Journal of Virological Methods, 16 (1987) l-11 Elsevier

JVM 00574

A biotin/avidin solid-phase sandwich enzyme immunoassay for the antibody to hepatitis B

surface antigen (anti-FIBS)

Isa K. Mushahwar Infectious Disease and Immunology Research, Abbott Laboratories, Abbott Park, Illinois, USA

(Accepted 26 November 1986)

Summary

A biotimavidin solid-phase enzyme immunoassay for the detection and quanti- tation of the antibody to hepatitis I3 surface antigen (anti-HBs) is described. The assay utilizes hepatitis B surface antigen (HBsAg) as a solid-phase ‘capture’ re- agent and a mixture of biotinylated HBsAg and avidin-conjugated horseradish peroxidase as a probe ‘detector’ reagent. The assay was compared to a commercial radioimmune assay for anti-HBs detection. The two assays were found to measure the same molecules and to correlate well regarding anti-HBs titers.

Anti-HBs; Enzyme immunoassay; Biotimavidin assay; Biotinylated HBsAg; Avi- din-HRP; Hepatitis B serologic marker

Introduction

The antibody to hepatitis B surface antigen (anti-HBs) is a long-persisting an- tibody associated with convalescence and recovery from an acute hepatitis B viral infection. The antibody also appears in serum after.a successful immune response to hepatitis B vaccine.

A commercially available direct solid-phase radioimmune assay (RIA) for anti- HBs detection and quantitation has been available since 1974 (AUSAB, Abbott Laboratories). In this assay, the serum specimen is incubated with a solid-phase (polystyrene bead) that has been coated with highly purified HBsAg. If anti-HBs

Correspondence to: I.K. Mushahwar, Infectious Disease and Immunology Research, Abbott Labora- tories, Abbott Park, IL 60064, USA.

~166-~34/87/$03.50 @ 1987 Etsevier Science Publishers B.V. (Biomedical Division)

2

is present in the serum, it will complex with the solid-phase antigen. In a second step, radiolabeled HBsAg (lz51-HBsAg) is added and reacts with the immobilized antibody. The amount of antibody in the original serum is in direct proportion to the amount of radiolabel fixed to the solid-phase.

This paper describes an enzyme-linked immunoassay (ELISA) for anti-HBs de- tection and quantitation similar to the RIA but in which lz51-HBsAg is replaced with a mixture of biotinylated HBsAg and an avidin-conjugated horseradish per- oxidase (avidin-HRP). The assay was compared with the RIA as to sensitivity, de-

tectability, and specificity and was found that both assays measure the same mol- ecules and correlate well regarding anti-HBs titers. Reproducible and consistent anti-HBs titers with the two assay procedures were obtained.

Materials and Methods

Reagents

Affinity purified egg white avidin, d-biotin, biotinyl-N-hydroxysuccinimide ester (BNHS) and horseradish peroxidase (HRP, RZ 2 3) were purchased from Sigma Chemical Co. (St Louis, MO).

Purification of HBsAg

Human plasma units containing HBsAg (adw or ayw subtype) with a rheopho- resis titer of 2 1:32 were used as starting material. The antigen was purified ac- cording to an established procedure (Gerin et al., 1975). The final product was di- alyzed overnight at 4°C against 0.1 M potassium phosphate-O.1 M NaCl buffer (pH

8).

Biotinylation of HBsAg

To 8 ml of 0.1 M potassium phosphate-O.1 M NaCl buffer (pH 8) containing 1 mg/ml purified HBsAg was added 0.4 ml of 6.9 - ~.LM solution of BNHS ester dis- solved in distilled dimethylformamide. The solution was rotated for 16 h at 4°C and then dialyzed overnight against 3 buffer changes. The biotinylated HBsAg was diluted to 2 kg/ml in a diluent of 0.1 M Tris-HCl-saline buffer (pH 7.5) containing 15% normal human serum and 50% newborn calf serum and stored at -20°C.

A vidin-HR P

Avidin preparations in 0.01 M potassium phosphate buffer (pH 8) were conju- gated with HRP at 1:2 ratio according to the method of Nakane and Kawaoi (1974). Avidin-HRP conjugate solution was diluted to 2 pg/ml in 0.05 M Tris-HC1-0.5 M NaCl-0.1% Tween 20 buffer (pH 8.5) containing 15% normal human serum and 50% newborn calf serum and stored at -20°C.

Solid-phase HBsAg

Polystyrene beads (6 mm in diameter) were coated with purified HBsAg, adw and ayw preparations (2 kg/ml each) in 0.10 M Tris-HCl buffer (pH 7.5) for 22 h at room temperature as described previously (Mushahwar et al., 1978).

EIA for anti-HBs utilizing the biotin-avidin system (BAV-EIA)

Serum anti-HBs was determined by a direct solid-phase (sandwich) assay. The sample (0.2 ml) was either incubated for 24 h with the HBsAg-coated bead at room temperature (Overnight Procedure) or for 2 h at 40°C (Short Procedure); after washing with water, the bead was further incubated with 0.2 ml of a freshly pre- pared mixture of biotinylated HBsAg and avidin-HRP conjugate at 40°C for 2 h.

The bead was then washed with water. A substrate solution (0.3 ml of 0.3% o- phenylenediamine 2 HCl in 0.1 M citrate phosphate buffer (pH 5.5) containing 0.02% H,Oz was added (Mushahwar and Overby, 1981). The enzymic reaction was allowed to proceed for 30 min at room temperature in the dark. The reaction was stopped by adding 1 ml of 1 N H,SO,. The intensity of the color that was devel- oped as a result of the enzymic catalysis of the substrate was measured at 492 nm by using Quantum II Spectrophotometer (Abbott Laboratories). Specimens with absorbancy equal to or greater than the cutoff (negative control mean absorbance plus 0.05) were considered to be reactive by the criteria of this test for the Over- night Procedure. For this calculation the mean of at least three sera was used as negative control (NC). The optimal dilution of biotinylated HBsAg and avidin-HRP used in the assay were determined by checkerboard titration.

Quantitation of anti-HBs

For routine quantitation, an unknown serum sample is compared with the WHO Reference Preparation of 26 January 1977. Anti-HBs concentration is calculated as described (Hollinger et al., 1982).

Confirmation of anti-HBs by a neutralization assay

The specificity of the EIA for anti-HBs was tested by comparing the inhibitory effect of an HBsAg pool (containing the adw and ayw subtypes) on the reactivity of the samples positive for anti-HBs by the EIA procedure. One set (A) of con- trols and the test samples were tested using the EIA reagents mixed with the HBsAg pool (4 and 1 part, respectively). The HBsAg pool had a titer of 10’ by AUSRIA II (Abbott Laboratories). Another set (B) of controls and samples were simultaneously tested using the EIA reagents mixed with normal human serum (negative for HBsAg and anti-HBs). Samples minus negative control absorbancy values at 492 nm were separately determined for samples in sets A and B. For as- say sample, if the absorbancy at 492 nm of (A) was 50% or less than the absorb- ancy at 492 nm of (B), the test was considered specific.

4

Other serologic procedures

Serum samples were tested with commercial RIA reagents supplied by Abbott Laboratories: AUSRIA II for HBsAg, CORAB for anti-HBc, and Abbott-HBe for HBeAg and anti-HBe. Rheophoresis tests for anti-HBs were performed with agar gel plates also supplied by Abbott Laboratories.

Results

Sensitivity and detectability

Titration of a vaccinee serum by RiA and BAV-EiA A vaccinee serum (recipient of H-B-VAX; Merck, FRG) reactive for anti-HBs

by AUSAB-RIA and BAV-EIA was serially diluted in normal human serum de- void of HBsAg and anti-HBs and assayed for anti-HBs by both procedures. For AUSAB, the results are expressed as cpm, and the ratio of these values to the negative control values (S/N). For BAV-EIA, the results are expressed as absorb- ance at 492 nm. These results are summarized in Table 1. The last concentration found unquestionably reactive by either RIA or EIA tests was 8 IUil, indicating equivalent sensitivity. The 4 IU/l concentration is marginally positive by both tests. Repeat testing of specimens near the cutoff may show these tests to be negative or positive by either test system.

US Bureau of Biologics Panel The detectability of the US Bureau of Biologics (USBB) Anti-HBs Reference

Panel No. 2 by both AUSAB and BAV-EIA procedures is summarized in Table 2. These results show that both procedures are of equivalent detectability.

TABLE 1

Comparison of AUSAB-RIA and BAV-EIA for the detection of anti-HBs of vaccinee.

AUSAB-RIA BAV-EIA

IUil CPM S/N Absorbance

120 60 30 1.5 8 4 2

NC 110 1.0 0.013 PC 6202 56.4 1.333

6120 55.6 > 2.000 3485 31.7 1.580 1753 15.9 0.785 1023 9.3 0.400 550 5.0 0.220 374 3.4 0.117 255 2.3 0.065 175 1.6 0.040

Cutoffs: RIA assay: S/N values of 3 2.1 are considered reactive; EIA assay: absorbancy values above 0.063 (NC mean absorbancy + 0.05) are considered reactive.

5

TABLE 2

Comparison of AUSAB-RIA and BAV-EIA for the detection of anti-HBs in the USBB Reference Panel

No. 2.

Panel/No.

AUSAB-RIA

CPM

BAV-EIA

Absorbance

USBB201 1794 22.4 0.797 202 1100 13.8 0.563

203 74 0.9 0.027

204 67 0.8 0.025

205 12482 156.0 2

206 237 3.0 0.089

207 148 1.9 0.055 208 6173 77.2 >2 209 77 1.0 0.032 210 311 3.9 0.173

211 658 8.2 0.243

NC Sd 1.0 0.018

PC 3670 45.9 1.636

Cutoffs: RIA assay: S/N values of 2 2.1 are considered reactive; EIA assay: absorbancy values above

0.068 (NC mean absorbancy + 0.05) are considered reactive.

24 r

Fig. 1. Histogram of the net absorbance (at 492 nm) values obtained when 55 anti-HBs reactive sera

were tested by BAV-EIA. The net absorbance for each specimen was calculated, and the results were grouped by number of sera in each percentile.

TABLE 3

Description of specimens used to evaluate the reproducibility of BAV-EIA

Panel member Dilution Exnected result

A 1:1600 Positive

B 1:3200 Positive

c 16400 Positive

D 1: 12stKl Borderline positive

E 1:256Otl Negative

F 1:51200 Negative

The Dilution Sensitivity Panel consisted of serial dilutions of a specimen from a patient whose serum

contained a high titer of anti-HBs. The results expected for the dilutions are indicated above.

Anti-HBs positive population The detectability of anti-HBs in 55 positive sera by BAV-EIA is shown in Fig.

1. The RIA test (data not shown) detects 2 more reactive specimens than the BAV- EIA. However, these two specimens had AUSAB S/N ratios of 2.5 and 2.6 and the BAV-EIA absorbancy was 0.065 and 0.062 which is just below cutoff 0.073. As pointed out above, specimens near the cutoff may be negative or positive by either test system. Thus, for all practical purposes the results are comparable.

The detectability of the same anti-HBs positive population by BAV-EIA Over- night Procedure (ONIRT; 2 hi40”C) compared to that of BAV-EIA Short Proce- dure (2 hi40”C; 2 hi4O”C) as described in the Materials and Methods section was also examined. When using a cutoff of the Negative Control mean absorbancy plus 0.03 for the Short Procedure, the Overnight Procedure detects 3 more specimens as reactive. These results show that the Short Procedure is 5.5% less sensitive than the Overnight Procedure.

Reproducibility of BAV-EiA In order to demonstrate the reproducibiIity of BAV-EIA test results by using

statistical analysis of repeat testing of positive and negative specimens, 30 repli-

TABLE 4

Estimated number of positive and negative test results if selected specimens were tested 1000 times for

anti-HBs with BAV-EIA.

Snecimens tested Dilution

Dilution sensitivity

Panel A

B C

D E

F Normal blood donor population

1: 1600 0 loo0 1:3200 0 1000

16400 0 1000

1:12800 0 1000

1:25600 775 22.5 1:51200 1000 0

None 1000 0

Number of specimens

Negative Positive

Absorbancy (492 nm)

Fig. 2. Histogram of the net absorbance (at 492 nm) values obtained when 400 HBsAg negative sera

were tested for anti-HBs by BAV-EIA. The net absorbance for each specimen was calculated, and re- sults were grouped by number of sera in each percentile.

cates of each member of a Dilution Sensitivity Panel (Table 3) for anti-HBs were tested by BAV-EIA. For each specimen, the probability of a positive or negative result was calculated using statistical methods. Additionally, data from the testing of specimens negative for anti-HBs from normal blood donors were examined to determine the frequency distribution of negative results. The results from this study are shown in Table 4. The data are expressed in terms of 1000 assays. Thus, for Dilution Sensitivity Panel members A, B, and C (Table 4), the frequency of po- sitive results is 1000 of 1000 assays. For Panel member D, which is a borderline positive sample, the data show that a positive value is also predicted in 1000 of 1000 assays. The last anti-HBs Panel members E and F, show the predicted neg- ative results. This statistical study shows that BAV-EIA appropriately and re- peatedly discriminates among anti-HBs positive and negative samples.

Specificity of BA V-EIA

Frequency distribution of anti-HBs in normal blood donor population The frequency distribution of anti-HBs in 400 HBsAg negative sera is shown in

Fig. 2. For anti-HBs, the analysis shows that 389 samples had a net absorbance (A492-A492 NC) of less than 0.05. Eleven samples had absorbance values above 0.09. Ten of these were also positive by AUSAB. Only one sample (0.25%) was unreactive for anti-HBs by BAV-EIA.

x

The serological profiles for a human accidentally infected with hepatitis B virus are shown in Table 5. During this study period, HBsAg, HBeAg, anti-H& and anti-HBs were detected, in this serological sequence as expected (Mushahwar et al., 1981). Anti-HBs was detected also by AUSAB-RIA as shown, indicating that both the RIA and BAV-EIA assays detected the same anti-HBs specimens re- gardless of the presence or absence of other hepatitis B virus serological markers.

Specimens from patients and animais with antibodies against a variety of viruses were tested by BAV-EIA and AUSAB. BAV-EIA and AUSAB did not detect anti-HBs in any of these specimens shown in Table 6.

Table 7 shows weak anti-HBs positive specimens which were negative by rheo- phoresis but positive by AUSAB and BAV-E.IA. The presence of anti-HBs was confirmed by neutralizing the anti-HBs reactivity with HBsAg. Normal sera gave no significant differences in absorbance in the confirmatory neutralization analysis

TABLE 5

Demonstration of hepatitis B markers in a ~em~dialysjs patient a&dentally expased to hepatitis ‘El vi- TUS.

iTate of Anti-HBs specimen collection HBsAg HBeAg Anti-HBc RIA EIA

41Il78 0 0 0 0 0 6/v?% 0 0 0 cl 0 71317% f + a if 0 8m78 i- + + 0 0 911.m + t i" 0 cf

11/t/78 + + + 0 0 1211178 + + + 0 0 113f79 + 0 + 0 0 212179 c 0 + 0 0 213!79 0 0 i 0 0

9

TABLE 6

Lack of cross-reactivity of sera positive for various viral antibodies and antigens with AUSAB and BAV- EIA.

Serum Source/ identification identi~cation

BAV-EIA

A492 +/0

AUSAB-RIA

CPM +iil

Rheumatoid factor (3-i-) Rheumatoid factor (2+) Rheumatoid factor (4+) Rheumatoid factor (1”) Immune complex disease Lupus erythematous Polymyositis Lupus erythematous IgG anti-HAV IgG anti-rubella IgM anti-rubella I&4 anti-HBc IgG anti-HBc Anti-HBe+/HBsAg+ HBeAg+/HBsAg+ Acute hepatitis A Acute hepatitis B Chronic hepatitis B Anti-varicella zoster Anti-measles Anti-mumps Anti-RSV Anti-adenovirus Anti-CMV NC PC Cutoff

Human/I‘M. Human/312 Humani269 Human!RND Human/68S~I Human/560~203 Human/8000367 R.E. 367 5605 Animal/~low Animal/Flow Animal/Flow Animai~o~ Animal/Flo~ Animal/Flow Human/32-439 Human/29-093

0.018 0.015 0.014 0.022 0.012 0.020 0.014 0.010 0.018 0.014 0.015 0.014 0.028 0.029 0.014 0.011 0.014 0.029 0.011 0.017 0.019 0.020 0.012 0,011 0.012 1.472

0 0 0 0 0

0 0

0

0 0 0 0 0 0 0 0 0

0 0 0 0

0 0

0 0 + 0

Not determined Not determined Not determined

86 0 81 0 86 0

110 0 76 0 86 0 89 0 58 0 69 0 82 0 72 0 89 0 70 0 89 0 72 0 80 0 83 0

110 0 121 0 64 0 84 0 77 0

3551 + 162 0 0.062

Discussion

This paper describes a BAV-EIA technique for the rapid and specific detection and yuantitation of anti-HBs in human and animal sera utilizing for the first time a biotinylated viral antigen (HBsAg) of an approximate molecular mass of 3.5 x

lo6 Da (Gerin et al., 1975). In 1982, this study resulted in the availability of a com- mercial assay (AUSAB-EIA, Abbott Laboratories) for anti-IIBs detection. Since then, an array of BAV assays utilizing biotinylated immunoglobulins as described originally (Guesdon et al., 1979) have been reported for the detection of a variety of viral and bacterial antigens and antibodies as well as other biological molecules (Kendall et al., 1983; Pillarisetti et al., 3983; Yolken et al., 1983; Chang et al., 1984; Plebani et al., 1984; Disco et al., 1985; Gupta et al., 1985; Vilja et al., 1985; Guesdon et al., 1986; Maldiney et al., 1986; Zrein et al., 1986).

BAV-EIA combines in one assay the amplification of the biotin-avidin system and the sensitivity afforded by AUSAB-RIA, This combination resulted in a unique

10

TABLE 7

Confirmation of anti-HBs positive analysis.

Serum

identification

Absorbancy A,,z of

serum added to specimen

Normal Neutralization

Rheophoresis AUSAB-RIA human HBsAg Conclusion

anti-HBs (cpm) serum + serum anti-HBs

(1) Anti-HBs positive

No.

6024 Negative

6056 Negative

3998 0.620 0.016 Positive

1989 0.312 0.028 Positive

6070 Negative 2136 0.407 0.060

6112 Negative 4270 0.690 0.028

Positive

Positive

6249 Negative 7665

6306 Positive 8995

0.914

1.573

0.109

0.022

Positive

Positive

6308 Positive 8430 1.465

6312 Positive 8029 0.926 0.017

0.019

Positive

Positive

6322 Negative 1623 0.254

(2) NC (anti- Negative

HBs negative) (3) PC (anti- Negative

HBs positive)

Cutoff

137 0.020

10273 1.150 0.019 Positive

288 0.070 0.070

0.019 Positive

0.020 Negative

assay for the detection of anti-HBs approximately .5-fold more sensitive than a di- rect sandwich assay utilizing HBsAg-HRP as the probe (data not shown). Fur-

thermore, numerous attempts to conjugate HRP to HBsAg by the glutaraldehyde method (Avrameas and Ternynck, 1979), the periodate method (Nakane and Ka- waoi, 1974), and a heterobifunctional reagent (Carlsson et al., 1978) were unsuc- cessful due to the poor yield, purity, and stability of the HBsAg-HRP conjugate. In contrast, biotinylated HBsAg was produced in high yield, high purity, excellent stability (6 months at 4°C when stored in its diluent in the presence of 0.1% thi- merosal), and with no loss in immunoreactivity. In agreement with Guesdon et al. (1979), however, it was found that a too high BNHS-protein ratio resulted in a decrease in immunological reactivity. When the degree of substitution of the free amino groups of HBsAg exceeded the optimal range of 20-25% as tested by the 2,4,6-trinitrobenzenesulfonic acid procedure (Habeeb, 1966), a dramatic decrease in HBsAg immunoreactivity was observed.

The high specificity of BAV-EIA was demonstrated by examining the frequency distribution of anti-HBs in a normal blood donor population (Fig. 2), human and animal studies (Tables 5 and 6), and neutralization experiments (Table 7). Its

11

comparative sensitivity to AUSAB-RIA was also demonstrated by titration studies (Table l), examination of anti-HBs panels (Table 2), and detectability comparison in an anti-HBs positive population (Fig. 1). BAV-EIA is inherently slightly less sensitive than AUSAB-RIA, but the two assays measure the same molecules and correlate well regarding titers. The lesser sensitivity of BAV-EIA minimizes the significance of borderline positives seen in AUSAB-RIA and many similar RIA assays described for other antiviral and antibacterial antibodies (~ushahwar and Brawner, 19X6).

Acknowledgement

I am grateful to Mr. Carlos M. Cabal for his competent technical assistance.

References

Avrameas, S. and Ternynck, T. (1971) Immunochemistry 8, 1175-1179. Carlsson, J., Drevin, H. and Axen, R. (1978) Biochem. J. 173, 723-737. Chang, H.-H., Takashima, I., Arikawa. J. and Hashimoto, N. (1984) J. Viral. Methods 9, 143-151. Disco, R., Hill, J.H., Hill, E.K., Tachibana, H. and Durand, D.P. (1985) J. Gen. Virol. 66, 20892094. Gerin, J.L., Faust, R.M. and Holland, P.V. (1975) J. Immunol. 115, 100-105. Guesdon. J.-L., Chevrier. D. ,. Mazie, J.-C., David, B. and Avrameas, S. (1986) J. Immunol. Methods

87, 69-78. Guesdon, J.-L.. Ternynck, T. and Avrameas, S. (1979) J. Histochem. Cytochem. 27, 1131-1139. Habeeb, A.F.S.A. (1966) Analyt. Biochem. 14, 328-336. Hollinger, F.B., Adam, E., Heiberg, D. and Melnick, J.L. (1982) In: Viral Hepatitis, 1981 Interna-

tional Symposium (Szmuness, W., Alter, H.J. and Maynard, J.E., eds.), pp. 451-466. Kendall, C., Ionescu-Matiu, I. and Dreesman, G.R. (1983) J. Immunol. Methods 56, 329-339. Maldiney. R., Leroux, B., Sabbagh, I., Sotta, B., Sossountzov, L. and Miginiac, E. (1986) J. Immu-

nol. Methods 90, 151-158. Mushahwar, I.K. and Overby, L.R. (1981) J. Virol. Methods 3, 89-97. Mushahwar, I.K., Overby. L.R., Frosner, G.. Deinhardt, F. and Ling, C.-M. (1978) J. Med. Viral. 2,

77-87. Mushahwar, I.K., Dienstag, J.L.. Polesky. H.F.. McGrath, L.C., Decker, R.H. and Gverby, L.R. (1981)

Am. J. Clin. Pathol. 76, 773-777. Mushahwar, I.K. and Brawner, T.A. (1986) In: Clinical Virology Manual, Chapter 8 (Specter, S. and

Lancz, G.J., eds.), Elsevier, New York. Nakane, P.K. and Kawaoi. A. (1974) J. Histochem. Cytochem. 22, 1084-1091. Pillarisetti. V., Rao, S., McCartney-Francis, N.L. and Metcalf, D.D. (1983) J. Immunol. Methods 57,

71-85. Pfebani, A., Avanzini, M.A., Massa, M. and Ugazio, A.G. (1984) J. Immunol. Methods 71, 133-140. Vilja, P., Krohn, K. and Tuohimaa, P. (1985) J. Immunol. Methods 76, 73-83. Yoiken, R.H., Leister, F.J., Whitcomb, L.S. and Santosham, M. (1983) J. Immunol. Methods 56,

319-327. Zrein, M., Buckard, J. and Van Regenmortel, M.H.V. (1986) J. Viral. Methods 13, 121-128.