FEMS Microbiology Immunology 47 (1988) 81-86 81 Published by Elsevier

FIM 00013

A rapid microassay for detecting hepatitis B surface antigen by dot enzyme immunoassay

K. Ma t sumura , S. Wakatsuki , A. Iwasaki , R. E n d o and K. T a n a k a

Yamaguchi Prefectural Research Institute of Health, Aoi Yamaguchi, Japan

Received 26 January 1988 Accepted 10 February 1988

1. SUMMARY

This study describes a dot enzyme immunoas- say (Dot-EIA) for detecting hepatitis B surface antigen (HBsAg). The results demonstrated that the detection level of this assay for HBsAg was 1.5 ng/ml ; no false-positive or -negative readings were observed. Also, this Dot-EIA had some ad- vantages over standard EIA: (1) antiserum could be directly and immediately bound on nitrocellu- lose paper set into microfiltration apparatus, (2) the paper could be easily washed under reduced pressure using a water aspirator, (3) all assay steps could be performed at room temperature within 2 h, (4) the well-defined brown spots could be evaluated by both visual observation and densito- metric reading. The Dot-EIA reported here may be useful for rapid diagnosis and screening of HBsAg in serum.

2. INTRODUCTION

Methods for detection of hepatitis B surface antigen (HBsAg) and the antibody in serum are widely available. Detection of HBsAg in serum is

now a routine procedure as a laboratory tech- nique. The most commonly used technique are enzyme inununoassay (standard EIA) and the re- verse passive haemagglutination (RPHA) test; however, these commercially available kits are very expensive. Although the EIA kit for HBsAg is available from many manufacturers, no descrip- tion of the dot enzyme immunoassay (Dot-EIA) using the biotin-avidin system for detecting HBsAg in serum has hitherto been published.

Recently, Hawkes et al. [1] reported a modifica- tion of the dot hybridization method where minute volumes of monoclonal antibodies were directly applied to nitrocellulose filter paper (NC paper). The Dot-EIA is a newly described technique using NC paper and reacted with precipitable chromo- genic substrate [2]. In this study we describe a Dot-EIA using the biotin-avidin system for the detection of HBsAg where rabbit anti-HBsAg serum was dotted on NC paper set into a micro- filtration apparatus. The assay is rapid, simple and economical and has a number of advantages over standard EIA.

3. MATERIALS A N D METHODS

Correspondence to: K. Matsumura, Yamaguchi Prefectural Re- search Institute of Health, 2-5-67 Aoi Yamaguchi 753, Japan.

3.1. Reagents Bovine serum albumin, fraction V (BSA) was

purchased from Miles Laboratories Inc., (Naper-

0920-8534/88/$03.50 © 1988 Federation of European Microbiological Societies

82

ville, IL). Purified immunoglobulin G from rabbit anti-HBsAg serum was obtained from the Medical and Biological Laboratories (Nagoya, Japan). NHS-Biotin (N-Hydroxysuccinimidobiotin) and horseradish peroxidase-Avidin A (avidin-per- oxidase conjugate) were purchased from Vector Laboratories Inc., (Burlingame, CA). Diamino- benzidine, O-phenylenediamine, hydrogen per- oxide and Tween 20 came from Wako Pure Chem- icals (Tokyo, Japan). HB vaccine was obtained from Green Cross K.K. (Osaka, Japan).

3.2. Sera Nine samples presumed to be positive were

provided from The Yamaguchi Blood Centre of The Red Cross. The negative samples were pre- pared in our laboratory. Positive and negative diagnosis of these sera was further evaluated by the identification of standard EIA developed in our laboratory as described below.

3.3. Dot-EIA An 8 × 11 cm NC paper (Nitrocellulose mem-

brane, Bio-Rad Laboratories, Richmond, CA) was carefully immersed in 0.02 M phosphate-buffered saline, p H 7.2 (PBS) and set into the ' D O T - PLATE' microfiltration apparatus (Advantec., Tokyo, Japan). 50/~1 of antiserum (IgG content 10 /~g/ml) prepared as above were pipetted into the wells under mildly reduced pressure using a water aspirator, and unbound sites on the NC paper were blocked with 50/~1 of 2% BSA in PBS. After incubation for 15 min at room temperature, the N C paper was washed three times with PBS under reduced pressure. Then, 50 /xl of serum were ad- ded to each well and incubated for 30 min at room temperature. After incubation, the NC paper was washed as above and then 50 /~1 of biotinylated antiserum (10 ~ g / m l ) diluted with 0.1% BSA were pipetted into each well and incubated for 30 rain at room temperature. The biotinylation of IgG from antiserum was performed by the method of Bayer et al [3]. The wells were then washed and 50 /~1 of avidin-peroxidase conjugate (1 ~ g / m l ) di- luted with 0.1% BSA were added to the wells. After incubation for 30 rain at room temperature, the wells were washed four times and 50 /~1 of substrate, freshly prepared diaminobenzidine/

hydrogen peroxide, were added to the wells. After the appearance of color reactions, the substrate was removed by reduced pressure and the NC paper was washed. The development of well-de- fined brown spots was considered to be positive by visual observation.

When the quantitative measurement of the stained spots is needed, a densitometric reading can be used on the spots, i.e., the NC paper was set into a densitometer (Dual-Wavelength TCL Scanner CS-910, Shimadzu Seisakusho, Tokyo, Japan) and the absorbance of the brown spot was measured using a 1-mm slit with 2-mm width at 470 nm.

3.4. Standard EIA Prepared antiserum ( IgG content 1 ~ g / m l ) was

diluted with 0.05 M carbonate buffer (pH 9.6) to coat a microtitration plate (Cook No. 223-24) which was sensitized with overnight incubation at 4 ° C with the addition of 50 /~1 of antiserum. It was removed and unbound sites in the wells were blocked with 100 /~1 of 2% BSA in PBS. After incubation for 1 h at 37 o C, the blocking buffer was removed and the plate was washed three times with PBS containing 0.05% Tween 20. Next, 40 ]~1 of 0.1% BSA were added to each well and then 10 /~1 of serum were added. After being mixed with microtitre mixer, the plate was incubated for 1 h at 37 ° C. It was then removed and the plate was washed as above. 50 ill of biotinylated antiserum (1 /~g/ml) were added to the wells and incubated for 1 h at 37 o C. After incubation antiserum was removed and the plate was washed again. The avidin-peroxidase conjugate (50 I~1) was added and incubated for 1 h at 37 ° C. Then, the con- jugate was removed and the plate was washed four times. The substrate (50 /~1), O-phenylenedia- m ine /hyd rogen peroxide, was then added and incubated for 10 min at room temperature. After stopping the enzyme reaction with 50/~1 of 2.5 M H2804, the plate was read in a spectrophotometer at 492 nm.

3.5. Additional techniques R P H A test (Reversecell *, Yamanouchi Seiyaku

K.K., Tokyo, Japan) was performed according to the manufacturer 's instructions. Presumptive-posi-

tive results were confirmed by the usual absorp- tion procedure.

4. RESULTS

4.1. Sensitivity and reproducibility The representative titration curve for the stan-

dard, HBsAg (ad) diluted in normal human serum, is shown in Fig. 1. In this assay, HB vaccine containing 20 /~g/ml of HBsAg was used as a standard. The curve was linear for an antigen dose between 40 and 1000 ng/ml . The sensitivity of the assay was 1.5 ng /ml of the standard. Intra-assay reproducibility of ten positive and negative sera tested in duplicate on the same day was < 8% and inter-assay reproducibility of the same samples tested on three different days was < 10%.

4.2. Typical example of Dot-EIA Typical Dot-EIA reactions using four positive

and four negative sera are shown in Fig. 2. After

83

incubation with avidin-peroxidase conjugate and then addition of precipitable substrate, positive reactions (upper row) appeared as brown-colored spots on the white background of the NC paper, which could be easily and clearly read by visual observation. However, when reading by eye was less reliable in borderline cases, densitometric reading was used for the quantitative measure- ment (right).

4.3. HBsAg levels by Dot-EIA and other methods The results obtained with positive and negative

sera are shown in Table 1. The absorbance value of positive and negative sera ranged from 0.05 to 0.39 and from 0 to 0.02, respectively. The mean + S.D. for negative sera was 0.01 + 0.01, i.e., even the lowest absorbance value in positive sera was beyond the 3 S.D. of the negative sera. Also, no false-positive or -negative readings were observed in this assay. Antigen levels for the positive sera varied from 30 n g / m l to > 5 ~tg/ml of the stan- dard. It is shown here that HBsAg could be de-

0 . 4

E

0

L I I I I 1 >~ K-

5000 200 8 ~ 0 . 2 G)

1000 40 l .5 E 0

A n t i g e n c o n c e n t r a t i o n ( n g / m l ) "~

O . l

0 .3

i I l I I •

5000 1000 200 40 8 1 . 5

A n t i g e n c o n c e n t r a t i o n ( n g / m l )

Fig. 1. Representative standard curve of Dot-EIA for the detection of HBsAg diluted in normal human serum. 5-fold serial dilutions of HBsAg ranging from 5000 to 1.5 ng/ml were precipitated on nitrocellulose paper (left) and their densitometries at 470 nm are presented (right).

84 0 . 4 - O

P o s i t i v e

N e g a t i v e

- 0 O

E c-

0.3 o

v

0.2 4J 0J E O

= 0 . l

o

O

O

L I

P 0 s i t i v e N e g a t i v e Fig. 2. Typical Dot-EIA reactions using positive and negative sera. Positive reactions developed as brown-colored spots (upper row) on the white background of nitrocellulose paper (left) and their densitometries are presented (right).

Table 1

The HBsAg levels by Dot-EIA compared with standard EIA and RPHA tests, in positive and negative sera

aHBsAg contents were calculated from the standard curve shown in Fig. 1. b and c These sera were identified to be negative by the

usual absorption method.

Serum No. A value HBsAg A value by Reciprocal titer by Dot-EIA content (ng /ml ) a standard EIA by RPHA test

Positive 1 0.34

2 0.12

3 0.26 4 0.05 5 0.39

6 0.20 7 0.07

8 0.13 9 0.28

Negative 10 0 11 0 .02 12 0

13 0 14 0 15 0.01 16 0.01 17 0.02 18 0.02

19 0

( > 5000) > 2.00 > X256

(110) 1.82 > ×256 (700) > 2.00 > × 256

(30) 0.18 ×8

( > 5000) > 2.00 > ×256 (290) > 2.00 × 256

(50) 0.56 × 16

(120) > 2.00 × 256 (1000) > 2.00 > x 256

0.02 × 4 0.04 > × 16 0.05 < × 4 0.05 < X 4 0.04 < × 4 0.05 < × 4

0.07 < × 4 0.07 < × 4 0.05 < × 4 0.05 < x 4

tected as easily by D o t - E I A as by the s tandard E IA and R P H A tests.

5. D I S C U S S I O N

This study describes a D o t - E I A technique for HBsAg using the biotin-avidin system. The results conf i rm the usefulness of the D o t - E I A for HBsAg detection in sera. The sensitivity of the technique was equal to the s t andard-EIA [4,5], whereas it offers some advantages over s tandard EIA. Anti- serum absorpt ion to the well surface must be performed with overnight incubat ion for the s tandard EIA. In contrast, in Dot -EIA, no incuba- t ion for antiserum binding is required, i.e., anti- serum can be directly and immediately bound on N C paper using a microfil tration apparatus. Fur- thermore, the unbound sites on N C paper can be easily blocked within a short period of time with 2% BSA. Also, the paper can be easily washed under reduced pressure using a water aspirator and all assay steps can be carried out at room temperature within 2 h. Therefore, a large number of sera and runs can be assayed in 1 day. The advantages of this D o t - E I A indicate that this tech- nique is suitable for rapid diagnosis and screening of HBsAg and probably for various infectious diseases.

A visual interpretat ion using reciprocal titers has been used for the quanti tat ive measurement in the Do t -E IA [1,2]. However, the expression of absorbance values had been used in the s tandard E I A [6] when the quanti tat ive measurement is required in a single serum dilution method. Simi-

85

larly, the absorbance value is required in the Dot- E IA when one serum dilution and serial serum dilution methods are used, particularly in the borderl ine cases. In the present study, the brown- colored spot could be quanti tat ively measured by a densi tometer with reflection.

Finally, testing a small number of samples, which is often required, can be as easily carried out as testing a large number of samples. After a small sheet of N C paper was set into microfiltra- t ion apparatus, unrequired wells were sealed. Also, we believe that the D o t - E I A is a rapid and inex- pensive method of detecting HBsAg in sera f rom donors and patients, a l though a small number of reference sera was evaluated in this study.

R E F E R E N C E S

[1] Hawkes, R., Niday, E. and Gordon, J. (1982) A dot-im- munoblotting assay for monoclonal and other antibodies. Anal. Biochem. 76, 142-147.

[2] Papas, M.G., Hajkowski, R. and Hockmeyer, W.T. (1983) Dot enzyme-linked immunosorbent assay (Dot-ELISA): a micro-technique for the rapid diagnosis of visceral leish- maniasis. J. Immunol. Methods 64, 205-214.

[3] Bayer, E.A., Skutelsky, E. and Eilchek, M. (1979) The avidin-biotin complex in affinity cytochemistry. Methods Enzymol. 62, 308-315.

[4] Wolters, G., Kuijpers, L., Kacaki, J. and Schuurs, A. (1976) Solid-phase enzyme-immunoassay for detection of hepatitis B surface antigen. J. Clin. Pathol. 29, 873-879.

[5] Adachi, H., Fukuda, T., Funahashi, S., Kurahori, T. and Ishikawa, E. (1978) Sandwich enzymoimmunoassay of hepatitis surface antigen (HBsAg). Vox Sang. 35, 219-223.

[6] World Health Organization (1976) The enzyme-linked im- munosorbent assay (ELISA). Bull. Wld. Org. 54, 129-139.