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Establishment and validation of an ELISA for the quantitation of HBsAg in recombinant hepatitis B vaccines
Journal of Virological Methods 172 (2011) 32–37
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Protocols
Establishment and validation of an ELISA for the quantitation of HBsAg in recombinant hepatitis B vaccines Catia Inês Costa a,b , Isabella Fernandes Delgado a,b , Jaline Alves Cabral da Costa a , Renata Faria de Carvalho a , Sérgio da Silva Mouta e Júnior c , Carlos Otávio Alves Vianna d , Márcia Terezinha Baroni de Moraes c,d,∗ a
Department of Immunology, National Institute of Quality Control in Health (INCQs), Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil Post Graduation Program in Health Surveillance, National Institute of Quality Control in Health (INCQs), Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil c Laboratory of Molecular Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil d Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil b
a b s t r a c t Article history: Received 7 May 2010 Received in revised form 13 December 2010 Accepted 16 December 2010 Available online 23 December 2010 Keywords: Hepatitis B virus Hepatitis B surface antigen Monoclonal antibody ELISA Potency test
Commercial enzyme-linked immunosorbent assay (ELISA) kits for the determination of the in vitro potency of recombinant hepatitis B vaccines, which detect hepatitis B surface antigen (HBsAg), have been used frequently as an alternative for traditional in vivo potency tests. With the constant need for validation procedures, an ELISA that could be employed to determine the in vitro potency of five recombinant hepatitis B vaccines simultaneously was established using two monoclonal antibodies. The use of two monoclonal antibodies produced “in house” specific for the small envelope protein S of the hepatitis B virus (HBV) resulted in the production of a highly specific, sensitive and stable ELISA. The standard ELISA parameters used in this study, considering the HBsAg content of each recombinant hepatitis B vaccine evaluated, resulted in a standard curve that could be applied for potency evaluations of different, commercial hepatitis B vaccine lots. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Worldwide, hepatitis B remains a major public health problem: two billion individuals have been infected with hepatitis B virus (HBV). Of these 350 million are persistent carriers of hepatitis B virus (Heathcote, 2008). Vaccination is fundamental to stopping the transmission of HBV (Gish and Locarnini, 2006; Mast et al., 2005, 2006). Several vaccine manufacturers have used recombinant deoxyribonucleic acid (DNA) technology to express HBsAg in HBV transfected yeast (i.e., Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha). These hepatitis B vaccines are recombinant hepatitis B vaccines used most commonly (Zanetti et al., 2008). The small envelope S protein expressed in transfected yeasts self-assembles into immunogenic, spherical particles almost identical to the 22 nm particles found in the serum of hepatitis B carriers. The expressed HBsAg is
∗ Corresponding author at: Laboratory of Molecular Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Av. Brasil, 4365 Manguinhos, Zipcode 926, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1891; fax: +55 21 2260 4866. E-mail address: baroni@ioc.fiocruz.br (M.T.B. de Moraes). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.12.010
purified to remove the yeast components by several physical separation techniques (i.e., filtration and chromatography). This technology has offered the potential for the unlimited production of vaccines. This vaccine is a part of the routine vaccination schedules of many of the infants and children of the world (Zanetti et al., 2008). Quality control for recombinant hepatitis B vaccines requires the determination of the potency of the vaccine by measuring its HBsAg content, as described by the World Health Organization (WHO, 1989). For this purpose, manufacturers have developed an in vitro assay that has been used by many national control laboratories for official testing. An ELISA that uses only two monoclonal antibodies is described. This ELISA could be used to determine simultaneously the comparative in vitro potency of five recombinant hepatitis B vaccines from different manufacturers that have been used in the Brazilian National Immunization Program. The ELISA standard parameters used in this study considered the HBsAg content of each recombinant hepatitis B vaccine evaluated and allowed for the construction of a standard curve. This standard curve could be used as an “in house” ELISA to replace commercial kits.
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Table 1 Summary of vaccines used in the study. Vaccine (®)
Manufacturer
Yeast used for expression
1. Butang 2. Engerix B 3. Euvax B 4. Hepavaxgene 5. Quinvaxem
Butantan Institute (São Paulo, Brazil) GlaxoSmithKline (Brentford, West London) LG Chemical (Seoul, South Korea) Green Cross (Crucell, Dutch) Berna Biotech (Crucell, Dutch)
Hansenula polymorpha Saccharomyces cervisiae Saccharomyces cervisiae Hansenula polymorpha Hansenula polymorpha
2. Materials and methods 2.1. Chemicals All reagents used in the study were of analytical grade and purchased from Sigma Chemical (MO, USA). 2.2. Recombinant hepatitis B vaccine samples The monovalent or combined (Quinvaxem® – diphtheria, tetanus, pertussis/whooping cough, hepatitis B and Haemophilus influenzae type B) hepatitis B vaccines used in this study were from different manufacturers, as described in Table 1. For the dissociation of aluminium hydroxide, the method described previously by Dobbelaer (1997), with slight modifications, was used. In brief, a volume of 500 L of each vaccine containing 20 g/mL of HBsAg was mixed with the dissociation solution (2.5% diethanolamine – DEA and 0.2% Triton X-100 in phosphate-buffered saline (PBS), pH 7.4). This mixture was incubated at room temperature for 30 min and centrifuged for 10 min at 231 × g. The supernatants were used directly in the ELISA as described below. To reduce their potency artificially, two lots of each vaccine sample (except for Heberbiovac® ) were stressed by incubation at 4 ◦ C (lot 1) and at 60 ◦ C (lot 2) separately for seven days. The vaccine was then used in the ELISA according to the standardised ELISA protocol described below in Section 2.7. 2.3. Reference vaccine The reference standard used was provided by each manufacturer, listed in Table 1, and contained 20 g/mL of HBsAg as measured by the Lowry protein assay. 2.4. Monoclonal antibodies Purified, murine monoclonal antibodies against HBsAg (antiHBs MAb) CG2 and AG9 were used as capture and conjugate, respectively. These anti-HBs MAbs were generated against HBsAg particles purified from pooled human plasma that contained HBV subtypes ad and ay, as previously described (Vianna et al., 2006). The final concentration of each purified MAb was determined by a modified Lowry method using bovine serum albumin (BSA) as a standard (Peterson, 1983). The anti-HBs MAb AG9 was peroxidaselabelled using a commercial kit, SureLink HRP conjugation from KPL (MD, USA), according to the manufacturer’s instructions. 2.5. Western blot analysis of samples of recombinant hepatitis B vaccine Gel electrophoresis, SDS-PAGE (12%), was performed with 2 and 5 g of each monovalent hepatitis B vaccine (Butang® , Engerix B® , Euvax B® , Hepavaxgene® ), which were dissociated previously from aluminium hydroxide (Section 2.2) according to the technique of Laemmli (1970). As a positive control for Western blotting, 5 L of concentrated HBsAg serum particles, prepared according to Moraes et al. (2006), was applied to the same gel. After electrophoresis,
the gel was blotted for 20 min at 16 V on a 0.22-m nitrocellulose membrane (BioRad, CA, USA) using a semi-dry system (BioRad). The membranes were incubated with a blocking reagent (5% non-fat dry milk and 0.05% Tween 20 in PBS (pH 7.4)) overnight at 4 ◦ C and subsequently processed for 1 h with anti-HBs MAb AG9 (Moraes et al., 2006) diluted in blocking reagent. After washing with PBS (pH 7.4) containing 0.05% Tween 20, the membranes were processed with an anti-mouse, alkaline phosphatase-AP conjugated immunoglobulin (Sigma) diluted in blocking buffer. Bands were detected using ditetrazolium salt nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates, according to the manufacturer’s instructions. 2.6. Parameters used for standardisation of the ELISA For standardisation of the assay, four parameters were considered. (1) Coating and conjugate concentrations, concentrations of 2 g, 4 g, 6 g and 8 g/mL of capture MAb and 1:500, 1:1000, 1:2500 and 1:5000 of conjugate MAb were evaluated. (2) Vaccine concentration. The tested concentrations were 0.005, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 and 0.81 g/mL. (3) Blocking solution: three different blocking solutions were tested. (i) PBS containing 0.05% Tween 20 (PBS-T) added to 5% (w/v) non-fat dry milk, 3% (v/v) fetal calf serum (FCS) and 0.5% (w/v) bovine serum albumin (BSA); (ii) PBS containing 5% (w/v) BSA and (iii) PBS containing 3% (w/v) BSA. (4) Time and temperature of incubation: the plates were incubated initially at 37 ◦ C for 1 h and at 4 ◦ C for 18 h. For the conjugate, incubation was at 37 ◦ C for 120, 60 and 30 min with and without rotation (63 × g) (blocking and vaccine addition steps). The presence of aluminium hydroxide in the vaccines was evaluated by testing the dissociated vaccine samples for aluminium hydroxide as described in Section 2.1. Two ELISA microplates from different manufacturers were evaluated: Nunc-Maxisorp (Roskilde, Denmark) and Microlom 600-Greiner Bio-one (Atlanta, USA). 2.7. Protocol for standardisation of the ELISA Anti-HBs MAb CG2 in coating buffer (0.1 M Na2 CO3 , 0.02% NaN3 , pH 9.6) was applied to an ELISA microplate (Nunc-Maxisorp) at a concentration of 8 g/mL. The microplate was then incubated at 37 ◦ C for 30 min and then moved to 4 ◦ C for 18 h. Next, the microplate was washed three times with PBS-T. To minimise nonspecific binding, the microplate was blocked for 1 h at 37 ◦ C on a rotational shaker (63 × g) with PBS containing 3% BSA. The same washing procedure was repeated, and the microplate was incubated at 37 ◦ C on a rotational shaker (63 × g) with 100 L of vaccine samples at different dilutions. After the same washing procedure, the microplate was incubated with peroxidase-labelled anti-HBs MAb AG9 diluted 1/2500 in PBS for 1 h at 37 ◦ C. Finally, the microplate was washed and incubated in the dark at room temperature with 3,3 ,5,5 -tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma). After 30 min, the reaction was stopped with 2 N sulphuric acid. The absorbance was measured at 450 nm with a Spectra Rainbow (Tecan, Crailsheim, Germany) microtiter plate reader within 30 min of adding the stop solution. The assays were carried out in triplicate.
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2.8. Evaluation of the stability of the ELISA Initially, ELISA microplates (Nunc-Maxisorp, Denmark) coated with anti-HBs MAb CG2 (8 g/mL) in buffer (0.1 M Na2 CO3 , 0.02% NaN3 , pH 9.6) were sealed and incubated at 4 ◦ C for 18 h in a humidified chamber. Next, the coating solution was discarded, and the microplate stabilised with 200 L of stabilisation buffer (0.1 M Na2 CO3 , 0.02% NaN3 (pH 9.6) containing 5% sucrose and 0.3% BSA) per well. The microplates were incubated for 45 min at 37 ◦ C, and the stabilisation buffer was discarded. To evaluate the stability in terms of analytical sensitivity, the microplates were stored dry and sealed with plastic film and aluminium foil packaging at 4 ◦ C and at −20 ◦ C for six months. Before each ELISA, the microplate was left at room temperature for 10 min and washed three times with PBS-T. The microplates were blocked for 1 h at 37 ◦ C on a rotational shaker (63 × g) with PBS containing 3% BSA. The ELISA followed the procedure described in Section 2.7. Variations of percent sensitivity were calculated based on the absorbance values obtained before (zero time = 100% sensitivity) and after storage of the microplate using the following equation: Sensitivity % =
Absorbance after storage × 100 Absorbance before storage
2.9. Evaluation of the validation parameters 2.9.1. Specificity Specificity was determined by testing the combined Quinvaxem® vaccine containing HBsAg in comparison with the combined (diphtheria, tetanus, pertussis/whooping cough) vaccine without HBsAg. 2.9.2. Limit of detection (LOD) The limit of detection was evaluated by analysing the absorbance values of a blank sample (PBS) and the negative IgG1 MAb interferon 1a (Cantelli et al., 2009) in multiple replicates of three different plate assays. The LOD was defined as being three times the standard deviation. 2.9.3. Limit of quantitation (LOQ) The limit of quantitation (LOQ) was calculated considering the average value of the absorbance obtained for the LOD added value of 10 standard deviations obtained from a blank sample (PBS). 2.9.4. Linearity The linearity of the ELISA was assessed by using the linear correlation coefficient of the standard curves established with the reference standard to determine the LOD. The HBsAg concentrations used in the standard curve were 0.2, 0.4, 0.6, 0.8 and 1 g/mL. The ELISA was repeated in triplicates. The linearity was calculated from the linear regression of the results of the standard curve. The linear, angular and variation coefficients were determined (R ≥ 0.98). Statistical analyses using ANOVA were performed considering p < 0.05 to the angular coefficient to be not null and p > 0.05 to be a straight line (SAS, 1990). 2.9.5. Precision The precision study was divided in repeatability (intra-assay) and intermediate (inter-assay) precision. To evaluate both parameters, vaccine samples diluted previously and containing a high, medium and low content of HBsAg (designed as lots 1, 2 and 3, respectively) were used. The HBsAg concentrations in the standard curve were 0.2, 0.4, 0.6, 0.8 and 1 g/mL. The ELISA was carried out in triplicate. To assess intermediate precision, the ELISA was executed independently by two technicians. The standard deviation was calculated. The coefficient of variation (CV) could not be higher
than 10%, whereas the CV of the intermediate precision could not exceed 20%. 2.9.6. Accuracy For evaluations of accuracy, three dilutions of each vaccine sample were prepared and tested in three replicates of each vaccine in the same run. The normal deviation could not exceed 20%. Accuracy values were determined using the following equation: Accuracy =
Experimental average value of concentration Theoretical value of concentration
3. Results 3.1. Recombinant hepatitis B vaccines and monoclonal anti-HBs antibodies used in the ELISA Five yeast cell-expressed recombinant hepatitis B vaccines were evaluated according to their reactivities with anti-HBs MAb CG2. Five were monovalent vaccines containing HBsAg and one was a combined vaccine (against diphtheria, tetanus, pertussis/whooping cough, hepatitis B and H. influenzae). All of the vaccine samples tested in the ELISA contained initially aluminium hydroxide as an adjuvant. To evaluate the influence of the adjuvant on ELISA sensitivity, these vaccine samples were also submitted to aluminium hydroxide dissociation and again evaluated by the ELISA (0.05, 0.25, 0.5 and 1 g/mL). The absorbance values obtained after aluminium hydroxide dissociation were higher than those obtained from aluminium hydroxide-associated vaccines (Fig. 1). The most effective concentration of anti-HBs MAb was determined by testing the ELISA protocol, described in Section 2.6. Three different concentrations of anti-HBs MAbCG2 (2, 4 and 8 g/mL) were used as capture in parallel with different concentrations of vaccine samples (0.005, 0.025, 0.050, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 g/mL). Different dilutions of anti-HBs MAb AG9 (1:500, 1:1000, 1:2500 and 1:5000) were used as conjugate. The optimal concentrations of anti-HBs MAb CG2 and AG9, which presented both high sensitivities, as measured by absorbance values of the different vaccine sample concentrations, and low backgrounds in the ELISA, were 8 g/mL and 1/2500, respectively. The sensitivity of the standardised ELISA for detection of the five recombinant hepatitis B vaccines was 0.2 g/mL (Fig. 2). The standardised ELISA protocol presented in a crescent order had higher sensitivity to Euvax® , Engerix B® , Hepavax-gene® and Butang® monovalent recombinant hepatitis B vaccines. The combined recombinant hepatitis B vaccine Quinvaxem® seemed to have very similar sensitivity to Euvax® . The monovalent recombinant hepatitis B vaccines were exposed to temperature stresses. These vaccines were incubated at 4 ◦ C and 60 ◦ C during a consecutive, 7-day period. The vaccines were then tested by the ELISA. The vaccines exposed to the 60 ◦ C temperature displayed a reduced sensitivity in comparison with the 4 ◦ C standard temperature, with p-values of 0.03 (Engerix B® ), 0.005 (Hepavaxgene® ) and 0.004 (Butang® ). The reduction of sensitivity for other vaccines was not significant (data not shown). 3.2. ELISA parameters tested The introduction of plate rotations to the ELISA procedure during the blocking and vaccine addition steps increased significantly the sensitivity of the assay. The blocking solution PBS containing 3% BSA showed the best results with the lowest background values and the highest sensibility (data not shown). Finally, the ELISA microplates from Nunc-Maxisorp (Denmark) showed significantly
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Fig. 3. Western blot analysis of monovalent recombinant hepatitis B vaccines. 2 and 5 g of each vaccine (respectively Butang® – lines 1, 2; Engerix B® – lines 3, 4; Euvax B® – lines 5, 6; Hepavaxgene® – lines 7, 8) and 5 L of concentrated HBsAg serum particles (Line 9) were reacted with anti-HBs MAb AG9.
The pattern of recombinant HBsAg on SDS-PAGE was compared to the HBsAg from human plasma particles (Fig. 3). 3.4. Robustness of the method
Fig. 1. ELISA analysis of recombinant hepatitis B vaccines associated with aluminium hydroxide (A) in comparison with recombinant hepatitis B vaccines dissociated from aluminium hydroxide (B). The concentration of each vaccine was adjusted to 0.05, 0.25, 0.5 and 1 g/mL.
higher sensitivities compared to the Microlom 600-Greiner Bio-one (Atlanta, USA) (data not shown). 3.3. Western blot analysis of recombinant hepatitis B vaccine samples using anti-HBs MAb AG9 The different forms of HBsAg from each monovalent vaccine produced by the yeast cells were recognised by the anti-HBs MAb AG9.
During the establishment of the ELISA, the BSA concentration in the blocking solution (5% and 3% tested) was not a significant factor for the recombinant hepatitis B vaccines (p > 0.05) evaluated. The absorbance values obtained in the ELISA were considerably higher after the introduction of incubation for 1 h and plate rotation (63 × g) during the blocking and vaccine addition steps. The dissociation of aluminium hydroxide from the evaluated vaccine samples significantly increased the sensitivity of the ELISA (data not shown). 3.5. Stability of the ELISA Fig. 4 shows the results of the ELISA using plate coating with anti-HBs MAb CG2 (8 g/mL) previously stored at 4 ◦ C or −20 ◦ C for six months (stability assay). The sensitivity results (%) obtained were above 90% for all tested samples at both temperatures. 3.6. Validation of the ELISA 3.6.1. Specificity of the ELISA The specificity of the ELISA was assessed by the absorbance values obtained for the combined vaccine containing HBsAg (Quinvaxem) in comparison with the combined vaccine (diphtheria, tetanus and pertussis/whooping cough), but without HBsAg, as shown in Table 2.
Fig. 2. Standardised ELISA. Different dilutions of four monovalent recombinant hepatitis B vaccines and one combined recombinant hepatitis B vaccine were analysed. The sensitivity of the ELISA was 0.2 g/mL.
Fig. 4. Stability of ELISA with three monovalent recombinant hepatitis B vaccines and one combined recombinant hepatitis B vaccine. The sensitivity (%) presented on a vertical axis corresponds to the standardised ELISA in comparison with plates coated with anti-HBs CG2 (8 g/mL) stored previously at 4 ◦ C for six months.
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Table 2 Specificity of the combined vaccines containing HBsAg in comparison with the combined vaccine without HBsAg content. Samples
Concentration (g/mL) expressed in OD value
Quinvaxem® combined vaccine Quinvaxem® combined vaccine without HBsAg content Buffer dilution (PBS)
0.2
0.4
0.6
0.8
1.0
0.436 0.060 0.059
0.684 0.059 0.059
0.775 0.061 0.060
0.836 0.063 0.062
0.894 0.064 0.063
3.6.2. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ were calculated using 3 and 10 standard deviations, respectively, of blank assay. Under the assay conditions described in this study, the LOD was 0.078 g/mL. The LOQ value (g/mL) found was below the lower curve point (absorbance value corresponded to 0.104). The analytical range observed corresponded to the lower limit of quantitation (LLOQ) and to the upper limit of quantitation (ULOQ), 0.2 g/mL and 1.0 g/mL, respectively. 3.6.3. Linearity of the ELISA The values of the correlation coefficients of the linear regression (R2 ) for three batches of each recombinant hepatitis B vaccine evaluated were all above 0.99. This demonstrated the linearity for the analytical range of 0.2, 0.4, 0.6, 0.8 and 1 g/mL. Using an ANOVA, values for the angular non null coefficient (with p < 0.05) and for the straight line (with p > 0.05) were obtained (data not shown). 3.6.4. Precision of the ELISA The results of the precision study are presented in Table 3. The CV for repeatability and intermediate precision for all vaccines were determined and found to be between 2 and 3% (below 10%) and 2 and 5% (below 20%), respectively. 3.6.5. Accuracy of the ELISA Variation coefficient values for all recombinant hepatitis B vaccines evaluated by the ELISA were between 2 and 15%. The accuracy percentage ranged between 97 and 100% for different
Table 3 Results of repeatability and intermediate precision studies. Each lot was tested three times in the same run in independent experiments involving two technicians. Vaccinea
Euvax® Lotb 1 Lot 2 Lot 3 Hepavax-gene® Lot 1 Lot 2 Lot 3 Engerix-B® Lot 1 Lot 2 Lot 3 Butang® Lot 1 Lot 2 Lot 3 Quinvaxem® Lot 1 Lot 2 Lot 3
Repeatability (intra-assay)
Intermediate precision (inter-assay)
SD
CVb
SD
CV
0.019 0.018 0.024
3.5 2.8 2.6
0.010 0.018 0.014
2.6 3.9 2.7
0.012 0.013 0.012
2.2 2.3 2.2
0.013 0.014 0.010
3.0 2.5 2.0
0.011 0.012 0.010
2.2 2.3 2.2
0.017 0.021 0.017
4.0 4.2 4.1
0.010 0.010 0.010
2.5 2.1 2.3
0.013 0.014 0.010
3.0 2.5 2.0
0.013 0.014 0.013
2.0 2.1 2.0
0.014 0.013 0.010
1.8 2.4 2.0
The coefficient of variation (CV) reported in each case corresponds to the media of triplicates for each batch. a Different types of vaccines. b Coefficient of variation = (standard deviation [SD]/mean) × 100.
vaccines containing different concentrations of HBsAg (data not shown). 4. Discussion The potency evaluation of hepatitis B vaccines used in Brazil’s National Immunization Program has led this group to develop and validate an alternative ELISA for commercial tests and to attempt to reduce the large number of animals needed as in vivo models. The ELISA was designed to allow for the determination of the HBsAg content of different recombinant hepatitis B vaccines from different manufacturers simultaneously. Thus, this ELISA will save time and money. Recombinant hepatitis B vaccines are produced using yeast expression technology, but each vaccine tested presented a characteristic sensitivity to the ELISA and a unique Western blotting expression pattern. These unique characteristics were probably observed because of the different yeast species used in the production of each vaccine, which leads to a different recognition pattern by the MAb used. The ELISA analyses using the recombinant hepatitis B vaccines associated to aluminium hydroxide in comparison to recombinant hepatitis B vaccines dissociated from aluminium hydroxide indeed show a considerable increase of sensitivity. This sensitivity difference will be important to the precise determination of potency. Despite the possibility of nonspecific binding between MAb and aluminium hydroxide, according to the results of Giffroy et al. (2006), the process of removing adsorbed aluminium hydroxide from the HBsAg probably exposed some other antigenic epitopes and increased the sensitivity of the ELISA. Even though the dissociation of aluminium hydroxide from HBsAg before carrying out the ELISA is an additional, necessary step that leads to better results, the protocol described by Dobbelaer (1997), which was performed in this study with slight modifications, is very simple. Otherwise, the sensitivity of the ELISA could be increased by introducing plate rotations, which apparently allow a correct interaction between the HBsAg and the anti-HBs MAbs. According to the validation study conducted, the performance characteristics of the ELISA were found to be satisfactory for all the recombinant hepatitis B vaccines (monovalent and combined) tested. It was shown that the proposed ELISA could measure specifically HBsAg in the presence of other vaccine antigens, as demonstrated using the combined vaccine against diphtheria, tetanus, pertussis/whooping cough, hepatitis B and H. influenzae. The sensitivity of this ELISA for the detection of HBsAg in human serum samples is lower (0.2 g/mL) compared to the commercial ELISA kit, which shows a sensitivity of about 0.5 ng/mL. For this ELISA to be applicable to human serum samples, an evaluation against an HBsAg reference panel will be necessary to measure HBsAg in human clinical samples. Combined, recombinant hepatitis B vaccines contain 20 g/mL of HBsAg. It has no impact on the intended use of the ELISA. As noted by Giffroy et al. (2006), lower sensitivity may confer an advantage by reducing the number of dilution steps required and thereby minimising potential error and variability. The validation parameters (specificity, LOD and LOQ, linearity, precision and accuracy) were conducted according to the International Conference Harmoniza-
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tion (ICH) and indicated that the results obtained in this study on the recombinant hepatitis B vaccines were satisfactory and useful for the national control laboratory of Brazil. 5. Conclusion The proposed ELISA is suitable for measuring the HBsAg content in monovalent and combined recombinant hepatitis B vaccines and provides a reliable method for determining potency. Acknowledgements We thank Vera Bongertz and Ana Cristina Martins de Almeida Nogueira for fruitful discussions. We thank Bruno Baroni de Moraes e Souza for reviewing this paper. This work was supported by the National Institute of Quality Control in Health (INCQS), BioManguinhos-Fiocruz and Oswaldo Cruz Institute-Fiocruz. Isabella Fernandes Delgado and Marcia Terezinha Baroni de Moraes are fellowship researchers from the National Research Council (CNPqBrazil). References Cantelli, C.P., Teixeira, M.G.M., Santos, E.A., da Silva Jr., H.C., e Mouta Jr., S.S., Pimenta, M.M.A., Vianna, C.O., Souza, N.P., Batoreu, N.M., Galler, R., Moraes, M.T.B., 2009. Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes. Hybridoma 28, 211–214.
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Dobbelaer, R., 1997. Collaborative study for the establishment of biological reference preparations for rDNA hepatitis B vaccine. Pharmeuropa 97, 3–18 (Special issue). Giffroy, D., Mazy, C., Duchêne, M., 2006. Validation of a new ELISA method for in vitro potency assay of hepatitis B-containing vaccines. Pharm. Biol., 7. Gish, R.G., Locarnini, S.A., 2006. Chronic hepatitis B: current testing strategies. Clin. Gastroenterol. Hepatol. 4, 666–676. Heathcote, E.J., 2008. Demography and presentation of chronic hepatitis B virus infection. Am. J. Med. 121 (12 Suppl.), S3–11. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685. Mast, E.E., Margolis, H.S., Fiore, A.E., et al., for the Advisory Committee on Immunization Practices (ACIP), 2005. A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) Part I: immunization of infants, children, and adolescents. MMWR Recomm. Rep. 54 (RR-16), 1–31. Mast, E.E., Weinbaum, C.M., Fiore, A.E., et al., For the Advisory Committee on Immunization Practices (ACIP) and the Centers for Disease Control and Prevention (CDC), 2006. A comprehensive strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP). Part II: immunization of adults. MMWR Recomm. Rep. 55 (RR-16), 1–33. Peterson, G.L., 1983. Determination of total protein. Methods Enzymol. 91, 95. SAS, 1990. SAS/Stat User’s Guide, Version 6, forth ed. SAS Institute Inc., Cary, NC. Vianna, C.O.A., Moraes, M.T.B., Mouta Junior, S.S., Martins, M.G.T., Batoreu, N.M., Queiroz, J.L., Gomes, S.A., Góes, A.C.M., Armoa, G.R.G., Marques, C.H., Moraes, M.T.B., 2006. Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches. J. Immunol. Methods 313, 38–47. World Health Organization-WHO, 1989. Requirements for hepatitis B vaccines made by recombinant DNA techniques in yeast. Technical Report Series v. 786. Zanetti, A.R., Van Damme, P., Shouval, D., 2008. The global impact of vaccination against hepatitis B: a historical overview. Vaccine 26, 6266–6273.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Protocols
Establishment and validation of an ELISA for the quantitation of HBsAg in recombinant hepatitis B vaccines Catia Inês Costa a,b , Isabella Fernandes Delgado a,b , Jaline Alves Cabral da Costa a , Renata Faria de Carvalho a , Sérgio da Silva Mouta e Júnior c , Carlos Otávio Alves Vianna d , Márcia Terezinha Baroni de Moraes c,d,∗ a
Department of Immunology, National Institute of Quality Control in Health (INCQs), Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil Post Graduation Program in Health Surveillance, National Institute of Quality Control in Health (INCQs), Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil c Laboratory of Molecular Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil d Laboratory of Recombinant Technology, Bio-Manguinhos, Oswaldo Cruz Foundation (FIOCRUZ), P.O. Box 926, Rio de Janeiro, Brazil b
a b s t r a c t Article history: Received 7 May 2010 Received in revised form 13 December 2010 Accepted 16 December 2010 Available online 23 December 2010 Keywords: Hepatitis B virus Hepatitis B surface antigen Monoclonal antibody ELISA Potency test
Commercial enzyme-linked immunosorbent assay (ELISA) kits for the determination of the in vitro potency of recombinant hepatitis B vaccines, which detect hepatitis B surface antigen (HBsAg), have been used frequently as an alternative for traditional in vivo potency tests. With the constant need for validation procedures, an ELISA that could be employed to determine the in vitro potency of five recombinant hepatitis B vaccines simultaneously was established using two monoclonal antibodies. The use of two monoclonal antibodies produced “in house” specific for the small envelope protein S of the hepatitis B virus (HBV) resulted in the production of a highly specific, sensitive and stable ELISA. The standard ELISA parameters used in this study, considering the HBsAg content of each recombinant hepatitis B vaccine evaluated, resulted in a standard curve that could be applied for potency evaluations of different, commercial hepatitis B vaccine lots. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Worldwide, hepatitis B remains a major public health problem: two billion individuals have been infected with hepatitis B virus (HBV). Of these 350 million are persistent carriers of hepatitis B virus (Heathcote, 2008). Vaccination is fundamental to stopping the transmission of HBV (Gish and Locarnini, 2006; Mast et al., 2005, 2006). Several vaccine manufacturers have used recombinant deoxyribonucleic acid (DNA) technology to express HBsAg in HBV transfected yeast (i.e., Pichia pastoris, Saccharomyces cerevisiae, and Hansenula polymorpha). These hepatitis B vaccines are recombinant hepatitis B vaccines used most commonly (Zanetti et al., 2008). The small envelope S protein expressed in transfected yeasts self-assembles into immunogenic, spherical particles almost identical to the 22 nm particles found in the serum of hepatitis B carriers. The expressed HBsAg is
∗ Corresponding author at: Laboratory of Molecular Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Av. Brasil, 4365 Manguinhos, Zipcode 926, Rio de Janeiro, RJ, Brazil. Tel.: +55 21 2562 1891; fax: +55 21 2260 4866. E-mail address: baroni@ioc.fiocruz.br (M.T.B. de Moraes). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.12.010
purified to remove the yeast components by several physical separation techniques (i.e., filtration and chromatography). This technology has offered the potential for the unlimited production of vaccines. This vaccine is a part of the routine vaccination schedules of many of the infants and children of the world (Zanetti et al., 2008). Quality control for recombinant hepatitis B vaccines requires the determination of the potency of the vaccine by measuring its HBsAg content, as described by the World Health Organization (WHO, 1989). For this purpose, manufacturers have developed an in vitro assay that has been used by many national control laboratories for official testing. An ELISA that uses only two monoclonal antibodies is described. This ELISA could be used to determine simultaneously the comparative in vitro potency of five recombinant hepatitis B vaccines from different manufacturers that have been used in the Brazilian National Immunization Program. The ELISA standard parameters used in this study considered the HBsAg content of each recombinant hepatitis B vaccine evaluated and allowed for the construction of a standard curve. This standard curve could be used as an “in house” ELISA to replace commercial kits.
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Table 1 Summary of vaccines used in the study. Vaccine (®)
Manufacturer
Yeast used for expression
1. Butang 2. Engerix B 3. Euvax B 4. Hepavaxgene 5. Quinvaxem
Butantan Institute (São Paulo, Brazil) GlaxoSmithKline (Brentford, West London) LG Chemical (Seoul, South Korea) Green Cross (Crucell, Dutch) Berna Biotech (Crucell, Dutch)
Hansenula polymorpha Saccharomyces cervisiae Saccharomyces cervisiae Hansenula polymorpha Hansenula polymorpha
2. Materials and methods 2.1. Chemicals All reagents used in the study were of analytical grade and purchased from Sigma Chemical (MO, USA). 2.2. Recombinant hepatitis B vaccine samples The monovalent or combined (Quinvaxem® – diphtheria, tetanus, pertussis/whooping cough, hepatitis B and Haemophilus influenzae type B) hepatitis B vaccines used in this study were from different manufacturers, as described in Table 1. For the dissociation of aluminium hydroxide, the method described previously by Dobbelaer (1997), with slight modifications, was used. In brief, a volume of 500 L of each vaccine containing 20 g/mL of HBsAg was mixed with the dissociation solution (2.5% diethanolamine – DEA and 0.2% Triton X-100 in phosphate-buffered saline (PBS), pH 7.4). This mixture was incubated at room temperature for 30 min and centrifuged for 10 min at 231 × g. The supernatants were used directly in the ELISA as described below. To reduce their potency artificially, two lots of each vaccine sample (except for Heberbiovac® ) were stressed by incubation at 4 ◦ C (lot 1) and at 60 ◦ C (lot 2) separately for seven days. The vaccine was then used in the ELISA according to the standardised ELISA protocol described below in Section 2.7. 2.3. Reference vaccine The reference standard used was provided by each manufacturer, listed in Table 1, and contained 20 g/mL of HBsAg as measured by the Lowry protein assay. 2.4. Monoclonal antibodies Purified, murine monoclonal antibodies against HBsAg (antiHBs MAb) CG2 and AG9 were used as capture and conjugate, respectively. These anti-HBs MAbs were generated against HBsAg particles purified from pooled human plasma that contained HBV subtypes ad and ay, as previously described (Vianna et al., 2006). The final concentration of each purified MAb was determined by a modified Lowry method using bovine serum albumin (BSA) as a standard (Peterson, 1983). The anti-HBs MAb AG9 was peroxidaselabelled using a commercial kit, SureLink HRP conjugation from KPL (MD, USA), according to the manufacturer’s instructions. 2.5. Western blot analysis of samples of recombinant hepatitis B vaccine Gel electrophoresis, SDS-PAGE (12%), was performed with 2 and 5 g of each monovalent hepatitis B vaccine (Butang® , Engerix B® , Euvax B® , Hepavaxgene® ), which were dissociated previously from aluminium hydroxide (Section 2.2) according to the technique of Laemmli (1970). As a positive control for Western blotting, 5 L of concentrated HBsAg serum particles, prepared according to Moraes et al. (2006), was applied to the same gel. After electrophoresis,
the gel was blotted for 20 min at 16 V on a 0.22-m nitrocellulose membrane (BioRad, CA, USA) using a semi-dry system (BioRad). The membranes were incubated with a blocking reagent (5% non-fat dry milk and 0.05% Tween 20 in PBS (pH 7.4)) overnight at 4 ◦ C and subsequently processed for 1 h with anti-HBs MAb AG9 (Moraes et al., 2006) diluted in blocking reagent. After washing with PBS (pH 7.4) containing 0.05% Tween 20, the membranes were processed with an anti-mouse, alkaline phosphatase-AP conjugated immunoglobulin (Sigma) diluted in blocking buffer. Bands were detected using ditetrazolium salt nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates, according to the manufacturer’s instructions. 2.6. Parameters used for standardisation of the ELISA For standardisation of the assay, four parameters were considered. (1) Coating and conjugate concentrations, concentrations of 2 g, 4 g, 6 g and 8 g/mL of capture MAb and 1:500, 1:1000, 1:2500 and 1:5000 of conjugate MAb were evaluated. (2) Vaccine concentration. The tested concentrations were 0.005, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 and 0.81 g/mL. (3) Blocking solution: three different blocking solutions were tested. (i) PBS containing 0.05% Tween 20 (PBS-T) added to 5% (w/v) non-fat dry milk, 3% (v/v) fetal calf serum (FCS) and 0.5% (w/v) bovine serum albumin (BSA); (ii) PBS containing 5% (w/v) BSA and (iii) PBS containing 3% (w/v) BSA. (4) Time and temperature of incubation: the plates were incubated initially at 37 ◦ C for 1 h and at 4 ◦ C for 18 h. For the conjugate, incubation was at 37 ◦ C for 120, 60 and 30 min with and without rotation (63 × g) (blocking and vaccine addition steps). The presence of aluminium hydroxide in the vaccines was evaluated by testing the dissociated vaccine samples for aluminium hydroxide as described in Section 2.1. Two ELISA microplates from different manufacturers were evaluated: Nunc-Maxisorp (Roskilde, Denmark) and Microlom 600-Greiner Bio-one (Atlanta, USA). 2.7. Protocol for standardisation of the ELISA Anti-HBs MAb CG2 in coating buffer (0.1 M Na2 CO3 , 0.02% NaN3 , pH 9.6) was applied to an ELISA microplate (Nunc-Maxisorp) at a concentration of 8 g/mL. The microplate was then incubated at 37 ◦ C for 30 min and then moved to 4 ◦ C for 18 h. Next, the microplate was washed three times with PBS-T. To minimise nonspecific binding, the microplate was blocked for 1 h at 37 ◦ C on a rotational shaker (63 × g) with PBS containing 3% BSA. The same washing procedure was repeated, and the microplate was incubated at 37 ◦ C on a rotational shaker (63 × g) with 100 L of vaccine samples at different dilutions. After the same washing procedure, the microplate was incubated with peroxidase-labelled anti-HBs MAb AG9 diluted 1/2500 in PBS for 1 h at 37 ◦ C. Finally, the microplate was washed and incubated in the dark at room temperature with 3,3 ,5,5 -tetramethylbenzidine (TMB) liquid substrate system for ELISA (Sigma). After 30 min, the reaction was stopped with 2 N sulphuric acid. The absorbance was measured at 450 nm with a Spectra Rainbow (Tecan, Crailsheim, Germany) microtiter plate reader within 30 min of adding the stop solution. The assays were carried out in triplicate.
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2.8. Evaluation of the stability of the ELISA Initially, ELISA microplates (Nunc-Maxisorp, Denmark) coated with anti-HBs MAb CG2 (8 g/mL) in buffer (0.1 M Na2 CO3 , 0.02% NaN3 , pH 9.6) were sealed and incubated at 4 ◦ C for 18 h in a humidified chamber. Next, the coating solution was discarded, and the microplate stabilised with 200 L of stabilisation buffer (0.1 M Na2 CO3 , 0.02% NaN3 (pH 9.6) containing 5% sucrose and 0.3% BSA) per well. The microplates were incubated for 45 min at 37 ◦ C, and the stabilisation buffer was discarded. To evaluate the stability in terms of analytical sensitivity, the microplates were stored dry and sealed with plastic film and aluminium foil packaging at 4 ◦ C and at −20 ◦ C for six months. Before each ELISA, the microplate was left at room temperature for 10 min and washed three times with PBS-T. The microplates were blocked for 1 h at 37 ◦ C on a rotational shaker (63 × g) with PBS containing 3% BSA. The ELISA followed the procedure described in Section 2.7. Variations of percent sensitivity were calculated based on the absorbance values obtained before (zero time = 100% sensitivity) and after storage of the microplate using the following equation: Sensitivity % =
Absorbance after storage × 100 Absorbance before storage
2.9. Evaluation of the validation parameters 2.9.1. Specificity Specificity was determined by testing the combined Quinvaxem® vaccine containing HBsAg in comparison with the combined (diphtheria, tetanus, pertussis/whooping cough) vaccine without HBsAg. 2.9.2. Limit of detection (LOD) The limit of detection was evaluated by analysing the absorbance values of a blank sample (PBS) and the negative IgG1 MAb interferon 1a (Cantelli et al., 2009) in multiple replicates of three different plate assays. The LOD was defined as being three times the standard deviation. 2.9.3. Limit of quantitation (LOQ) The limit of quantitation (LOQ) was calculated considering the average value of the absorbance obtained for the LOD added value of 10 standard deviations obtained from a blank sample (PBS). 2.9.4. Linearity The linearity of the ELISA was assessed by using the linear correlation coefficient of the standard curves established with the reference standard to determine the LOD. The HBsAg concentrations used in the standard curve were 0.2, 0.4, 0.6, 0.8 and 1 g/mL. The ELISA was repeated in triplicates. The linearity was calculated from the linear regression of the results of the standard curve. The linear, angular and variation coefficients were determined (R ≥ 0.98). Statistical analyses using ANOVA were performed considering p < 0.05 to the angular coefficient to be not null and p > 0.05 to be a straight line (SAS, 1990). 2.9.5. Precision The precision study was divided in repeatability (intra-assay) and intermediate (inter-assay) precision. To evaluate both parameters, vaccine samples diluted previously and containing a high, medium and low content of HBsAg (designed as lots 1, 2 and 3, respectively) were used. The HBsAg concentrations in the standard curve were 0.2, 0.4, 0.6, 0.8 and 1 g/mL. The ELISA was carried out in triplicate. To assess intermediate precision, the ELISA was executed independently by two technicians. The standard deviation was calculated. The coefficient of variation (CV) could not be higher
than 10%, whereas the CV of the intermediate precision could not exceed 20%. 2.9.6. Accuracy For evaluations of accuracy, three dilutions of each vaccine sample were prepared and tested in three replicates of each vaccine in the same run. The normal deviation could not exceed 20%. Accuracy values were determined using the following equation: Accuracy =
Experimental average value of concentration Theoretical value of concentration
3. Results 3.1. Recombinant hepatitis B vaccines and monoclonal anti-HBs antibodies used in the ELISA Five yeast cell-expressed recombinant hepatitis B vaccines were evaluated according to their reactivities with anti-HBs MAb CG2. Five were monovalent vaccines containing HBsAg and one was a combined vaccine (against diphtheria, tetanus, pertussis/whooping cough, hepatitis B and H. influenzae). All of the vaccine samples tested in the ELISA contained initially aluminium hydroxide as an adjuvant. To evaluate the influence of the adjuvant on ELISA sensitivity, these vaccine samples were also submitted to aluminium hydroxide dissociation and again evaluated by the ELISA (0.05, 0.25, 0.5 and 1 g/mL). The absorbance values obtained after aluminium hydroxide dissociation were higher than those obtained from aluminium hydroxide-associated vaccines (Fig. 1). The most effective concentration of anti-HBs MAb was determined by testing the ELISA protocol, described in Section 2.6. Three different concentrations of anti-HBs MAbCG2 (2, 4 and 8 g/mL) were used as capture in parallel with different concentrations of vaccine samples (0.005, 0.025, 0.050, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 g/mL). Different dilutions of anti-HBs MAb AG9 (1:500, 1:1000, 1:2500 and 1:5000) were used as conjugate. The optimal concentrations of anti-HBs MAb CG2 and AG9, which presented both high sensitivities, as measured by absorbance values of the different vaccine sample concentrations, and low backgrounds in the ELISA, were 8 g/mL and 1/2500, respectively. The sensitivity of the standardised ELISA for detection of the five recombinant hepatitis B vaccines was 0.2 g/mL (Fig. 2). The standardised ELISA protocol presented in a crescent order had higher sensitivity to Euvax® , Engerix B® , Hepavax-gene® and Butang® monovalent recombinant hepatitis B vaccines. The combined recombinant hepatitis B vaccine Quinvaxem® seemed to have very similar sensitivity to Euvax® . The monovalent recombinant hepatitis B vaccines were exposed to temperature stresses. These vaccines were incubated at 4 ◦ C and 60 ◦ C during a consecutive, 7-day period. The vaccines were then tested by the ELISA. The vaccines exposed to the 60 ◦ C temperature displayed a reduced sensitivity in comparison with the 4 ◦ C standard temperature, with p-values of 0.03 (Engerix B® ), 0.005 (Hepavaxgene® ) and 0.004 (Butang® ). The reduction of sensitivity for other vaccines was not significant (data not shown). 3.2. ELISA parameters tested The introduction of plate rotations to the ELISA procedure during the blocking and vaccine addition steps increased significantly the sensitivity of the assay. The blocking solution PBS containing 3% BSA showed the best results with the lowest background values and the highest sensibility (data not shown). Finally, the ELISA microplates from Nunc-Maxisorp (Denmark) showed significantly
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Fig. 3. Western blot analysis of monovalent recombinant hepatitis B vaccines. 2 and 5 g of each vaccine (respectively Butang® – lines 1, 2; Engerix B® – lines 3, 4; Euvax B® – lines 5, 6; Hepavaxgene® – lines 7, 8) and 5 L of concentrated HBsAg serum particles (Line 9) were reacted with anti-HBs MAb AG9.
The pattern of recombinant HBsAg on SDS-PAGE was compared to the HBsAg from human plasma particles (Fig. 3). 3.4. Robustness of the method
Fig. 1. ELISA analysis of recombinant hepatitis B vaccines associated with aluminium hydroxide (A) in comparison with recombinant hepatitis B vaccines dissociated from aluminium hydroxide (B). The concentration of each vaccine was adjusted to 0.05, 0.25, 0.5 and 1 g/mL.
higher sensitivities compared to the Microlom 600-Greiner Bio-one (Atlanta, USA) (data not shown). 3.3. Western blot analysis of recombinant hepatitis B vaccine samples using anti-HBs MAb AG9 The different forms of HBsAg from each monovalent vaccine produced by the yeast cells were recognised by the anti-HBs MAb AG9.
During the establishment of the ELISA, the BSA concentration in the blocking solution (5% and 3% tested) was not a significant factor for the recombinant hepatitis B vaccines (p > 0.05) evaluated. The absorbance values obtained in the ELISA were considerably higher after the introduction of incubation for 1 h and plate rotation (63 × g) during the blocking and vaccine addition steps. The dissociation of aluminium hydroxide from the evaluated vaccine samples significantly increased the sensitivity of the ELISA (data not shown). 3.5. Stability of the ELISA Fig. 4 shows the results of the ELISA using plate coating with anti-HBs MAb CG2 (8 g/mL) previously stored at 4 ◦ C or −20 ◦ C for six months (stability assay). The sensitivity results (%) obtained were above 90% for all tested samples at both temperatures. 3.6. Validation of the ELISA 3.6.1. Specificity of the ELISA The specificity of the ELISA was assessed by the absorbance values obtained for the combined vaccine containing HBsAg (Quinvaxem) in comparison with the combined vaccine (diphtheria, tetanus and pertussis/whooping cough), but without HBsAg, as shown in Table 2.
Fig. 2. Standardised ELISA. Different dilutions of four monovalent recombinant hepatitis B vaccines and one combined recombinant hepatitis B vaccine were analysed. The sensitivity of the ELISA was 0.2 g/mL.
Fig. 4. Stability of ELISA with three monovalent recombinant hepatitis B vaccines and one combined recombinant hepatitis B vaccine. The sensitivity (%) presented on a vertical axis corresponds to the standardised ELISA in comparison with plates coated with anti-HBs CG2 (8 g/mL) stored previously at 4 ◦ C for six months.
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Table 2 Specificity of the combined vaccines containing HBsAg in comparison with the combined vaccine without HBsAg content. Samples
Concentration (g/mL) expressed in OD value
Quinvaxem® combined vaccine Quinvaxem® combined vaccine without HBsAg content Buffer dilution (PBS)
0.2
0.4
0.6
0.8
1.0
0.436 0.060 0.059
0.684 0.059 0.059
0.775 0.061 0.060
0.836 0.063 0.062
0.894 0.064 0.063
3.6.2. Limit of detection (LOD) and limit of quantitation (LOQ) The LOD and LOQ were calculated using 3 and 10 standard deviations, respectively, of blank assay. Under the assay conditions described in this study, the LOD was 0.078 g/mL. The LOQ value (g/mL) found was below the lower curve point (absorbance value corresponded to 0.104). The analytical range observed corresponded to the lower limit of quantitation (LLOQ) and to the upper limit of quantitation (ULOQ), 0.2 g/mL and 1.0 g/mL, respectively. 3.6.3. Linearity of the ELISA The values of the correlation coefficients of the linear regression (R2 ) for three batches of each recombinant hepatitis B vaccine evaluated were all above 0.99. This demonstrated the linearity for the analytical range of 0.2, 0.4, 0.6, 0.8 and 1 g/mL. Using an ANOVA, values for the angular non null coefficient (with p < 0.05) and for the straight line (with p > 0.05) were obtained (data not shown). 3.6.4. Precision of the ELISA The results of the precision study are presented in Table 3. The CV for repeatability and intermediate precision for all vaccines were determined and found to be between 2 and 3% (below 10%) and 2 and 5% (below 20%), respectively. 3.6.5. Accuracy of the ELISA Variation coefficient values for all recombinant hepatitis B vaccines evaluated by the ELISA were between 2 and 15%. The accuracy percentage ranged between 97 and 100% for different
Table 3 Results of repeatability and intermediate precision studies. Each lot was tested three times in the same run in independent experiments involving two technicians. Vaccinea
Euvax® Lotb 1 Lot 2 Lot 3 Hepavax-gene® Lot 1 Lot 2 Lot 3 Engerix-B® Lot 1 Lot 2 Lot 3 Butang® Lot 1 Lot 2 Lot 3 Quinvaxem® Lot 1 Lot 2 Lot 3
Repeatability (intra-assay)
Intermediate precision (inter-assay)
SD
CVb
SD
CV
0.019 0.018 0.024
3.5 2.8 2.6
0.010 0.018 0.014
2.6 3.9 2.7
0.012 0.013 0.012
2.2 2.3 2.2
0.013 0.014 0.010
3.0 2.5 2.0
0.011 0.012 0.010
2.2 2.3 2.2
0.017 0.021 0.017
4.0 4.2 4.1
0.010 0.010 0.010
2.5 2.1 2.3
0.013 0.014 0.010
3.0 2.5 2.0
0.013 0.014 0.013
2.0 2.1 2.0
0.014 0.013 0.010
1.8 2.4 2.0
The coefficient of variation (CV) reported in each case corresponds to the media of triplicates for each batch. a Different types of vaccines. b Coefficient of variation = (standard deviation [SD]/mean) × 100.
vaccines containing different concentrations of HBsAg (data not shown). 4. Discussion The potency evaluation of hepatitis B vaccines used in Brazil’s National Immunization Program has led this group to develop and validate an alternative ELISA for commercial tests and to attempt to reduce the large number of animals needed as in vivo models. The ELISA was designed to allow for the determination of the HBsAg content of different recombinant hepatitis B vaccines from different manufacturers simultaneously. Thus, this ELISA will save time and money. Recombinant hepatitis B vaccines are produced using yeast expression technology, but each vaccine tested presented a characteristic sensitivity to the ELISA and a unique Western blotting expression pattern. These unique characteristics were probably observed because of the different yeast species used in the production of each vaccine, which leads to a different recognition pattern by the MAb used. The ELISA analyses using the recombinant hepatitis B vaccines associated to aluminium hydroxide in comparison to recombinant hepatitis B vaccines dissociated from aluminium hydroxide indeed show a considerable increase of sensitivity. This sensitivity difference will be important to the precise determination of potency. Despite the possibility of nonspecific binding between MAb and aluminium hydroxide, according to the results of Giffroy et al. (2006), the process of removing adsorbed aluminium hydroxide from the HBsAg probably exposed some other antigenic epitopes and increased the sensitivity of the ELISA. Even though the dissociation of aluminium hydroxide from HBsAg before carrying out the ELISA is an additional, necessary step that leads to better results, the protocol described by Dobbelaer (1997), which was performed in this study with slight modifications, is very simple. Otherwise, the sensitivity of the ELISA could be increased by introducing plate rotations, which apparently allow a correct interaction between the HBsAg and the anti-HBs MAbs. According to the validation study conducted, the performance characteristics of the ELISA were found to be satisfactory for all the recombinant hepatitis B vaccines (monovalent and combined) tested. It was shown that the proposed ELISA could measure specifically HBsAg in the presence of other vaccine antigens, as demonstrated using the combined vaccine against diphtheria, tetanus, pertussis/whooping cough, hepatitis B and H. influenzae. The sensitivity of this ELISA for the detection of HBsAg in human serum samples is lower (0.2 g/mL) compared to the commercial ELISA kit, which shows a sensitivity of about 0.5 ng/mL. For this ELISA to be applicable to human serum samples, an evaluation against an HBsAg reference panel will be necessary to measure HBsAg in human clinical samples. Combined, recombinant hepatitis B vaccines contain 20 g/mL of HBsAg. It has no impact on the intended use of the ELISA. As noted by Giffroy et al. (2006), lower sensitivity may confer an advantage by reducing the number of dilution steps required and thereby minimising potential error and variability. The validation parameters (specificity, LOD and LOQ, linearity, precision and accuracy) were conducted according to the International Conference Harmoniza-
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tion (ICH) and indicated that the results obtained in this study on the recombinant hepatitis B vaccines were satisfactory and useful for the national control laboratory of Brazil. 5. Conclusion The proposed ELISA is suitable for measuring the HBsAg content in monovalent and combined recombinant hepatitis B vaccines and provides a reliable method for determining potency. Acknowledgements We thank Vera Bongertz and Ana Cristina Martins de Almeida Nogueira for fruitful discussions. We thank Bruno Baroni de Moraes e Souza for reviewing this paper. This work was supported by the National Institute of Quality Control in Health (INCQS), BioManguinhos-Fiocruz and Oswaldo Cruz Institute-Fiocruz. Isabella Fernandes Delgado and Marcia Terezinha Baroni de Moraes are fellowship researchers from the National Research Council (CNPqBrazil). References Cantelli, C.P., Teixeira, M.G.M., Santos, E.A., da Silva Jr., H.C., e Mouta Jr., S.S., Pimenta, M.M.A., Vianna, C.O., Souza, N.P., Batoreu, N.M., Galler, R., Moraes, M.T.B., 2009. Generation of monoclonal antibodies against human recombinant interferon beta using genetic immunization with simultaneous expression of IgM and IgG isotypes. Hybridoma 28, 211–214.
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