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Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg
Journal of Virological Elsevier VIRMET
Methods,
211
25 (1989) 211-232
00907
Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg Larry Mimms, Andrew Goetze, Suzanne Swanson, Marco Floreani, Brook Edwards, Jerzy Macioszek, Greg Okasinski and Willie Kiang HepatitislAIDS
Research,
Abbott
Laboratories,
(Accepted
31 March
Abbott
Park, IL 60064, U.S.A.
1989)
Summary
A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 1251was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples. Anti-HBs; Enzyme-linked immunoassay; HBsAg; Hepatitis B serological marker
Radioimmunoassay;
Correspondence U.S.A.
Abbott
to;
0166~0934/89/$03.50
L. Mimms,
Hepatitis/AIDS
6J 1989 Elsevier
Science
Research,
Publishers
B.V.
Laboratories,
(Biomedical
Abbott
Division)
Recombinant
Park, IL 60064,
212
Introduction The antibody to hepatitis B surface antigen (anti-HBs) is a long-persisting antibody associated with convalescence and recovery from an acute hepatitis B viral infection. The antibody is also detectable in serum after an immune response to hepatitis B vaccine. A commercially available direct solid-phase radioimmunoassay (RIA) for antiHBs detection and quantitation has been available since 197.5 (AUSAB, Abbott Laboratories). In this assay, the serum or plasma specimen is incubated with a solidphase (polystyrene bead) that has been coated with human plasma-derived HBsAg (hpHBsAg). If anti-HBs is present in the serum, it will bind to the solid-phase antigen. In a second step, radiolabeled HBsAg (‘251-HBsAg) is added and reacts with the immobilized antibody. The amount of radiolabel bound to the solid phase within limits is in direct proportion to the amount of anti-HBs in the original specimens. An enzyme-linked immunoassay (EIA) version of this test (AUSAB EIA) has been available since 1983 and uses a biotinylated HBsAg, horseradish peroxidase (HRP) conjugated avidin mixture as a probe ‘detecting’ reagent (Mushahwar, 1987). In an improved version of this test, a biotinjanti-biotin detection system was substituted for the biotimavidin detection reagent (Mushahwar and Spiezia, 1987). In this paper we describe a second generation RIA and EIA for the detection of anti-HBs in which the hpHBsAg is replaced with recombinant DNA-derived HBsAg (rHBsAg). Purified hpHBsAg consists primarily of 22 nm spherical particles with an estimated molecular weight of 2-5 x lo3 kDa. The major protein constituent is 226 amino acids long, is encoded by the hepatitis B virus (HBV) S gene, and exists in the particle as two species: glycosylated, gp27 (27 kDa) and nonglycosylated, p24 (24 kDa) (Tiollais et al., 1985). The hepatitis S gene for both adw, and ayw subtypes was cloned and expressed separately in mouse L cells which can secrete HBsAg particles 22 nm in diameter and morphologically indistinguishable from hpHBsAg particles. The rHBsAg particles were affinity purified and shown to contain the important HBsAg epitopes as determined by reactivity with monoclonal anti-HBs antibodies. Although several rHBsAg containing vaccines have been produced (Gerety, 1988; Andre and Stafary, 1988; Yano and Tashiro, 1988; Adamowicz et al., 1988), no rHBsAg based anti-HBs assays have been described for monitoring vaccinees. rHBsAg offers advantages over hpHBsAg in ease of purification, greater consistency in production and elimination of the risks associated with working with potentially infectious human plasma. The performance of the recombinant anti-HBs assays was compared to AUSAB RIA and showed equivalent sensitivity and detectability. Data indicate that these assays may provide improved specificity compared to AUSAB RIA.
213
Materials and Methods Reagents
Horseradish peroxidase (HRP, RZ B 3) was purchased from Sigma (St. Louis, MO) or from Toyoba Chemical (New York, NY). All SDS PAGE and Western blot chemicals and lactoperoxidase/glu~ose oxidase beads were obtained from BioRad tRi~hmond, CA). All ~hromatographic media were from Pharmacia (Piscatoway, NJ). Naiz51 was obtained from Amersham (Arlington Heights, IL). All monoclonai antibodies against HBsAg were from Abbott Laboratories (N. Chicago, IL). Production of rHBsAg
Both major subtypes of HBsAg (adw, and ayw) were cloned and expressed separately in mouse L cells. HBV DNA was isolated from HBV particles purified from human plasma. A 1.37 kb BamHl fragment containing the HBV S gene was inserted into plasmid containing thymidine kinase or neomycin as selectable markers. The herpes simplex virus ICP4 promoter was used to direct synthesis of the S gene (Post et al., 1980). The BamHl fragment of expression plasmids containing the HBV S gene was sequenced by the method of Maxam and Gilbert (1980). Nucleotide sequences for subtype ayw and adw, coded for amino acid sequences identical to those published by Galibert et al. (1979) and Valenzuela et al. (1980). Secreted rHBsAg levels as high as 500 wg/ml were obtained in hollow fiber tissue culture systems. Pooled cell media containing rHBsAg was clarified by centrifugation and applied to an anti-HBs affinity column. After binding to the column, rHBsAg was specifically eluted with 0.02 M citrate, 0.5 N NaCl, pH 2.3. The pH of the eluate was immediately adjusted to neutral using 1.0 M borate buffer. Peak fractions were pooled, then concentrated and dialyzed against phosphate buffered saline. rHBsAg adw, and ayw subtypes were purified separately on immuno affinity columns dedicated to each subtype. Purification of hpHBsAg
Human plasma units containing HBsAg (adw, or ayw subtype) with a rheophoresis titer of 2 1:32 were used as starting material. The antigen was purified according to an established procedure (Gerin et al., 1975). The final product was dialyzed overnight at 4°C against 0.1 M potassium phosphate, 0.1 M NaCl buffer (PH 8). Characterization
of HBsAg
Purified HBsAg was evaluated by SDS polyacrylamide gel electrophoresis by the method of Laemmli (1970). Gels were stained with Coomassie blue and purity was
214
quantified using a gel scanning densitometer. cording to Towbin et al. (1979). rHBsAg
Western
blotting
was performed
ac-
coated beads (subtype ad and ay)
Polystyrene beads (6 mm in diameter) were coated with rHBsAg (1:l ratio of ay and ad subtypes) at total concentration of 1 kg/ml for 2 h at 40°C. The beads were rinsed with PBS, then incubated with a PBS solution containing 3% BSA or less for 1 h at 40°C. Beads were rinsed with PBS then allowed to air dry. Radioiodination
of rHBsAg
Affinity purified rHBsAg was radioiodinated using a lactoperoxidase catalyzed reaction (Newman et al., 1981). In this procedure, Na1251, immobilized lactoperoxidase/glucose oxidase and HBsAg were allowed to react for 20-25 min, and the reaction mixture was passed over a Sephadex G-50 column to separate free from bound [ 1251]. Peak fractions of “‘1 rHBsAg were pooled then applied to a Sephacryl S-300 column. Peak fractions were counted for radioactivity and pooled. Recombinant HBsAg ad and ay were iodinated and purified separately, diluted into tracer diluent containing animal sera and plasma to produce solutions with radioactivity levels between 0.3-0.8 t.rCi/ml and then pooled at 1:l v/v ratio producing a final tracer solution. rHBsAg:HRP
conjugate
Affinity-purified rHBsAg ad and ay were each conjugated separately to horseradish peroxidase (HRP) as described by Nakane and Kawaoi (1974). Conjugated rHBsAg ad and ay were then mixed 1: 1 (w/w protein) and diluted into a diluent containing animal sera and human plasma to produce a final conjugate solution of approximately 1 &ml total conjugate concentration. RIA for anti-HBs using rHBsAg
(AUSAB
rDNA RIA)
Two procedures for AUSAB rDNA RIA were developed. Sample (0.2 ml) was either incubated for 16-20 h with the rHBsAg-coated bead at room temperature (procedure B) or for 2 h at 40°C (procedure A); after washing with water, the bead was incubated with 0.2 ml of 1251-labeled rHBsAg for 2 h at 40°C. The bead was then washed with water and counted for radioactivity in an ANSR@ gamma counter (Abbott Laboratories). Specimens with cpm greater than or equal to the cut-off (negative control mean (NCx) times 2.7 for procedure A and NCx times 4.0 for procedure B) were considered to be reactive by the criteria of this test. For the calculation of the NCx, eight replicates of the negative control (NC) were used.
215
EIA for anti-HBs using rHBsAg
(AUSAB
rDNA EIA)
The sample (0.2 ml) was either incubated for 16-20 h with the rHBsAg-coated bead at room temperature (procedure B) or for 2 h at 40°C (procedure A). After washing with water, the bead was further incubated with 0.2 ml of rHBsAg:HRP conjugate at 40°C for 2 h. The bead was then washed and a substrate solution (0.3 ml of 0.3% Q-phenyle~ediamine 2 HCI (OPD) in 0.1 M citrate phosphate buffer (pH 5.5) containing 0.02% H202) was added (Mushahwar and Overby, 1981). The enzymatic 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 developed as a result of the enzymatic catalysis of the substrate was measured at 492 nm by using the Quantum II Spectrophotometer (Abbott Laboratories). Specimens with absorbancy equal to or greater than the cut-off (NCx absorbance + 0.03 or NCx + 0.05 for procedures A or B, respectively) were considered to be reactive. For the calculation of the NCx, three replicates of the NC were used. ~0~o~lonaL anti-H&3 reactivity
HBsAg protein concentration in mg/ml was calculated from the following formula: (absorbance at 280 nm - absorbance at 320 nm)/2.5. To determine reactivity of HBsAg with various mon~lonal anti-I-Es antibo~es, HBsAg preparations were diluted quantitatively into human plasma which was negative for HBsAg and antiHBs (NHP) to approximately 1 nglml and these solutions were then tested for antigenic activity. The specimen (0.2 ml) was incubated with a Monoclonal Auszyme bead (Abbott Laboratories) for 2 h at 40°C. The bead was washed and then incubated for 2 h at 40°C with 0.2 ml of the detecting reagent which contained appropriate concentrations of an HRP conjugated or radioiodinated monoclonal antiHE& diluted into NHP. The bead was washed and counted sedately in a gamma counter for tests using a radiolabeled antibody or the bead was incubated with OPD substrate solution for tests using an HRP conjugated antibody. A HBsAg ad sensitivity panel (Abbott Laboratories) was used as a calibration standard for evaluating HBsAg ad subtype preparations, and a HBsAg ay sensitivity panel (Abbott Laboratories) was used for ay preparations. The specific antigenic activity was calculated as relative antigenic activity (determined from the immunoassay described above) divided by the protein concentration. A specific antigenic activity for various HBsAg preparations was evaluated using seven different monoclonal antibodies as detecting reagents. ~~~~titatio~
of anti-NBs
A quantitation panel was made by diluting an anti-HBs positive plasma into negative human plasma. The quantitation panel consisted of members with antiHBs concentrations of 150, 75, 40, 15 and 6 mIU/ml, respectively. These values were assigned by calibration against a World Health Organization Reference
216
Preparation of 26 January 1977 using the AUSAB RIA assay. The 150 mIU/ml panel member gave absorbances greater than 2.0 in AUSAB rDNA EIA and was not routinely used. Linear correlation coefficients were better than 0.99. The anti-HBs concentration (mIU/ml) in reactive specimens was determined from the best fit line generated from the quantitation panel. Sensitivity of AUSAB assays were calculated in mIU/ml by determining the point at which the assay cut-off value intercepts this line. Other serologic procedures Serum samples were tested with commercial RIA and EIA reagents supplied by Abbott Laboratories: AUSAB and AUSAB EIA for anti-HBs, Monoclonal Auszyme and AUSRIA II for HBsAg, and CORAB for anti-HBc.
-
97.4
--_)
66.2 -_) 42.7 --f
21.5 +
14.4 --_)
1
2
3
4
5
6
7
8
9
Fig. 1. SDS PAGE of hpHBsAg ad (lane 5), hpHBsAg ay (lane 6), three preparations of rHBsAg ad (lanes 2-4). and three preparations of rHBsAg ay (lanes 7-9). Molecular weight markers are shown in lane 1. Running gel of 12% polyacrylamide was used.
217
Results
Characterization
of rHBsAg
Affinity-purified rHBsAg lots were evaluated by SDS polyacrylamide gel electrophoresis. Greater than 95% of all staining was observed in bands at 27 and 24 kDa, co-migrating with gp27 and p24 of hpHBsAg (Fig. 1). Bovine serum albumin from tissue culture media was the major contaminant and represents less than 4% of all staining. Purified hpHBsAg also contains some human serum albumin and IgG as contaminants. A monoclonal antibody, H166, (Peterson et al., 1984) recognizes a unique epitope which is present on hpHBsAg and which is not irreversibly destroyed by reduction/SDS treatment. Using this monoclonal antibody in Western blot experiments, bands at 24 and 27 kDa of rHBsAg reacted strongly corresponding to the gp27 and p24 bands of hpHBsAg. When rHBsAg or hpHBsAg were applied to the gel at high concentrations, higher molecular weight species (corresponding to dimers) are seen (Fig. 2). Purified rHBsAg was negatively stained with 2% phosphotungstic acid (PTA) and examined by transmission electron microscopy (typical lots are shown in Fig. 3). In all preparations, 22 nm rHBsAg particles were indistinguishable from
130 + 75 3
50 3
9P27 ~24 17+
1
2
3
4
Fig. 2. Western blotting of hpHBsAg ad ad (lanes l-3), and three preparations of tibody (IgM), H166, was used to detect chain) conjugated to HRP.
5
8
7
8
9
(lane 2), hpHBsAg ay (lane 6), three preparations of rHBsAg rHBsAg ay (lanes 7-9). An anti-HBs specific monoclonal anHBsAg followed by reaction with goat anti-mouse IgM (mu Precipitating substrate was 4 chloro-1-naphthol.
Fig. 3.
Transmission electron HBsAg preparations
micrographs of hpHBsAg (A), rHBsAg were negatively stained with 2% PTA.
ay (B), and rHBsAg The bar is 100 nm.
ad (C).
219
hpHBsAg preparations in size and morphology. Slightly elongated mo~hological forms could be visualized with low frequency. These morphological features were also seen in hpHBsAg preparations (Fig. 3). Amino and carboxy terminal amino acid sequencing of rHBsAg gave results consistent with those predicted from the S gene DNA sequence. Immunological
reactivity
The specific antigenic activity of purified rHBsAg was evaluated by determining the relative reactivity of these preparations in the Monoclonal Auszyme assay as a function of the protein concentration (determined from absorbance spectra) and was calculated as Monoclonal Auszyme activity (mg/ml)lHBsAg protein concentration (mglml). Three preparations of each of rHBsAg ad and ay subtypes yielded specific antigenic activities of 1.9 ‘- 0.2 which was in good agreement with specific activities of highly purified hpHBsAg. To demonstrate that important S gene antigenic epitopes were present in rHBsAg, the reactivity of rHBsAg and hpHBsAg with seven distinct monoclonal antibodies binding different HBsAg epitopes (five common to ad and ay subtypes (H5, H12, H53, H35, H166), one reactive only with ay (HlO), and one reactive only with ad (H95), were evaluated as described in Methods. A description of these monoclonals is given in Peterson et al. (1984). Results shown in Table 1 demonstrate that all important group specific and ad and ay subtype determinants are quantitatively expressed in rHBsAg. Also anti-y (HIO) and anti-d (H95) monoclonals reacted only with the appropriate ad or ay rHBsAg subtype. To demonstrate the immunological identity of hpHBsAg and rHBsAg, human sera from vaccinees and individuals with natural immunity were adsorbed with rHBsAg (ad and ay) which completely blocked their ability to bind to hpHBsAg in the AUSAB RIA assay. Goats immunized with rHBsAg developed high titers against HBsAg as measured by AUSAB RIA or by AUSAB rDNA assays. This goat sera reactivity against rHBsAg as measured by AUSAB rDNA was completely abolished by preadsorption of the sera with hpHBsAg (data not shown). No unique epitopes in rHBsAg were detectable by these competition studies. Sensitivity and detectability
The performance of AUSAB rDNA RIA and EIA were compared to AUSAB RIA in terms of sensitivity, detectability, and specificity. The detectability of the US Office of Biological Research and Review (OBRR) Anti-HBs reference panel by AUSAB RIA, AUSAB rDNA RIA, and EIA is shown in TabIe.2. In the RIA assays, results are expressed as counts per minute (cpm) and as sample (cpm)/cutoff (cpm), and in the EIA are expressed as absorbance at 492 run and as sample absorbance/cut-off absorbance. These results indicate that all three assays show equivalent detectability. Panel member 9 contains anti-HBs at about 1 mIU/ml and is elevated above the negative control mean for all assays. This member occasionally may give borderline reactivity for these anti-HBs assays.
Antibody gate/label
1.00 1.00 1.00 1.00 0.00 1.00 1.00
02758
ad
1.39 1.14 1.07 0.70 0.00 0.55 0.71
90152
preparations
and rHBsAg
hpHBsAg
Subtype
of hpHBsAg
H166
Relative
H5:HRP H12:HRP H53:HRP H35:HRP HlO:HRP H95:HRP Radioiodinated
conju-
1
reactivity
TABLE (subtypes
1.67 1.75 1.04 1.25 0.00 2.19 1.46
11
rHBsAg
1.45, 1.48 1.55 1.22 0.00 1.75 0.90
12
preparations
ad and CIY)with monoclonal
1.00 1.00 1.00 1.00 1.00 0.00 1.00
95458
hpHBsAg
Subtype
anti-HBs ay
1.10 0.64 0.56 1.46 0.78 0.00 1.00
96565
preparations
1.00 0.52 0.48 0.93 0.77 0.00 0.74
88371
1.34 0.69 1.07 0.57 1.49 0.00 1.91
Y7
rHBsAg
1.36 0.47 1.48 0.77 1.46 0.00 1.26
Y8
preparations
+ + t + + t + + +/t + + + + t + + + -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
%CO=sampIe/cut-off.
Negative control (NCx) Positive control (PCx) Cut-off
AUSAB RIA
Panel member
63 8382 170 Cut-off=2.7 0.4 49.3 1 x NCx
133.3 55.4 34.9 17.4 10.6 6.6 4.0 1.7 1.2 353.6 273.2 204.6 104.1 68.7 29.9 14.0 7.1 3.5 0.4 62 11977 248 Cut-off=4.0
19440 11059 5811 2719 1265 647 288 183 65940 51084 47250 28631 18422 8160 4170 2011 866 48 0.25 48.3 1 x NCx
158.3 78.4 44.6 23.4 11.0 5.1 2.6 1.2 0.7 265.9 206.0 190.5 115.4 74.3 32.9 16.8 8.1 3.5 0.2
S/CO
0.025 0.983 0.055 Cut-off=NCx
>2 1.669 1.078 0.588 0.311 0.172 0.105 0.068 0.047 >2 >2 >2 >2 1.468 0.887 0.418 0.214 0.129 0.029
Absorbance
0.45 39.3 1 + 0.03
36.4 30.3 19.6 10.7 5.7 3.1 1.9 1.2 0.9 36.4 36.4 36.4 36.4 26.7 16.1 7.6 3.9 2.3 0.5
S/CO
CPM
CPM
S/CO”
Procedure A
Procedure A
22662 9418 5936 2953 1801 1121 683 291 197 60120 46443 34784 17692 11676 5089 2377 1199 591 60
AUSAB rDNA EIA Procedure B
AUSAB rDNA RIA
24.7 24.7 22.3 12.2 6.2 3.5 1.8 1.1 0.8 24.7 24.7 24.7 24.7 24.7 19.1 10.0 5.1 2.5 0.4
S/CO
0.031 0.38 1.436 17.7 0.081 1 Cut-off= NCx + 0.05
>2 >2 1.803 0.99 0.499 0.28 ‘0.143 0.089 0.061 >2 >2 >2 >2 >2 1.551 0.813 0.413 0.201 0.032
Absorbance
Procedure B
Comparison of AUSAB RIA and AUSAB rDNA RIA and EIA sensitivity for the detection of anti-HBs in the Ol3RR reference panel
TABLE 2
6 4 2 1 0.6
Bl 2 3 4 5
Negative control (NCx) Positive control (PCx) Cut-off
16 9 3 2 1
Al 2 3 4 5
[Anti-HBs] mIU/ml
Sensitivity panel member
f + +I-
+ + + +/-
59 8459 159
459 340 191 126 85 0.37 53.2 1
2.9 2.1 1.2 0.8 0.5
8.3 4.3 1.8 1.2 0.6
68 11986 272
632 480 240 140 100
1718 1005 353 2.58 1.54
CPM
1323 677 288 192 103
Procedure B s/co
Procedure A CPM
AUSAB RIA AUSAB rDNA RIA
0.25 44.1 1
2.3 1.8 0.9 0.5 0.4
6.3 3.7 1.3 0.9 0.6
s/co
0.02s 0.983 0.055
0.144 0.099 0.058 0.038 0.034
0.334 0.175 0.082 0.06 0.043
Absorbance
Procedure A
0.45 39.3 1
2.6 1.8 1.1 0.7 0.6
6.1 3.2 1.5 1.1 0.8
S/CO
AUSAB rDNA EIA
Comparison of AUSAB RIA and AUSAB rDNA RIA and EIA sensitivity in dilutions of two anti-NBS reactive individuals
TABLE 3
0.031 1.436 0.081
0.175 0.117 0.074 0.053 0.043
0.444 0.235 0.105 0.077 0.067
Absorbance
Procedure B
0.38 17.7 1
2.2 1.4 0.9 0.7 0.5
5.5 2.9 1.3 1.0 0.8
S/CO
223
Two specimens reactive for anti-HBs were diluted into normal human sera devoid of HBsAg and anti-HBs and assayed for anti-HBs by rDNA based assays and AUSAB RIA. Results are shown in Table 3 for AUSAB RIA and both procedure A (2 h/2 h) and procedure B (overnight/2 h) for AUSAB rDNA RIA and AUSAB rDNA EIA. The 3 or 4 mIU/ml specimens were slightly positive for all assays and the 2 mIU/ml specimen was borderline reactive for all assays. Repeat testing of specimens near the cut-off may show these tests to be negative or positive by the three test systems. Sensitivities for all assay runs were routinely calculated using a quantitation panel as described in Materials and Methods. Values for sensitivity were generally 1.5-3.0 mIU/ml for all three anti-HBs assays. Serial bleeds from three vaccinees (‘Heptavax’ or ‘Recombivax’, Merck) and two HBV infected individuals were also tested by all three AUSAB assays and results showed good qualitative and quantitative agreement among assays (Tables 4, 5). Anti-HBs positive population
The detectability of anti-HBs in a population of 225 hepatitis B vaccinees is shown in Fig. 4 AC. All vaccinees were reactive by all three AUSAB assays. Forty-three percent, 40% and 44% of specimens were greater than 100 mIU/ml by AUSAB RIA, AUSAB rDNA RIA, and AUSAB rDNA EIA, respectively. These specimens were not diluted to determine an actual anti-HBs concentration. Thirty to thirty-seven percent of all reactives were less than 10 mIU/ml. These data indicate equivalent detectability between rHBsAg and hpHBsAg based anti-HBs assays. Specificity
A random population (N=385) was evaluated using AUSAB RIA, AUSAB EIA procedure B, and both procedures of AUSAB rDNA RIA and EIA. Overall agreement among the assays was 97.2% (376/385). The nine discrepant results are shown in Table 6. Seven specimens were low level reactive by AUSAB RIA, negative by AUSAB EIA and AUSAB rDNA assays. No reliable confirmatory assay for the detection of low anti-HBs concentrations exists. In the absence of such an assay, we have tested the specimens for the presence of anti-HBc (CORAB) as an indicator of possible previous exposure to hepatitis B. Only one of these seven was also reactive by CORAB. One specimen negative by AUSAB EIA was positive by all other assays including CORAB. One specimen was positive by all AUSAB rDNA procedures, but negative by AUSAB RIA and EIA. This specimen was also positive for anti-HBc. A random serum population (N=lOO) drawn from hospital patients was tested by AUSAB RIA and both procedures of AUSAB rDNA RIA and EIA. Overall agreement was 95% with 14 specimens positive and 81 specimens negative by all assays. A 100% agreement among AUSAB rDNA assays was observed. Discrepant results are shown in Table 6. Similar to the plasma population, four of five discrepant specimens were positive by AUSAB RIA but unreactive in AUSAB rDNA assays and unreactive by CORAB. One discrepant was reactive by AU-
224 TABLE
4
Comparison
of AUSAB
Vaccinee
Time (months)
F (Heptavax)
59 (Recombivax)
64 (Recombivax)
Vaccinees
assays
0 0.5 1 1.5 2 12 0 1 2 3 6 8 12 0 1 2 3 6 8 12
were inoculated
for the detection AUSAB RIA (mIU/ml)
of anti-HBs AUSAB Procedure
in serial bleeds
rDNA
RIA
of three vaccinees
AUSAB
A Procedure
B Procedure
14 >150 >150 >150
13 >150 >150 >150
3 >150 >150 >150
22 44 56 >150 >i50
ND ND ND ND ND ND ND
rDNA
EIA
A Procedure
B
_
_ 44 33 91 >150 >150 with vaccine
16 36 47 >150 138
_ 32 30 67 >150 >150 at 0, 1, and 6 months;
ND ND ND ND ND ND ND
15 50 60 >150 >lSO
ND ND ND ND ND ND ND
35 34 104 >150 >150
ND ND ND ND ND ND ND
_
ND, not done
SAB RIA and CORAB, but unreactive in the AUSAB rDNA assay. These data show good agreement between AUSAB RIA and AUSAB rDNA and indicate no false reactives by AUSAB rONA assays. Most discrepants were less than 10 mIU/ml.
Autoimmune
and disease sera
Specimens from patients with a variety of diseases and viral infections were tested by AUSAB RIA and all procedures of AUSAB rDNA RIA and EIA (Table 7). All specimens were negative for all AUSAB procedures. Also tested by AUSAB RIA, AUSAB rDNA RIA and EIA were specimens from patients with toxoplasmosis (9 samples), Epstein-Barr virus (3 samples), and E. cofi infection (3), IgM anti hepatitis A reactive (6) or specimens containing high titer IgA (5), high titer IgE (3), high titer IgG (5), and high titer IgM (5). All were negative by all three assays.
225 TABLE
5
Comparison uals
of AUSAB
assays
Date
HBV-infected individuals
for the detection
of anti-HBs
AUSAB
AUSAB RIA
RIA
22 13 27 19 22 3
May Jun Jun Jul Aug Jan
+ +
2 May 9 May
_
6
Analysis
of specimens
Specimen
AUSAB RIA (mIU,m,)
_ _ _ -
+ + + + + _
+ + + + + +
_ _ _
+ + +
+ + +
12 19 35
AUSAB
rDNA
RIA
ydure
pdure
17 3 _ _
15 4 +I_
-
+ + + + + +
B
3 65
12 Jul 18 Jul 1 Aug
AUSAB
CORAB
7 101
_ _ _
12 19 46
results
AUSAB ydure
EIA
AUSAB
rDNA
ydure
y
_ _ -
9 +I_
14 3 _ _
_
-
_
-
Plasma P12-31 P12-151 P12-179 13-72 13-84 13-94 13-109 13-118 13-146
2 15 3 22 3
_ _
_ -
_ -
_ _
_ -
-
_
_
_
_
Serum N-15 N-23 N-40 N-48 N-77
3 32 6 9 12
_
_
_ -
_ _ _
ND ND ND ND ND
_ _ _
_ _ -
QNS,
insufficient
quantity.
individ-
Auszyme
+ + + _ _ _
May Jun Jun Jul
giving discordant
B Procedure (mIUim1) _ _ _ -
_ _ _ _ _
23 6 13 5
TABLE
_ _ _
_ _
rDNA
of HBV infected
EIA
Procedure (mIU/ml) A
in serial bleeds
EIA
CORAB
+ _ -
QNS _
_ QNS _
_ _ + _
226 TABLE
7
Lack of AUSAB Disease
crossreactivity ID
type
of sera positive AUSAB
RIA
for various AUSAB
auto-immune rDNA
RIA
and viral antibodies AUSAB Procedure
rDNA
EIA
A
CPM
S/CO”
CPM
s/co
OD
s/co
Y70 Y87 Y77 3118188
130 109 102 128
0.44 0.37 0.35 0.44
34 48 49 48
0.27 0.39 0.40 0.39
0.021 0.026 0.026 0.036
0.28 0.34 0.34 0.47
Y46 YlOo Y93 25
118 140 122 111
0.40 0.48 0.42 0.38
44 35 41 31
0.35 0.28 0.33 0.25
0.022 0.021 0.025 0.022
0.29 0.28 0.33 0.29
Anti-nuclear antibody (ANA)
34-9 140-21 140-13 142-7 142-14 86-12 34-20 140-11 F-130 G623 3860BS
82 108 116 87 137 109 114 102 99 94 117
0.28 0.37 0.40 0.30 0.47 0.37 0.39 0.35 0.34 0.32 0.40
38 38 33 39 70 60 59 64 47 38 51
0.31 0.31 0.27 0.31 0.56 0.48 0.48 0.52 0.38 0.31 0.41
0.047 0.027 0.028 0.027 0.023 0.03 0.02 0.02 0.03 0.029 0.038
0.62 0.36 0.37 0.36 0.30 0.39 0.26 0.26 0.39 0.38 0.50
Rheumatoid arthritis
S121087 S121188
95 110
0.32 0.38
52 48
0.42 0.39
0.034 0.032
0.45 0.42
Anti-CMV
1 4
107 102
0.37 0.35
34 52
0.27 0.42
0.036 0.033
0.47 0.43
Systemic lupus erythrematosus (SLE)
Rheumatoid
factor
“S/CO=Specimen
value/Cut-off
value.
S/CO
are considered
negative.
Clinical studies Three different laboratories tested a total of 1711 specimens by AUSAB RIA and by both procedures of AUSAB rDNA and AUSAB rDNA EIA. These specimens were derived from random donors, individuals previously determined to be reactive by AUSAB RIA, patients with autoimmune disease, hospital employees, residents of mental institutions, patients diagnosed with hepatitis, vaccine recipients, dialysis patients, medical students, and patients with other diseases. Total agreement among all assays was 98.6% ([1711-24]/1711) with 24 discordants. All 24 discordants were less than 10 mIU/ml. Nine specimens were low level positive only by AUSAB RIA but negative by all rDNA assays and also negative by CORAB. Nine specimens were marginally positive for all AUSAB RIA procedures but negative by rDNA EIA assays. Almost all of these specimens were
227
120 110
AUSAB
120
<30
c50
c40
120
AUSAB
<70
c60
[Anti-HRs]
110
A
RIA
‘~1
-30
490
>I00
-330
<90
,100
mllliml
rDNA
RIA
100
<2Q
<30
c40
c50
[Anti-HBsl
120 110
AUSAB
<60
<70
mlU/ml
rDNA
EIA
_
c20
<30
<70 c40 <50 ~60 IAnti-HBsl mlU/ml
CEO
<90
c
4
>I00
Fig. 4. Frequency histogram of anti-HBs concentration in a population of 225 hepatitis B vaccine recipients measured by AUSAB RIA (A), AUSAB rDNA RIA (B), and AUSAB rDNA EIA (C). Procedure A was used for both rDNA based assays. Specimens were not diluted prior to testing and specimens with anti-HBs concentrations greater than 100 mIU/ml gave absorbances greater than 2.0 in the EIA and are reported as > 100 mIU/mi. Anti-HBs concentrations below this value were calculated for each specimen using a quantitation standard panel as described in Methods.
very low level reactives (less than 5 mIU/ml). Six specimens were repeatably borderline reactive in one or more anti-HBs assays. Overall there was almost 100% agreement among all assays for specimens above 10 mIU/ml by AUSAB and 73% ([90-24]/90) agreement for samples greater than the cut-off but less than 10 mIUlm1.
228 8) AUSAB
RIA
A
SAMPWCPM)/NCx(CPM)
AUSAB rDNA RIA-Procedure
AUSAB
rONA EIA-Procedure
B
B
C
iuz
a-
a a
0” 91. 5 g 43. 0 30. al-
e w
F1 i;
NET ABSORBANCE
(492 nm)
229
The frequency distribution of anti-HBs in 292 HBsAg negative plasma by AUSAB and AUSAB rDNA RIA and EIA (procedure B only) is shown in Fig. 5. Seven specimens (2.4% of total specimens) were reactive and the remainder were negative for anti-HBs in all three assays. The negative population mean was close to the negative control mean and well separated from the calculated cut-off value.
Second generation assays which use rHI3sAg for the detection and quantitation of anti-HBs in human sera were developed. Recombinant HBsAg has been cloned successfully and expressed in a variety of eukaryotic cells ~~lu~g CHO and yeast. In this report the rHBsAg was produced in mouse L cells and was antigenically indistinguishable from purified hpHBsAg. Recombinant HBsAg provides several advantages over the hpI-IBsAg used in previously described anti-HBs assays (~ushahwar, 1987; ~ushahwar and Spiezia, 1987). Recombinant rHBsAg is produced at high concentrations (up to 500 pglml) in tissue culture hollow fiber systems, may be easily purified to greater than 95% homogeneity by affinity methods, gives better reproducibility in purity than hpHBsAg, and is not infectious and dues not require BL3 biosafety containment facilities for production. The rHBsAg also shows excellent stability and storage characteristics comparable or superior to purified hpHBsAg (data not shown). The high specificity of rHBsAg based anti-HBs assays was demonstrated by examining large populations of random blood donors, hospital patients, and autoimmune and disease specimens, and comparing these results with AUSAB RIA. The AUSAB RIA and AUSAB rDNA assays gave similar sensitivity and detectability when anti-HBs panels, hepatitis B vaecinees, and HBV infected individuals were tested. These assays measure the same molecules and give good quantitative agreement. One hundred percent a~eement among anti-N& assays was observed for specimens having anti-HBs concentrations greater than 10 mIU/ml. The few discrepancies occurred with specimens having less than 10 mIU/ml, although there was 73% agreement among all assays for specimens less than 10 mIU/ml. The discordant specimens generally fell into two groups: specimens reactive by AUSAB-RIA only and negative by all other tests including CORAB and speci-
Fig, 5. Histogram of sample (~Pm)/negative control mean (cpm) and net absorbance (492 nm) values obtained when plasma from 292 random blood donors was tested by AUSAB RIA (A), AUSAB rDNA RIA (B), and AUSAB rDNA EIA (C). Sample (cpm) divided by NCx (cpm) for each specimen was calculated for RIA and the net absorbance (sample absorbance - NCx absorbance) for each specimen was calculated for the EIA. Procedure B was used for the rDNA based assays. Specimens giving sample!NCx or net absorbance values greater than the cut-off (indicated by arrow) are considered reactive in the assay.
230
mens with borderline reactivity by AUSAB RIA and AUSAB rDNA RIA but negative by AUSAB rDNA EIA. Since the specimens reactive by AUSAB RIA only were also unreactive for anti-HBc, it is unlikely that these specimens were derived from individuals with previous exposure to hepatitis B virus. In the absence of a reliable confirmatory procedure for these low level AUSAB reactives, it is not possible to determine whether the reactivity may result from a nonspecific binding component. Kessler et al. (1985) demonstrated that a small number of specimens contained a reduction sensitive component that reacted in the AUSAB assay. These individuals with low AUSAB reactivity may not have protective levels of anti-HBs (Hadler et al., 1986). Results from the second class of discordants which are marginally reactive by RIA assays but negative by rDNA EIA procedures suggest that for a small percentage of specimens the RIA procedure may be slightly more sensitive than the EIA. Our results indicate that most of these specimens have primarily antibody against the d epitope since they are not reactive with HBsAg (uy subtype) (Decker et al., 1988). The protective efficacy of anti-HBs subtype antibodies is presently unclear (Shiels et al., 1987). Most importantly in these studies, no apparent false reactives were observed by rDNA based assays and overall agreement with AUSAB RIA was greater than 98%.
Acknowledgements We are grateful to Jeff Farmer, Kashfinur Heine, Carlos Maldonado and Bernie Schleicher for their help in production of rHBsAg. We also thank Laurie Metz for typing this manuscript and Mahlon Miller for electron microscopy work. We thank Robert Penillo, Gary Tegtmeier, Eugene Schiff and Maria DeMedina for their help in clinical trials.
References Adamowicz, Ph., Tron, F., Vinas, R., Mevelec, M., Diaz, I., Courouce, A., Mazert, M., Lagarde, D. and Girard, M. (1988) Hepatitis B vaccine containing the S and preS-2 antigens produced in Chinese hamsters ovary cells. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 1087-1090. Andre, F. and Safary, A. (1988) Clinical experience with a yeast derived hepatitis B vaccine. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 1025-1030. Decker, R., Kuhns, M., Brawner, T. and Mimms, L. (1988) Future advanced diagnostic techniques for hepatitis B. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 231-236. Galibert, F., Mandart, E., Fitoussi, F., Tiollais, P. and Charnay, P. (1979) Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature (London) 281, 64&650. Gerety, R. (1988) Recombinant hepatitis B vaccine. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 1017-1024. Gerin, J.L., Faust, R.M. and Holland, P.V. (1975) Biophysical characterization of the adr subtype of hepatitis B antigen and preparation of anti-r sera in rabbits. J. Immunol. 115, 100-105. Hadler, S., Francis, D., Maynard, J. et al. (1986) Long term immunogenicity and efficacy of hepatitis B vaccine in homosexual men. N. Engl. J. Med. 315, 209-214.
231 Kessler, H., Harris, A., Payner, J., Hudson, E., Potkin, B. and Levin, S. (1985) Antibodies to hepatitis B surface antigen as the sole hepatitis B marker in hospital personnel. Ann. Intern. Med. 103, 21-26. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature (London) 227, 680-685. Maxam, A. and Gilbert, W. (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Methods Enzymol. 65, 499-560. Mushahwar, I.K. and Overby, L.R. (1981) An immunoassay for hepatitis Be antigen and antibody. J. Virol. Methods 3, 89-97. Mushahwar, I. (1987) A biotimavidin solid-phase sandwich enzyme immunoassay for the antibody to hepatitis B surface antigen (anti-HBs). J. Virol. Methods 16, l-11. Mushahwar, I. and Spiezia, K. (1987) The utilization of biotimanti-biotin interaction for the detection of antibody to hepatitis B surface antigen (anti-HBs). J. Viral. Methods 16, 45-54. Nakane, P.K. and Kawaoi, A. (1974) Peroxidase labeled antibody: a new method of conjugation. J. Histochem. Cytochem. 22, 1084-1091. Newman, P., Kahn, R. and Hines, A. (1981) Detection and characterization of monoclonal antibodies to platelet membrane proteins. J. Cell Biol. 90, 249-253. Peterson, D., Paul, D., Lam, J., Tribby, I. and Achord, D. (1984) Antigenic structure of hepatitis B surface antigen: identification of the ‘d’ subtype determinant by clinical modification and use of monoclonal antibodies. J. Immunol. 132, 920-927. Post, L., Conley, A., Mocarski, E. and Roizman, B. (1980) Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences and BamHl fragments. Proc. Natl. Acad. Sci. USA 77, 4201-4205. Shiels, M., Taswell, H., Czaja, A., Nelson, C. and Swenke, P. (1987) Frequency and significance of concurrent hepatitis B surface antigen and antibody in acute and chronic hepatitis B. Gastroenterology 93, 675-680. Tiollais, P., Pourcel, C. and Dejean, A. (1985) The hepatitis B virus. Nature (London) 317, 489-495. Towbin, H., Staehelin and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4353. Valenzuela, P., Quiroga, M., Zalvidar, J., Gray, P. and Rutter, W.J. (1980) The nucleotide sequence of the hepatitis B viral genome and the identification of the major viral genes. In: B. Fields and R. Jaenisch (Eds.), Animal Virus Genetics, pp. 57-70. Yano, M. and Tashiro, A. (1988) Clinical evaluation of recombinant yeast derived hepatitis B vaccine. In: A.J. Zuckerman (Ed.), Viral Hepatitis and Liver Disease, pp. 1050-1052.
Methods,
211
25 (1989) 211-232
00907
Second generation assays for the detection of antibody to HBsAg using recombinant DNA-derived HBsAg Larry Mimms, Andrew Goetze, Suzanne Swanson, Marco Floreani, Brook Edwards, Jerzy Macioszek, Greg Okasinski and Willie Kiang HepatitislAIDS
Research,
Abbott
Laboratories,
(Accepted
31 March
Abbott
Park, IL 60064, U.S.A.
1989)
Summary
A second generation radioimmunoassay (RIA) and enzyme-linked immunoassay (EIA) for the detection and quantitation of the antibody to hepatitis B surface antigen (anti-HBs) was developed which utilizes recombinant DNA-derived HBsAg (rHBsAg) in place of human plasma derived HBsAg. In these sandwich assays, rHBsAg immobilized on a solid phase was used to capture anti-HBs from the specimen and rHBsAg conjugated to horseradish peroxidase or radiolabeled with 1251was used as a detecting reagent. These rHBsAg-based assays were compared to a commercial radioimmunoassay for anti-HBs detection (AUSAB RIA). For a population of 1711 sera and plasma specimens, 99.2% overall agreement was demonstrated between the recombinant RIA and EIA and 98.6% agreement was observed between the recombinant assays and AUSAB-RIA. The recombinant assays demonstrated equivalent sensitivity and detectability to AUSAB RIA. Most discrepant samples were low-level reactive by AUSAB-RIA, generally less than 10 mIU/ml, and likely represent nonspecific reactivity since no other marker for hepatitis B infection was detected in these samples. Anti-HBs; Enzyme-linked immunoassay; HBsAg; Hepatitis B serological marker
Radioimmunoassay;
Correspondence U.S.A.
Abbott
to;
0166~0934/89/$03.50
L. Mimms,
Hepatitis/AIDS
6J 1989 Elsevier
Science
Research,
Publishers
B.V.
Laboratories,
(Biomedical
Abbott
Division)
Recombinant
Park, IL 60064,
212
Introduction The antibody to hepatitis B surface antigen (anti-HBs) is a long-persisting antibody associated with convalescence and recovery from an acute hepatitis B viral infection. The antibody is also detectable in serum after an immune response to hepatitis B vaccine. A commercially available direct solid-phase radioimmunoassay (RIA) for antiHBs detection and quantitation has been available since 197.5 (AUSAB, Abbott Laboratories). In this assay, the serum or plasma specimen is incubated with a solidphase (polystyrene bead) that has been coated with human plasma-derived HBsAg (hpHBsAg). If anti-HBs is present in the serum, it will bind to the solid-phase antigen. In a second step, radiolabeled HBsAg (‘251-HBsAg) is added and reacts with the immobilized antibody. The amount of radiolabel bound to the solid phase within limits is in direct proportion to the amount of anti-HBs in the original specimens. An enzyme-linked immunoassay (EIA) version of this test (AUSAB EIA) has been available since 1983 and uses a biotinylated HBsAg, horseradish peroxidase (HRP) conjugated avidin mixture as a probe ‘detecting’ reagent (Mushahwar, 1987). In an improved version of this test, a biotinjanti-biotin detection system was substituted for the biotimavidin detection reagent (Mushahwar and Spiezia, 1987). In this paper we describe a second generation RIA and EIA for the detection of anti-HBs in which the hpHBsAg is replaced with recombinant DNA-derived HBsAg (rHBsAg). Purified hpHBsAg consists primarily of 22 nm spherical particles with an estimated molecular weight of 2-5 x lo3 kDa. The major protein constituent is 226 amino acids long, is encoded by the hepatitis B virus (HBV) S gene, and exists in the particle as two species: glycosylated, gp27 (27 kDa) and nonglycosylated, p24 (24 kDa) (Tiollais et al., 1985). The hepatitis S gene for both adw, and ayw subtypes was cloned and expressed separately in mouse L cells which can secrete HBsAg particles 22 nm in diameter and morphologically indistinguishable from hpHBsAg particles. The rHBsAg particles were affinity purified and shown to contain the important HBsAg epitopes as determined by reactivity with monoclonal anti-HBs antibodies. Although several rHBsAg containing vaccines have been produced (Gerety, 1988; Andre and Stafary, 1988; Yano and Tashiro, 1988; Adamowicz et al., 1988), no rHBsAg based anti-HBs assays have been described for monitoring vaccinees. rHBsAg offers advantages over hpHBsAg in ease of purification, greater consistency in production and elimination of the risks associated with working with potentially infectious human plasma. The performance of the recombinant anti-HBs assays was compared to AUSAB RIA and showed equivalent sensitivity and detectability. Data indicate that these assays may provide improved specificity compared to AUSAB RIA.
213
Materials and Methods Reagents
Horseradish peroxidase (HRP, RZ B 3) was purchased from Sigma (St. Louis, MO) or from Toyoba Chemical (New York, NY). All SDS PAGE and Western blot chemicals and lactoperoxidase/glu~ose oxidase beads were obtained from BioRad tRi~hmond, CA). All ~hromatographic media were from Pharmacia (Piscatoway, NJ). Naiz51 was obtained from Amersham (Arlington Heights, IL). All monoclonai antibodies against HBsAg were from Abbott Laboratories (N. Chicago, IL). Production of rHBsAg
Both major subtypes of HBsAg (adw, and ayw) were cloned and expressed separately in mouse L cells. HBV DNA was isolated from HBV particles purified from human plasma. A 1.37 kb BamHl fragment containing the HBV S gene was inserted into plasmid containing thymidine kinase or neomycin as selectable markers. The herpes simplex virus ICP4 promoter was used to direct synthesis of the S gene (Post et al., 1980). The BamHl fragment of expression plasmids containing the HBV S gene was sequenced by the method of Maxam and Gilbert (1980). Nucleotide sequences for subtype ayw and adw, coded for amino acid sequences identical to those published by Galibert et al. (1979) and Valenzuela et al. (1980). Secreted rHBsAg levels as high as 500 wg/ml were obtained in hollow fiber tissue culture systems. Pooled cell media containing rHBsAg was clarified by centrifugation and applied to an anti-HBs affinity column. After binding to the column, rHBsAg was specifically eluted with 0.02 M citrate, 0.5 N NaCl, pH 2.3. The pH of the eluate was immediately adjusted to neutral using 1.0 M borate buffer. Peak fractions were pooled, then concentrated and dialyzed against phosphate buffered saline. rHBsAg adw, and ayw subtypes were purified separately on immuno affinity columns dedicated to each subtype. Purification of hpHBsAg
Human plasma units containing HBsAg (adw, or ayw subtype) with a rheophoresis titer of 2 1:32 were used as starting material. The antigen was purified according to an established procedure (Gerin et al., 1975). The final product was dialyzed overnight at 4°C against 0.1 M potassium phosphate, 0.1 M NaCl buffer (PH 8). Characterization
of HBsAg
Purified HBsAg was evaluated by SDS polyacrylamide gel electrophoresis by the method of Laemmli (1970). Gels were stained with Coomassie blue and purity was
214
quantified using a gel scanning densitometer. cording to Towbin et al. (1979). rHBsAg
Western
blotting
was performed
ac-
coated beads (subtype ad and ay)
Polystyrene beads (6 mm in diameter) were coated with rHBsAg (1:l ratio of ay and ad subtypes) at total concentration of 1 kg/ml for 2 h at 40°C. The beads were rinsed with PBS, then incubated with a PBS solution containing 3% BSA or less for 1 h at 40°C. Beads were rinsed with PBS then allowed to air dry. Radioiodination
of rHBsAg
Affinity purified rHBsAg was radioiodinated using a lactoperoxidase catalyzed reaction (Newman et al., 1981). In this procedure, Na1251, immobilized lactoperoxidase/glucose oxidase and HBsAg were allowed to react for 20-25 min, and the reaction mixture was passed over a Sephadex G-50 column to separate free from bound [ 1251]. Peak fractions of “‘1 rHBsAg were pooled then applied to a Sephacryl S-300 column. Peak fractions were counted for radioactivity and pooled. Recombinant HBsAg ad and ay were iodinated and purified separately, diluted into tracer diluent containing animal sera and plasma to produce solutions with radioactivity levels between 0.3-0.8 t.rCi/ml and then pooled at 1:l v/v ratio producing a final tracer solution. rHBsAg:HRP
conjugate
Affinity-purified rHBsAg ad and ay were each conjugated separately to horseradish peroxidase (HRP) as described by Nakane and Kawaoi (1974). Conjugated rHBsAg ad and ay were then mixed 1: 1 (w/w protein) and diluted into a diluent containing animal sera and human plasma to produce a final conjugate solution of approximately 1 &ml total conjugate concentration. RIA for anti-HBs using rHBsAg
(AUSAB
rDNA RIA)
Two procedures for AUSAB rDNA RIA were developed. Sample (0.2 ml) was either incubated for 16-20 h with the rHBsAg-coated bead at room temperature (procedure B) or for 2 h at 40°C (procedure A); after washing with water, the bead was incubated with 0.2 ml of 1251-labeled rHBsAg for 2 h at 40°C. The bead was then washed with water and counted for radioactivity in an ANSR@ gamma counter (Abbott Laboratories). Specimens with cpm greater than or equal to the cut-off (negative control mean (NCx) times 2.7 for procedure A and NCx times 4.0 for procedure B) were considered to be reactive by the criteria of this test. For the calculation of the NCx, eight replicates of the negative control (NC) were used.
215
EIA for anti-HBs using rHBsAg
(AUSAB
rDNA EIA)
The sample (0.2 ml) was either incubated for 16-20 h with the rHBsAg-coated bead at room temperature (procedure B) or for 2 h at 40°C (procedure A). After washing with water, the bead was further incubated with 0.2 ml of rHBsAg:HRP conjugate at 40°C for 2 h. The bead was then washed and a substrate solution (0.3 ml of 0.3% Q-phenyle~ediamine 2 HCI (OPD) in 0.1 M citrate phosphate buffer (pH 5.5) containing 0.02% H202) was added (Mushahwar and Overby, 1981). The enzymatic 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 developed as a result of the enzymatic catalysis of the substrate was measured at 492 nm by using the Quantum II Spectrophotometer (Abbott Laboratories). Specimens with absorbancy equal to or greater than the cut-off (NCx absorbance + 0.03 or NCx + 0.05 for procedures A or B, respectively) were considered to be reactive. For the calculation of the NCx, three replicates of the NC were used. ~0~o~lonaL anti-H&3 reactivity
HBsAg protein concentration in mg/ml was calculated from the following formula: (absorbance at 280 nm - absorbance at 320 nm)/2.5. To determine reactivity of HBsAg with various mon~lonal anti-I-Es antibo~es, HBsAg preparations were diluted quantitatively into human plasma which was negative for HBsAg and antiHBs (NHP) to approximately 1 nglml and these solutions were then tested for antigenic activity. The specimen (0.2 ml) was incubated with a Monoclonal Auszyme bead (Abbott Laboratories) for 2 h at 40°C. The bead was washed and then incubated for 2 h at 40°C with 0.2 ml of the detecting reagent which contained appropriate concentrations of an HRP conjugated or radioiodinated monoclonal antiHE& diluted into NHP. The bead was washed and counted sedately in a gamma counter for tests using a radiolabeled antibody or the bead was incubated with OPD substrate solution for tests using an HRP conjugated antibody. A HBsAg ad sensitivity panel (Abbott Laboratories) was used as a calibration standard for evaluating HBsAg ad subtype preparations, and a HBsAg ay sensitivity panel (Abbott Laboratories) was used for ay preparations. The specific antigenic activity was calculated as relative antigenic activity (determined from the immunoassay described above) divided by the protein concentration. A specific antigenic activity for various HBsAg preparations was evaluated using seven different monoclonal antibodies as detecting reagents. ~~~~titatio~
of anti-NBs
A quantitation panel was made by diluting an anti-HBs positive plasma into negative human plasma. The quantitation panel consisted of members with antiHBs concentrations of 150, 75, 40, 15 and 6 mIU/ml, respectively. These values were assigned by calibration against a World Health Organization Reference
216
Preparation of 26 January 1977 using the AUSAB RIA assay. The 150 mIU/ml panel member gave absorbances greater than 2.0 in AUSAB rDNA EIA and was not routinely used. Linear correlation coefficients were better than 0.99. The anti-HBs concentration (mIU/ml) in reactive specimens was determined from the best fit line generated from the quantitation panel. Sensitivity of AUSAB assays were calculated in mIU/ml by determining the point at which the assay cut-off value intercepts this line. Other serologic procedures Serum samples were tested with commercial RIA and EIA reagents supplied by Abbott Laboratories: AUSAB and AUSAB EIA for anti-HBs, Monoclonal Auszyme and AUSRIA II for HBsAg, and CORAB for anti-HBc.
-
97.4
--_)
66.2 -_) 42.7 --f
21.5 +
14.4 --_)
1
2
3
4
5
6
7
8
9
Fig. 1. SDS PAGE of hpHBsAg ad (lane 5), hpHBsAg ay (lane 6), three preparations of rHBsAg ad (lanes 2-4). and three preparations of rHBsAg ay (lanes 7-9). Molecular weight markers are shown in lane 1. Running gel of 12% polyacrylamide was used.
217
Results
Characterization
of rHBsAg
Affinity-purified rHBsAg lots were evaluated by SDS polyacrylamide gel electrophoresis. Greater than 95% of all staining was observed in bands at 27 and 24 kDa, co-migrating with gp27 and p24 of hpHBsAg (Fig. 1). Bovine serum albumin from tissue culture media was the major contaminant and represents less than 4% of all staining. Purified hpHBsAg also contains some human serum albumin and IgG as contaminants. A monoclonal antibody, H166, (Peterson et al., 1984) recognizes a unique epitope which is present on hpHBsAg and which is not irreversibly destroyed by reduction/SDS treatment. Using this monoclonal antibody in Western blot experiments, bands at 24 and 27 kDa of rHBsAg reacted strongly corresponding to the gp27 and p24 bands of hpHBsAg. When rHBsAg or hpHBsAg were applied to the gel at high concentrations, higher molecular weight species (corresponding to dimers) are seen (Fig. 2). Purified rHBsAg was negatively stained with 2% phosphotungstic acid (PTA) and examined by transmission electron microscopy (typical lots are shown in Fig. 3). In all preparations, 22 nm rHBsAg particles were indistinguishable from
130 + 75 3
50 3
9P27 ~24 17+
1
2
3
4
Fig. 2. Western blotting of hpHBsAg ad ad (lanes l-3), and three preparations of tibody (IgM), H166, was used to detect chain) conjugated to HRP.
5
8
7
8
9
(lane 2), hpHBsAg ay (lane 6), three preparations of rHBsAg rHBsAg ay (lanes 7-9). An anti-HBs specific monoclonal anHBsAg followed by reaction with goat anti-mouse IgM (mu Precipitating substrate was 4 chloro-1-naphthol.
Fig. 3.
Transmission electron HBsAg preparations
micrographs of hpHBsAg (A), rHBsAg were negatively stained with 2% PTA.
ay (B), and rHBsAg The bar is 100 nm.
ad (C).
219
hpHBsAg preparations in size and morphology. Slightly elongated mo~hological forms could be visualized with low frequency. These morphological features were also seen in hpHBsAg preparations (Fig. 3). Amino and carboxy terminal amino acid sequencing of rHBsAg gave results consistent with those predicted from the S gene DNA sequence. Immunological
reactivity
The specific antigenic activity of purified rHBsAg was evaluated by determining the relative reactivity of these preparations in the Monoclonal Auszyme assay as a function of the protein concentration (determined from absorbance spectra) and was calculated as Monoclonal Auszyme activity (mg/ml)lHBsAg protein concentration (mglml). Three preparations of each of rHBsAg ad and ay subtypes yielded specific antigenic activities of 1.9 ‘- 0.2 which was in good agreement with specific activities of highly purified hpHBsAg. To demonstrate that important S gene antigenic epitopes were present in rHBsAg, the reactivity of rHBsAg and hpHBsAg with seven distinct monoclonal antibodies binding different HBsAg epitopes (five common to ad and ay subtypes (H5, H12, H53, H35, H166), one reactive only with ay (HlO), and one reactive only with ad (H95), were evaluated as described in Methods. A description of these monoclonals is given in Peterson et al. (1984). Results shown in Table 1 demonstrate that all important group specific and ad and ay subtype determinants are quantitatively expressed in rHBsAg. Also anti-y (HIO) and anti-d (H95) monoclonals reacted only with the appropriate ad or ay rHBsAg subtype. To demonstrate the immunological identity of hpHBsAg and rHBsAg, human sera from vaccinees and individuals with natural immunity were adsorbed with rHBsAg (ad and ay) which completely blocked their ability to bind to hpHBsAg in the AUSAB RIA assay. Goats immunized with rHBsAg developed high titers against HBsAg as measured by AUSAB RIA or by AUSAB rDNA assays. This goat sera reactivity against rHBsAg as measured by AUSAB rDNA was completely abolished by preadsorption of the sera with hpHBsAg (data not shown). No unique epitopes in rHBsAg were detectable by these competition studies. Sensitivity and detectability
The performance of AUSAB rDNA RIA and EIA were compared to AUSAB RIA in terms of sensitivity, detectability, and specificity. The detectability of the US Office of Biological Research and Review (OBRR) Anti-HBs reference panel by AUSAB RIA, AUSAB rDNA RIA, and EIA is shown in TabIe.2. In the RIA assays, results are expressed as counts per minute (cpm) and as sample (cpm)/cutoff (cpm), and in the EIA are expressed as absorbance at 492 run and as sample absorbance/cut-off absorbance. These results indicate that all three assays show equivalent detectability. Panel member 9 contains anti-HBs at about 1 mIU/ml and is elevated above the negative control mean for all assays. This member occasionally may give borderline reactivity for these anti-HBs assays.
Antibody gate/label
1.00 1.00 1.00 1.00 0.00 1.00 1.00
02758
ad
1.39 1.14 1.07 0.70 0.00 0.55 0.71
90152
preparations
and rHBsAg
hpHBsAg
Subtype
of hpHBsAg
H166
Relative
H5:HRP H12:HRP H53:HRP H35:HRP HlO:HRP H95:HRP Radioiodinated
conju-
1
reactivity
TABLE (subtypes
1.67 1.75 1.04 1.25 0.00 2.19 1.46
11
rHBsAg
1.45, 1.48 1.55 1.22 0.00 1.75 0.90
12
preparations
ad and CIY)with monoclonal
1.00 1.00 1.00 1.00 1.00 0.00 1.00
95458
hpHBsAg
Subtype
anti-HBs ay
1.10 0.64 0.56 1.46 0.78 0.00 1.00
96565
preparations
1.00 0.52 0.48 0.93 0.77 0.00 0.74
88371
1.34 0.69 1.07 0.57 1.49 0.00 1.91
Y7
rHBsAg
1.36 0.47 1.48 0.77 1.46 0.00 1.26
Y8
preparations
+ + t + + t + + +/t + + + + t + + + -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
%CO=sampIe/cut-off.
Negative control (NCx) Positive control (PCx) Cut-off
AUSAB RIA
Panel member
63 8382 170 Cut-off=2.7 0.4 49.3 1 x NCx
133.3 55.4 34.9 17.4 10.6 6.6 4.0 1.7 1.2 353.6 273.2 204.6 104.1 68.7 29.9 14.0 7.1 3.5 0.4 62 11977 248 Cut-off=4.0
19440 11059 5811 2719 1265 647 288 183 65940 51084 47250 28631 18422 8160 4170 2011 866 48 0.25 48.3 1 x NCx
158.3 78.4 44.6 23.4 11.0 5.1 2.6 1.2 0.7 265.9 206.0 190.5 115.4 74.3 32.9 16.8 8.1 3.5 0.2
S/CO
0.025 0.983 0.055 Cut-off=NCx
>2 1.669 1.078 0.588 0.311 0.172 0.105 0.068 0.047 >2 >2 >2 >2 1.468 0.887 0.418 0.214 0.129 0.029
Absorbance
0.45 39.3 1 + 0.03
36.4 30.3 19.6 10.7 5.7 3.1 1.9 1.2 0.9 36.4 36.4 36.4 36.4 26.7 16.1 7.6 3.9 2.3 0.5
S/CO
CPM
CPM
S/CO”
Procedure A
Procedure A
22662 9418 5936 2953 1801 1121 683 291 197 60120 46443 34784 17692 11676 5089 2377 1199 591 60
AUSAB rDNA EIA Procedure B
AUSAB rDNA RIA
24.7 24.7 22.3 12.2 6.2 3.5 1.8 1.1 0.8 24.7 24.7 24.7 24.7 24.7 19.1 10.0 5.1 2.5 0.4
S/CO
0.031 0.38 1.436 17.7 0.081 1 Cut-off= NCx + 0.05
>2 >2 1.803 0.99 0.499 0.28 ‘0.143 0.089 0.061 >2 >2 >2 >2 >2 1.551 0.813 0.413 0.201 0.032
Absorbance
Procedure B
Comparison of AUSAB RIA and AUSAB rDNA RIA and EIA sensitivity for the detection of anti-HBs in the Ol3RR reference panel
TABLE 2
6 4 2 1 0.6
Bl 2 3 4 5
Negative control (NCx) Positive control (PCx) Cut-off
16 9 3 2 1
Al 2 3 4 5
[Anti-HBs] mIU/ml
Sensitivity panel member
f + +I-
+ + + +/-
59 8459 159
459 340 191 126 85 0.37 53.2 1
2.9 2.1 1.2 0.8 0.5
8.3 4.3 1.8 1.2 0.6
68 11986 272
632 480 240 140 100
1718 1005 353 2.58 1.54
CPM
1323 677 288 192 103
Procedure B s/co
Procedure A CPM
AUSAB RIA AUSAB rDNA RIA
0.25 44.1 1
2.3 1.8 0.9 0.5 0.4
6.3 3.7 1.3 0.9 0.6
s/co
0.02s 0.983 0.055
0.144 0.099 0.058 0.038 0.034
0.334 0.175 0.082 0.06 0.043
Absorbance
Procedure A
0.45 39.3 1
2.6 1.8 1.1 0.7 0.6
6.1 3.2 1.5 1.1 0.8
S/CO
AUSAB rDNA EIA
Comparison of AUSAB RIA and AUSAB rDNA RIA and EIA sensitivity in dilutions of two anti-NBS reactive individuals
TABLE 3
0.031 1.436 0.081
0.175 0.117 0.074 0.053 0.043
0.444 0.235 0.105 0.077 0.067
Absorbance
Procedure B
0.38 17.7 1
2.2 1.4 0.9 0.7 0.5
5.5 2.9 1.3 1.0 0.8
S/CO
223
Two specimens reactive for anti-HBs were diluted into normal human sera devoid of HBsAg and anti-HBs and assayed for anti-HBs by rDNA based assays and AUSAB RIA. Results are shown in Table 3 for AUSAB RIA and both procedure A (2 h/2 h) and procedure B (overnight/2 h) for AUSAB rDNA RIA and AUSAB rDNA EIA. The 3 or 4 mIU/ml specimens were slightly positive for all assays and the 2 mIU/ml specimen was borderline reactive for all assays. Repeat testing of specimens near the cut-off may show these tests to be negative or positive by the three test systems. Sensitivities for all assay runs were routinely calculated using a quantitation panel as described in Materials and Methods. Values for sensitivity were generally 1.5-3.0 mIU/ml for all three anti-HBs assays. Serial bleeds from three vaccinees (‘Heptavax’ or ‘Recombivax’, Merck) and two HBV infected individuals were also tested by all three AUSAB assays and results showed good qualitative and quantitative agreement among assays (Tables 4, 5). Anti-HBs positive population
The detectability of anti-HBs in a population of 225 hepatitis B vaccinees is shown in Fig. 4 AC. All vaccinees were reactive by all three AUSAB assays. Forty-three percent, 40% and 44% of specimens were greater than 100 mIU/ml by AUSAB RIA, AUSAB rDNA RIA, and AUSAB rDNA EIA, respectively. These specimens were not diluted to determine an actual anti-HBs concentration. Thirty to thirty-seven percent of all reactives were less than 10 mIU/ml. These data indicate equivalent detectability between rHBsAg and hpHBsAg based anti-HBs assays. Specificity
A random population (N=385) was evaluated using AUSAB RIA, AUSAB EIA procedure B, and both procedures of AUSAB rDNA RIA and EIA. Overall agreement among the assays was 97.2% (376/385). The nine discrepant results are shown in Table 6. Seven specimens were low level reactive by AUSAB RIA, negative by AUSAB EIA and AUSAB rDNA assays. No reliable confirmatory assay for the detection of low anti-HBs concentrations exists. In the absence of such an assay, we have tested the specimens for the presence of anti-HBc (CORAB) as an indicator of possible previous exposure to hepatitis B. Only one of these seven was also reactive by CORAB. One specimen negative by AUSAB EIA was positive by all other assays including CORAB. One specimen was positive by all AUSAB rDNA procedures, but negative by AUSAB RIA and EIA. This specimen was also positive for anti-HBc. A random serum population (N=lOO) drawn from hospital patients was tested by AUSAB RIA and both procedures of AUSAB rDNA RIA and EIA. Overall agreement was 95% with 14 specimens positive and 81 specimens negative by all assays. A 100% agreement among AUSAB rDNA assays was observed. Discrepant results are shown in Table 6. Similar to the plasma population, four of five discrepant specimens were positive by AUSAB RIA but unreactive in AUSAB rDNA assays and unreactive by CORAB. One discrepant was reactive by AU-
224 TABLE
4
Comparison
of AUSAB
Vaccinee
Time (months)
F (Heptavax)
59 (Recombivax)
64 (Recombivax)
Vaccinees
assays
0 0.5 1 1.5 2 12 0 1 2 3 6 8 12 0 1 2 3 6 8 12
were inoculated
for the detection AUSAB RIA (mIU/ml)
of anti-HBs AUSAB Procedure
in serial bleeds
rDNA
RIA
of three vaccinees
AUSAB
A Procedure
B Procedure
14 >150 >150 >150
13 >150 >150 >150
3 >150 >150 >150
22 44 56 >150 >i50
ND ND ND ND ND ND ND
rDNA
EIA
A Procedure
B
_
_ 44 33 91 >150 >150 with vaccine
16 36 47 >150 138
_ 32 30 67 >150 >150 at 0, 1, and 6 months;
ND ND ND ND ND ND ND
15 50 60 >150 >lSO
ND ND ND ND ND ND ND
35 34 104 >150 >150
ND ND ND ND ND ND ND
_
ND, not done
SAB RIA and CORAB, but unreactive in the AUSAB rDNA assay. These data show good agreement between AUSAB RIA and AUSAB rDNA and indicate no false reactives by AUSAB rONA assays. Most discrepants were less than 10 mIU/ml.
Autoimmune
and disease sera
Specimens from patients with a variety of diseases and viral infections were tested by AUSAB RIA and all procedures of AUSAB rDNA RIA and EIA (Table 7). All specimens were negative for all AUSAB procedures. Also tested by AUSAB RIA, AUSAB rDNA RIA and EIA were specimens from patients with toxoplasmosis (9 samples), Epstein-Barr virus (3 samples), and E. cofi infection (3), IgM anti hepatitis A reactive (6) or specimens containing high titer IgA (5), high titer IgE (3), high titer IgG (5), and high titer IgM (5). All were negative by all three assays.
225 TABLE
5
Comparison uals
of AUSAB
assays
Date
HBV-infected individuals
for the detection
of anti-HBs
AUSAB
AUSAB RIA
RIA
22 13 27 19 22 3
May Jun Jun Jul Aug Jan
+ +
2 May 9 May
_
6
Analysis
of specimens
Specimen
AUSAB RIA (mIU,m,)
_ _ _ -
+ + + + + _
+ + + + + +
_ _ _
+ + +
+ + +
12 19 35
AUSAB
rDNA
RIA
ydure
pdure
17 3 _ _
15 4 +I_
-
+ + + + + +
B
3 65
12 Jul 18 Jul 1 Aug
AUSAB
CORAB
7 101
_ _ _
12 19 46
results
AUSAB ydure
EIA
AUSAB
rDNA
ydure
y
_ _ -
9 +I_
14 3 _ _
_
-
_
-
Plasma P12-31 P12-151 P12-179 13-72 13-84 13-94 13-109 13-118 13-146
2 15 3 22 3
_ _
_ -
_ -
_ _
_ -
-
_
_
_
_
Serum N-15 N-23 N-40 N-48 N-77
3 32 6 9 12
_
_
_ -
_ _ _
ND ND ND ND ND
_ _ _
_ _ -
QNS,
insufficient
quantity.
individ-
Auszyme
+ + + _ _ _
May Jun Jun Jul
giving discordant
B Procedure (mIUim1) _ _ _ -
_ _ _ _ _
23 6 13 5
TABLE
_ _ _
_ _
rDNA
of HBV infected
EIA
Procedure (mIU/ml) A
in serial bleeds
EIA
CORAB
+ _ -
QNS _
_ QNS _
_ _ + _
226 TABLE
7
Lack of AUSAB Disease
crossreactivity ID
type
of sera positive AUSAB
RIA
for various AUSAB
auto-immune rDNA
RIA
and viral antibodies AUSAB Procedure
rDNA
EIA
A
CPM
S/CO”
CPM
s/co
OD
s/co
Y70 Y87 Y77 3118188
130 109 102 128
0.44 0.37 0.35 0.44
34 48 49 48
0.27 0.39 0.40 0.39
0.021 0.026 0.026 0.036
0.28 0.34 0.34 0.47
Y46 YlOo Y93 25
118 140 122 111
0.40 0.48 0.42 0.38
44 35 41 31
0.35 0.28 0.33 0.25
0.022 0.021 0.025 0.022
0.29 0.28 0.33 0.29
Anti-nuclear antibody (ANA)
34-9 140-21 140-13 142-7 142-14 86-12 34-20 140-11 F-130 G623 3860BS
82 108 116 87 137 109 114 102 99 94 117
0.28 0.37 0.40 0.30 0.47 0.37 0.39 0.35 0.34 0.32 0.40
38 38 33 39 70 60 59 64 47 38 51
0.31 0.31 0.27 0.31 0.56 0.48 0.48 0.52 0.38 0.31 0.41
0.047 0.027 0.028 0.027 0.023 0.03 0.02 0.02 0.03 0.029 0.038
0.62 0.36 0.37 0.36 0.30 0.39 0.26 0.26 0.39 0.38 0.50
Rheumatoid arthritis
S121087 S121188
95 110
0.32 0.38
52 48
0.42 0.39
0.034 0.032
0.45 0.42
Anti-CMV
1 4
107 102
0.37 0.35
34 52
0.27 0.42
0.036 0.033
0.47 0.43
Systemic lupus erythrematosus (SLE)
Rheumatoid
factor
“S/CO=Specimen
value/Cut-off
value.
S/CO
are considered
negative.
Clinical studies Three different laboratories tested a total of 1711 specimens by AUSAB RIA and by both procedures of AUSAB rDNA and AUSAB rDNA EIA. These specimens were derived from random donors, individuals previously determined to be reactive by AUSAB RIA, patients with autoimmune disease, hospital employees, residents of mental institutions, patients diagnosed with hepatitis, vaccine recipients, dialysis patients, medical students, and patients with other diseases. Total agreement among all assays was 98.6% ([1711-24]/1711) with 24 discordants. All 24 discordants were less than 10 mIU/ml. Nine specimens were low level positive only by AUSAB RIA but negative by all rDNA assays and also negative by CORAB. Nine specimens were marginally positive for all AUSAB RIA procedures but negative by rDNA EIA assays. Almost all of these specimens were
227
120 110
AUSAB
120
<30
c50
c40
120
AUSAB
<70
c60
[Anti-HRs]
110
A
RIA
‘~1
-30
490
>I00
-330
<90
,100
mllliml
rDNA
RIA
100
<2Q
<30
c40
c50
[Anti-HBsl
120 110
AUSAB
<60
<70
mlU/ml
rDNA
EIA
_
c20
<30
<70 c40 <50 ~60 IAnti-HBsl mlU/ml
CEO
<90
c
4
>I00
Fig. 4. Frequency histogram of anti-HBs concentration in a population of 225 hepatitis B vaccine recipients measured by AUSAB RIA (A), AUSAB rDNA RIA (B), and AUSAB rDNA EIA (C). Procedure A was used for both rDNA based assays. Specimens were not diluted prior to testing and specimens with anti-HBs concentrations greater than 100 mIU/ml gave absorbances greater than 2.0 in the EIA and are reported as > 100 mIU/mi. Anti-HBs concentrations below this value were calculated for each specimen using a quantitation standard panel as described in Methods.
very low level reactives (less than 5 mIU/ml). Six specimens were repeatably borderline reactive in one or more anti-HBs assays. Overall there was almost 100% agreement among all assays for specimens above 10 mIU/ml by AUSAB and 73% ([90-24]/90) agreement for samples greater than the cut-off but less than 10 mIUlm1.
228 8) AUSAB
RIA
A
SAMPWCPM)/NCx(CPM)
AUSAB rDNA RIA-Procedure
AUSAB
rONA EIA-Procedure
B
B
C
iuz
a-
a a
0” 91. 5 g 43. 0 30. al-
e w
F1 i;
NET ABSORBANCE
(492 nm)
229
The frequency distribution of anti-HBs in 292 HBsAg negative plasma by AUSAB and AUSAB rDNA RIA and EIA (procedure B only) is shown in Fig. 5. Seven specimens (2.4% of total specimens) were reactive and the remainder were negative for anti-HBs in all three assays. The negative population mean was close to the negative control mean and well separated from the calculated cut-off value.
Second generation assays which use rHI3sAg for the detection and quantitation of anti-HBs in human sera were developed. Recombinant HBsAg has been cloned successfully and expressed in a variety of eukaryotic cells ~~lu~g CHO and yeast. In this report the rHBsAg was produced in mouse L cells and was antigenically indistinguishable from purified hpHBsAg. Recombinant HBsAg provides several advantages over the hpI-IBsAg used in previously described anti-HBs assays (~ushahwar, 1987; ~ushahwar and Spiezia, 1987). Recombinant rHBsAg is produced at high concentrations (up to 500 pglml) in tissue culture hollow fiber systems, may be easily purified to greater than 95% homogeneity by affinity methods, gives better reproducibility in purity than hpHBsAg, and is not infectious and dues not require BL3 biosafety containment facilities for production. The rHBsAg also shows excellent stability and storage characteristics comparable or superior to purified hpHBsAg (data not shown). The high specificity of rHBsAg based anti-HBs assays was demonstrated by examining large populations of random blood donors, hospital patients, and autoimmune and disease specimens, and comparing these results with AUSAB RIA. The AUSAB RIA and AUSAB rDNA assays gave similar sensitivity and detectability when anti-HBs panels, hepatitis B vaecinees, and HBV infected individuals were tested. These assays measure the same molecules and give good quantitative agreement. One hundred percent a~eement among anti-N& assays was observed for specimens having anti-HBs concentrations greater than 10 mIU/ml. The few discrepancies occurred with specimens having less than 10 mIU/ml, although there was 73% agreement among all assays for specimens less than 10 mIU/ml. The discordant specimens generally fell into two groups: specimens reactive by AUSAB-RIA only and negative by all other tests including CORAB and speci-
Fig, 5. Histogram of sample (~Pm)/negative control mean (cpm) and net absorbance (492 nm) values obtained when plasma from 292 random blood donors was tested by AUSAB RIA (A), AUSAB rDNA RIA (B), and AUSAB rDNA EIA (C). Sample (cpm) divided by NCx (cpm) for each specimen was calculated for RIA and the net absorbance (sample absorbance - NCx absorbance) for each specimen was calculated for the EIA. Procedure B was used for the rDNA based assays. Specimens giving sample!NCx or net absorbance values greater than the cut-off (indicated by arrow) are considered reactive in the assay.
230
mens with borderline reactivity by AUSAB RIA and AUSAB rDNA RIA but negative by AUSAB rDNA EIA. Since the specimens reactive by AUSAB RIA only were also unreactive for anti-HBc, it is unlikely that these specimens were derived from individuals with previous exposure to hepatitis B virus. In the absence of a reliable confirmatory procedure for these low level AUSAB reactives, it is not possible to determine whether the reactivity may result from a nonspecific binding component. Kessler et al. (1985) demonstrated that a small number of specimens contained a reduction sensitive component that reacted in the AUSAB assay. These individuals with low AUSAB reactivity may not have protective levels of anti-HBs (Hadler et al., 1986). Results from the second class of discordants which are marginally reactive by RIA assays but negative by rDNA EIA procedures suggest that for a small percentage of specimens the RIA procedure may be slightly more sensitive than the EIA. Our results indicate that most of these specimens have primarily antibody against the d epitope since they are not reactive with HBsAg (uy subtype) (Decker et al., 1988). The protective efficacy of anti-HBs subtype antibodies is presently unclear (Shiels et al., 1987). Most importantly in these studies, no apparent false reactives were observed by rDNA based assays and overall agreement with AUSAB RIA was greater than 98%.
Acknowledgements We are grateful to Jeff Farmer, Kashfinur Heine, Carlos Maldonado and Bernie Schleicher for their help in production of rHBsAg. We also thank Laurie Metz for typing this manuscript and Mahlon Miller for electron microscopy work. We thank Robert Penillo, Gary Tegtmeier, Eugene Schiff and Maria DeMedina for their help in clinical trials.
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