I, and Western blotting, appeared to be false-negative by the present assay as described in the Discussion. Group 2-2 (70 sera, serum nos. 120- 189) was divided into two groups by the present assay: 5 positive sera (group 2-2- 1, serum nos. 120- 124)and 65 negative sera (group 2-2-2, serum nos. 125-189). In group 2-2-1 (five sera, serum nos. 120-124), all the sera were positive by the present assay, but were positive, negative, or indeterminate by ELISA using HTLV-I or Western blotting. Notably, high fluorescence intensities for bound P-D-galactosidase activity were observed with some sera (serum nos. 120-122), which were indeterminate by Western blotting. In group 2-2-2 (65 sera, serum nos. 125-189), all the sera were negative by Western blotting and the present assay. The
TABLE 2. Test Results by Various Methods for Groups 1,2-1, and 2-2-1
Group Group 1 (90sera)
Serum no. 1-87 88 89
90 Group 2 OrOup2-1 (29 sera) Group 2-2-1
(5sera)
91-117 118; 119 120 121 122 123 124
Gelatin particle agglutination using HTLV-I +++a
+++ +++ +++ + + + + + + +
Optical density at 405 nm by ELISA using HTLV-I with cutoff valueof0.62 1.9-14 3.1 0.21
Western blotting using four 'Ore proteins Of HTLv-l p19 p24 p28 p53
NTb NT
NT NT
NT NT
0.16
+
-
-
-
2.7; 1.1-15 1.2; 1.5
+ +
+ +
+ +
+ +
+
*
*
2
*
5.5 1.3 0.94 0.17 0.13
2
+
-
NT NT
+
-
-
-
-
-
?
-
-
*
4
-
Fluorescence intensity for bound P-D-galactosidase activity by the present assay using recombinant gag p24( 14-214) with a cutoff value of 40
Inhibition by excess of recombinant gag p24( 14-214)with a cutoff value of 73(%)
230-2,300,OOO 25 7.1 14
81-100 NT
150; 49-46,OOO
74; 84-100
22; 31 38,000 140 2,100 64 51
NT
NT NT
100 89 99 93 40
"The maximal dilution of serum samples with the diluent, included in the kit to cause gelatin particle agglutination was 128- to 16,384-fold for serum nos. 1-90and 16-to 64-fold for serumnos. 91-124. bNT, not tested.
308
Kohno et el.
TABLE 3. Test Results by Various Methods for Groups 2-2-2, and 3
Group Group 2 Group 2-2-2 (65 sera) Group 3 ( 1 16 sera)
Serum no. 125- 186 187; 188 189 190-299 300; 301 302-304 305
Gelatin particle agglutination using HTLV-I
+= +
+
-
-
Optical density at 405 nm by ELlSA using HTLV-I with cutoff valueof0.62
Westem blotting using four core proteins Of HTLv-l p19 p24 p28 p53
0.10-0.58
-
-
1.1;0.82 I .3 0.12-0.43 0.69; 0.97 0.19-0.46 0.14
-
-
+ NT NT NT NT
-
-
-
-
-
NT NT NT NT
NT NT NT NT
NT NT NT NT
-
Fluorescence intensity for bound P-D-galactosidase activity by the present assay using recombinant gag p24( 14-214) with a cutoff value of 40
Inhibition by excess of recombinant gag p24(14-214) with a cutoff value of 73(%)
5.7-35 12; 19 33 3.0-38 22; 11 64-99 1.ooo
NT~ NT NT NT NT 34-70 95
"The maximal dilution of serum samples with the diluent included in the kit to cause gelatin particle agglutination was 8- to 64-fold for serum nos. 125-189. bNT, not tested
results by gelatin particle agglutination for most of these sera might have been false-positive, which was consistent with a previous report that the gelatin particle agglutination kit used in this study often gave false-positiveresults (28). However, some of these sera should be examined more carefully for reliable results as described in the Discussion. In group 3 (1 16 sera, serum nos. 190-305), 110 sera (serum nos. 190-299) were consistently negative by gelatin particle agglutination, ELISA using HTLV-I, and the present assay. However, six sera (serum nos. 300-305) appeared to be falsepositive by ELISA using HTLV-I or the present assay.
Specificity of the Present Assay Using Recombinantgag p24(14-214) In order to test the specificity of the present assay using recombinant gag p24( 14-214), sera, which showed fluorescence intensities for bound P-D-galactosidase activity over the tentative cutoff value of 40, were preincubated with excess of recombinant gag p24( 14-214) and subjected to the present assay (Tables 2 and 3). In most of sera, which were positive by both gelatin particle agglutination and ELISA using HTLV-I (group 1) or by all of gelatin particle agglutination, ELISA using HTLV-I, and Western blotting (group 2-1), the degree of inhibition by the preincubation was more than 80%. When the cutoff value was tentatively taken to be 73%, one serum in group 2-2-1 (serum no. 124) and three sera in group 3 (serum nos. 302-304) were negative, although these sera were positive on the basis of the fluorescence intensity for bound P-D-galactosidase activity by the present assay as described above.
DISCUSSION The results obtained by the present assay using recombinant gag p24( 14-214) may be summarized as follows:
1. The present assay was more sensitive than other methods. 2. Anti-p24 IgG was detected in most of positive sera by ELISA using HTLV-I and Western blotting using four core proteins (p19, p24, p28, and p53) of HTLV-I. Notably, anti-p24 IgG was detected in four sera (serum nos. 120-123) which were indeterminateor negative by Western blotting. 3. However, anti-p24 IgG was not detected in one serum in group 1 (serum no. 88) which was strongly positive by gelatin particle agglutination and ELISA using HTLV-I and in two sera in group 2- 1 (serum nos. 118 and 119) which were positive by gelatin particle agglutination, ELISA using HTLV-I, and Western blotting. 4. False-positiveresults for some sera in group 3 (serum nos. 302-304) which were negative by gelatin particle agglutination and ELISA using HTLV-I may be corrected by the inhibition degree with excess of recombinant gag p24( 14-214). When test results by various methods for serodiagnosis are inconsistent, it is difficult to decide which results are more reliable. This difficulty may be overcome at least in part by separately demonstrating antibodies against as many different epitopes of HTLV-I as possible. From such consideration, sensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) using three peptides, Cys-gag p 19(100130), Cys-env gp46( 188-224), and Ala-Cys-env gp46(237262), have been developed ( 14- 16). Since test results by these three assays were mostly consistent with those by other methods, the 305 sera used in this study were tested by these three assays. Eighty-eight sera in group 1 (serum nos. 1-88), 29 sera in group 2- 1 (serum nos. 9 1- 119), and 4 sera in group 2-2-1 (serum nos. 120-123) were positive by at least three of the four assays using the three peptides and recombinant gag p24( 14-214) and could be taken as positive with high probability, although some of these sera (serum nos. 88, 118, 119, and 120- 123) were negativehdeterminate by ELISA using HTLV-I, Western blotting, and/or the present assay. Two sera
Enzyme lrnrnunoassayfor Anti-HTLV-l IgG
in group 1 (serum nos. 89 and 90), 1 serum in group 2-2-1 (serum no. 124), 61 serain group 2-2-2 (serumnos. 125-183, 187, and 188), and 115 sera in group 3 (serum nos. 190-303 and 305) were negative by at least three of the four assays and could be taken as negative with high probability, although some of these sera (serum nos. 89,90, 125-183, 187, 188, 300, 301, and 305) were positive by gelatin particle agglutination, ELISA using HTLV-I, and/or the present assay. Two sera in group 2-2-2 (serum nos. 186 and 189) were positive by at least three of the four assays and could be taken as positive with high probability, although the two sera were negative by the present assay, negative/positive by ELISA using HTLV-I, and indeterminate by Western blotting. However, two sera in group 2-2-2 (serum nos. 184 and 185) and one serum in group 3 (serum no. 3 0 4 ) , which were positive by two of the four assays, required the use of many other different epitopes of HTLV-I as possible to improve the reliability of serodiagnosis. Thus, recombinant gag p24( 14-214) was suggested to be useful, when used with other peptides and/or recombinant proteins, for improving the reliability of serodiagnosis of HTLV-I infection by separately demonstratingantibodies against different epitopes of HTLV-I. Finally, recombinant gag p24( 14-214) remains to be replaced by an appropriate synthetic peptide(s), since it is not easy to completely separate recombinant proteins from proteins of E. coli, against which antibodies are often present in serum.
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