Vox Sang. 35: 219-223 (1978)

Sandwich Enzymoimmunoassay of Hepatitis B Surface Antigen (HBsAg) Hidenari A dachi, Toshiaki Fukuda, Shuji Funahashi, Tomohiro Kurahori and Eiji Ishikawa Meguro Institute, Osaka National Hospital; Research Institute for Microbial Diseases, Osaka University, and Department of Biochemistry, Medical College of Miyazaki, Miyazaki

Abstract. A sandwich enzymoimmunoassay (EIA) procedure was developed for the detection of HBsAg using Fab’ of anti-HBsAg conjugated with p-D-galactosidase from Escherichia coli with anti-HBsAg-coated silicone rubber discs as a solid phase. EIA could detect 1 ng/ml of HBsAg. It was as sensitive as radioimmunoassay (RIA, AusRia 11) and about 30-fold more sensitive than reversed passive hemagglutination assay RPHA, Reversecell). EIA and RIA could detect more HBsAg-positive sera than RPHA.

Introduction Since the discovery of the relationships between hepatitis B surface antigen (HBsAg) and serum hepatitis (type B), various immunological methods for the detection of HBsAg have been described. A survey of these methods is given in the WHO Technical Report (No. 602) entitled ‘Viral Hepatitis’ (1977). Radioimmunoassay (RIA) is the most sensitive. However, it has some disadvantages: these are the hazard and short life of radionuclides used and the high cost of assay kit and equipment. To overcome these disadvantages, enzymoimmunoassay (EIA) procedures have been developed [14]. The present paper describes a sandwich EIA procedure for the detection of HBsAg. The enzyme used is P-D-galactosidase from Escherichia coEi conjugated with Fab’ fragments of rabbit anti-HBsAg, and

the solid phase used is a silicone rubber disc coated with IgG of rabbit anti-HBsAg.

Materials and Methods Purified HBsAg (subtype adr) was kindly provided from the Virology Division, National Cancer Center Research Institute, Tokyo. It was obtained from human sera by affinity chromatography [41 and purified by ultracentrifugation in CsCI, [l]. Its concentration was estimated by the absorbance at 280 nm (El.$$ = 30) 1101. Antiserum against HBsAg was obtained by repeated injections of 2 female rabbit with purified HBsAg (adr) in incomplete Freund’s adjuvant at intervals of a month until a reciprocal titer of antibody to HBsAg of 106 or more in the passive hemagglutination test [12] was attained. The antiserum was absorbed three times with 1/20vol of human serum which was shown to be free of HBsAg by RIA. After the absorption an IgG fraction was prepared by fractionation with (NH,),SO, [6] followed by passage through a column of

220

DEAE-cellulose [8]. Purified anti-HBsAg was obtained by affinity chromatography. After the absorption the antiserum applied to an affinity column of purified HBsAg coupled to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals AB, Uppsala). The column had been equilibrated with 0.01 M Tris-HC1 buffer, pH 7.4, and antiHBsAg was eluted with 5 M MgCI, and dialyzed against the same buffer. The amount of IgG was determined by the absorbance at 280 nm (E!& -15) ~91. The IgG fraction of anti-HBsAg, which was not purified by affinity chromatography, was digested with pepsin followed by reduction with 2-mercaptoethylamine to obtain Fab’ fragments as described previously [6]. The Fab’ fragments were treated with excess N,N-o-phenylenedimaleimide (Aldrich Chemicals Company, Inc., Milwaukee) and the maleimide residues introduced into the Fab’ fragments were allowed to react with sulfhydryl groups of B-D-galactosidase from E. coli (Boehringer, Mannheim) [ 5 ] . The amount of the conjugate was expressed as units of fl-D-galactosidase activity, and 1U of the activity is defined as that which forms 1 pmol of 4-methylumbelliferone/min when the enzyme was incubated with 0.1 mM 4-methylumbelliferyl-~-Dgalactoside at 30 OC as described previously [S]. Silicone rubber discs ( 5 mm in diameter and 3 mm thick; Yamada Rubber Co., Ltd., Osaka) were coated with anti-HBsAg by simple adsorption as described previously [3]. The discs were washed with a nonionic detergent, immersed into a solution of the anti-HBsAg (50pg/ml 0.25M sodium phosphate buffer, pH 7.5) which had been purified by affinity chromatography, and washed with 0.01 M sodium phosphate buffer, pH 7.0 containing 0.1 M NaCI, 1 mM MgCI,, 0.1% bovine serum albumin (fraction V, Armour Pharmaceutical Co., Chicago) and 0.1% NaN, (buffer A). The discs were stored in buffer A until use. EIA Procedure EIA was performed by a sandwich procedure essentially in the same manner as described previously [5]. Various amounts of HBsAg in 0.2 ml of buffer A or 0.2 ml of serum to be tested were incubated with anti-HBsAg discs at 37 OC for 6 h with shaking, followed by standing at 4 OC overnight. Each disc was washed with 1 ml of buffer A and incubated with 5,000 p U of anti-HBsAg-fl-D-

Adachi/Fukuda/Funahashi/Kurahori/Ishi kawa

galactosidase in 0.2 ml of buffer A at 37 O C for 4 h with shaking. After incubation, each disc was washed with 1.0ml of buffer A and transfered to another tube. To assay B-D-galactosidase activity bound, each disc was incubated with 0.1 mM 4methylumbelliferyl-8-D-galactoside in 0.2 ml of buffer A with shaking at 3OoC for 20min. The enzyme reaction was stopped by adding 2.5 ml of 0.1 M glycine-NaOH buffer, pH 10.3 and 4-methylumbelliferone formed was determined fluorometrically. The wave lengths for excitation and emission analysis were 360 and 450nm, respectively. R I A cind Reversed Passive Henmgglutinntion (RPHA) RIA and RPHA were performed using AusRIA I1 (Abbot Laboratories, Chicago) and Reversecell (Yamanouchi Pharmaceutical Co., Ltd., Tokyo), respectively, according to the instruction of the manufacturers.

Results Measurable Range, Serum Interference, Specificity and Sensitivity (fig. 1) Purified HBsAg (adr) was diluted 100fold or more with either buffer A or 5 normal human sera which were shown to be free of HBsAg by RIA, and subjected to EIA. P-D-Galactosidase activity bound to the solid phase increased as the amount of HBsAg increased, and the measurable range was between 1 and 100 ng/ml. The addition of 5 normal human sera produced little difference in the binding of the enzyme activity both in the presence and absence of HBsAg. A typical result of such experiments is illustrated in figure 1. Furthermore, 60 human serum samples which were shown to be free of HBsAg by RIA were subjected to EIA without adding purified HBsAg and P-D-galactosidase activity bound was 4.6 f0.12 p U (meanf SE). This value is even lower than 5.0 kO.00 pU obtained from 6

Enzyrnoinirnunoassay of HBsAg

22 1

Ii E

4

s

c ._ c

HBsAg, respectively, while the corresponding ratio in EIA was 1.4, 7.0, 55.8 and 116. So, EIA was as sensitive as RIA. In RPHA, the reciprocal of highest dilution giving positive result was 64 against 1 puglml of purified HBsAg. Normal serum interfered with RPHA and significant results were obtained only after diluting 4.5-fold or more with buffer. So, EIA was about 30-fold more sensitive than RPHA.

.-

lo*

3 . . .

3 -

0 1 HBsAg, nglrnl

10

10’

2

103

Fig. 1. Calibration curves of HBsAg by EIA and RIA. Purified HBsAg was subjected to EIA in the presence (A) or absence (0)of normal human serum or after the incubation with antiHBsAg (m). 0= RIA.

experiments in the absence of serum. These findings indicate that normal human serum did not interfere with EIA and that EIA did not measure normal components in human scrum but measured specifically HBsAg. To test further the specificity of EIA, purified HBsAg was diluted with normal human serum containing 50 pl/ml of antiHBsAg IgG and incubated at 37°C for 2 h, and subjected to EIA. P-D-Galactosidase activity bound to the solid phase did not increase with increasing amounts of HBsAg added, confirming the specificity of the present EIA. For comparison, various amounts of purified HBsAg in buffer A were subjected to RIA. The ratio of the radioactivity bound to the solid phase in the presence of HBsAg to that in its absence was 1.1, 5.0, 28.5 and 41.0 for 1, 10, 100 and 1,00Ong/ml of

Detection of HBsAg in Sera from Patients The serum panel consisted of 119 sera from 101 patients with suspected hepatitis B infection, or recovering from hepatitis B, or suffering from liver diseases. These samples were subjected to EIA, RIA and RPHA. The results of EIA and RIA were expressed as the ratio of the enzyme activity or radioactivity bound to the solide phase in the presence of sera to be tested to that in the presence of control serum which was attached to the RIA kit. When sera showing the ratio of over 2.1 were taken as positive, 60 samples out of 119 were shown to be negative by EIA, RIA and RPHA and 49 samples were positive by all the methods. The other 10 samples were shown to be positive by RIA and negative by RPHA. Six of these were negative and four were positive by EIA (table I). So, these 10 samples were again subjected to EIA and RIA. Three out of these 10 were shown to be positive by both EIA and RIA. Five were negative by both EIA and RIA. One was negative by RIA and positive by EIA. The last one was positive by RIA and negative by EIA. In summary (tableII), 49 and 65 were positive and negative, respectively, by all the three methods. Three were positive by both EIA and RIA. Two were positive

Adachi/Fukuda!Funahashi/Kurahori/Ishikawa

222

Table 1. Comparison of 10 of 119 patient sera giving equivocal results by EIA, RIA and RPHA Serum No.

Ratio

RPHA

EIA

1 2 3 4 6 8 9 16 19 40

RIA

1st

2nd

preincubated with anti-HB,Ag

1st

2nd

1.o

1 .o 1 .o 1.4 2.1 1.6 4.8 2.4 5.6 1.8

_.

2.3 2.3 2.3 2.3 3.6 2.5 4.6 2.7 9.2 8.4

1 .o 1.3 1 .o 1.3 1.3 10.5 9.9 6.7 1.5 2.3

1 .o 1.2 1.2 1.5 8.0 2.4 5.0 1.2 2.1

1.o 1.o 1.o 1.1

1.o 1 .o 1.1 1 .o

1.6

Table II. Detection of HBsAg in 119 sera by EIA, RIA and RPHA EIA

RIA

RPHA

+ + +

+ + + -

+ -

-

-

-

-

First assay

Second assay

49 4 6

49 3 1 I

0 60

65

either by EIA or RIA. The correlation coefficient between RIA and EIA for 49 samples which were shown to be positive by all the methods and 10 samples subjected to the second assays was calculated to be 0.92.

Discussion EIA was demonstrated to be as sensitive as RIA and much more sensitive than RPHA. In the present experiments, anti-HBsAg was purified for the preparation of the solid phase but not for the preparation of antiHBsAg-p-D-galactosidase conjugate. With

neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.

purified anti-HBsAg for both the solide phase and conjugate, EIA may became much more sensitive. The sensitivity of the present EIA cannot be compared directly with that of other EIA that have been reported [2, 7, 13, 151, since the sensitivity of EIA with purified HBsAg has not been described. EIA with antibodies against different subtypes remains to be tested with reference to the sensitivity of the assay for different subtypes. EIA described in the present paper took 6 hour-incubation with HBsAg at 37 "C and 4 hour-incubation with anti-HBsAg-PD-galactosidase, while RIA requires 2 and 1 h for incubation with HBsAg and radiolabelled anti-HBsAg, respectively. (It has recently been shown that in EIA 2 hourincubation with HBsAg at 40 "C in place of 6 hour-incubation gives the same sensitivity as described above.) Moreover, EIA requires more than 20 min for enzyme assay, while radioactivity can be measured automatically. However, fluorometers are usually much less expensive than automatic radiocounters.

Enzymoimmunoassay of HBsAg

The ratio of P-D-galactosidase activity bound in the presence of samples to that in the presence of control serum was used for expressing results of EIA, and the ratio of over 2.1 was taken as positive tentatively as in RIA. However, the ratio less than 2.1 does not always correspond to the absence of HBsAg. Eight samples in table I which showed smaller ratios but over 1.O were subjected to inhibition tests. Samples were preincubated with anti-HBsAg at 37 "C for 2 h and then subjected to EIA. As a result, the ratio of all the samples tested decreased to 1.0 or 1.1. So, sera No. 3, 4,6, 19 and 40 in table I are considered to be positive, although the ratio is less than 2.1. Repeated assays in combination with inhibition test may detect more positive sera, although it remains to be decided carefully what ratio should be taken as positive in routine assays.

Acknowledgements We are grateful to Dr. T. Sekine, National Cancer Center Research Institute and Drs. Ozawa and Y . Yamashila, Meguro Institute for their kind criticism and advices.

References 1 Bond, H. E. and Hall, W. R.: Separation and purification of hepatitis-associated antigen into morphologic types by zonal ultracentrifugation. J. infect. Dis. 125: 263-268 (1972). 2 Castro, A.; Wu, M.; Risenthal, C.; Cleary, T.; Wunsch, C., and Malkus, H.: Enzyme immunoassay of hepatitis associated antigen (HAA). Res. Commun. Chem. Pathol. Pharmacol. 16: 199-202 (1977). 3 Hamaguchi, Y.; Kato, K.; Ishikawa, E.; Kobayashi, K., and Katunuma, N.: Enzyme-linked sandwich immunoassay of macromolecular antigens using the rabbit antibody-loaded silicone piece as a solid phase. FEBS Lett. 69: 11-14 (1976).

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4 Jozwiak, W. and Koscielak, J.: Affinity chromatography of RNA-containing preparation of Au antigen. J. Immun. 110: 1151-1153 (1973). 5 Kato, K.; Fukui, H.; Hamaguchi, Y., and Ishikawa, E.: Enzyme-linked immunoassay. Conjugation of Fab' fragment of rabbit IgG with ,4-D-galactosidase from E.coZi and its use for immunoassay. J. Immun. 116: 1154-1160 (1976). 6 Kato, K.; Hamaguchi, Y.; Fukui, H., and Ishikawa, E.: Coupling Fab' fragment of rabbit anti-human IgG antibody to @-D-galactosidase and a highly sensitive immunoassay of human IgG. FEBS Lett. 56: 370-372 (1975). 7 Kekwich, R. A.: The serum protein in multiple myelomatosis. Biochem. J. 34: 1248-1257 (1940). 8 Levy, H. B. and Sober, H. A.: A sample chromatographic method for preparation of gamma globulin. Proc. SOC.exp. Biol. Med. 103: 250252 (1960). 9 Palmen, J. L. and Nisonoff, A.: Dissociation of rabbit y-globulin into half-molecules after reduction of one labile disulfide bond. Biochemistry 3: 863-869 (1964). 10 Takahashi, T.: Physico-chemical properties of hepatitis B antigen. J. Jap. med. Ass. 73: 225234 (1975). 11 Ukkonen, P.; Koistinen, V., and Penttinen, K.: Enzyme-immunoassay in the detection of hepatitis B surface antigen. J. immunol. Methods 15: 343-353 (1977). 12 Vyas, G. N. and Shalman, N. R.: Haemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332333 (1970). 13 Wel, R.; Knight, G. J.; Zimmerman, D. H., and Bond, H. E.: Solid-phase enzyme immunoassay for hepatitis B surface antigen. Clin. Chem. 23: 813-815 (1977). 14 Widsom, G. B.: Enzyme-immunoassay. Clin. Chem. 22: 1243-1255 (1976). 15 Wolters, G.; Kuijpers, L.; Kancaki, J., and Schuurs, A.: Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J. clin. Path. 29: 873-879 (1976). Received: September 26, 1977 Accepted: January 5, 1978 Hidenari Adachi, Meguro Institute, Masumi-cho 7-29, Ikeda, Osaka 563 (Japan)

Sandwich enzymoimmunoassay of hepatitis B surface antigen (HBsAg).

Vox Sang. 35: 219-223 (1978) Sandwich Enzymoimmunoassay of Hepatitis B Surface Antigen (HBsAg) Hidenari A dachi, Toshiaki Fukuda, Shuji Funahashi, To...
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