JIM-11834; No of Pages 5 Journal of Immunological Methods xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroups A, C, W and Y in multivalent vaccines Fátima Reyes ⁎, Oscar Otero, Maribel Cuello, Nevis Amin, Luis García, Daniel Cardoso, Frank Camacho Research and Development Vice Presidency, Finlay Institute, A.P. 16017, La Habana Cod. 11600, Cuba
a r t i c l e
i n f o
Article history: Received 31 January 2014 Received in revised form 21 March 2014 Accepted 25 March 2014 Available online xxxx Keywords: Quantitation ELISA Capsular polysaccharide Meningococcal vaccines
a b s t r a c t Neisseria meningitidis is a Gram negative bacterium that has been classified in 13 serogroups according to the biochemical composition of the capsular polysaccharide (CP). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY). Individual CP quantitation in multivalent meningococcal CP-based vaccines is required for quality control testing of these products. In this regard, four sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the quantitation of CP. The quantitation and detection limits of the four ELISAs were below 1 ng/mL. The assays showed good reproducibility and repeatability as calculated for each point of the standard curve (CV b 15%). In addition, five multivalent meningococcal CP-based vaccines were evaluated and the proposed ELISAs showed that these vaccines were found into the accepted range (±30%) of CP content. These assays are suitable for screening multiple plain or conjugated meningococcal CP-based vaccines and could be useful for monitoring lot-to-lot consistency and stability analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Meningococcal disease, caused by Neisseria meningitidis, occurs mainly as either septicemia or meningitis, and is a worldwide health problem (Chang et al., 2012). N. meningitidis strains have been classified into at least 13 serogroups on the basis of the structure of the capsular polysaccharide (CP)
Abbreviations: CP, capsular polysaccharide; MenA, N. meningitidis serogroup A; MenC, N. meningitidis serogroup C; MenW, N. meningitidis serogroup W; MenY, N. meningitidis serogroup Y; ELISA, enzyme-linked immunosorbent assay; CV, coefficient of variation; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection; MAbs, monoclonal antibodies; TT, tetanus toxoid; HRP, horseradish peroxidase; IC, internal controls; OD, optical density; 4PL, four-parameter logistic equation; QCs, quality control; LOD, limit of detection; LOQ, limit of quantification; SD, standard deviation; CZE, capillary zone electrophoresis. ⁎ Corresponding author at: Finlay Institute, Calle 27, No. 19805, La Lisa, A.P. 16017, Cod. 11600 Havana, Cuba. Tel.: +53 7 2716911; fax: +53 7 2731218. E-mail addresses: [email protected], freyes@finlay.edu.cu (F. Reyes).
(Harrison et al., 2009). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY) (Stephens et al., 2007). Most meningococcal vaccines are monovalent or multivalent mixtures of either polysaccharides or polysaccharides conjugated to carrier proteins (Tan et al., 2010). Quantitative determination of the individual polysaccharide components in multivalent meningococcal vaccines is an important step in manufacturing and regulatory control (Cook et al., 2013a). Several methods for individual quantitation of meningococcal polysaccharides have been described. Determination of total phosphorus content (Chen et al., 1956) has been employed for measurement of MenA and MenX, while sialic acid content (Svennerholm, 1957) can be used for MenC, MenW, and MenY. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been used for the quantitation of polysaccharides in multivalent meningococcal CP-based vaccines (Cook et al., 2013b). These methods can provide accurate and sensitive measurement of meningococcal
http://dx.doi.org/10.1016/j.jim.2014.03.020 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
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F. Reyes et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
polysaccharides but require prior conversion to monosaccharides by acid digestion. The use of strong acids, often at elevated temperature, represents a potential safety concern for laboratory staff and has a cost associated with material disposal (Lamb et al., 2005). Simple and affordable techniques to quantitate polysaccharides in meningococcal vaccines are essential for the evaluation of antigen content and lot-to-lot consistency of manufacture. Immunological assays such as rate nephelometry or inhibition enzyme-linked immunosorbent assay (ELISA) may be used (WHO Expert Committee on Biological Standardization, 2006). In addition, sandwich ELISA is a common tool for antigen quantitation (Trad et al., 2011; Suzaki et al., 2006; Ma et al., 2013). Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. However, monoclonal antibodies (MAbs) recognize a single epitope that allows fine detection and quantitation of small differences in antigen. In a previous study, our group obtained MAbs for MenA, MenC, MenW and MenY CPs. The selectivity of these MAbs was confirmed for each CP, with no cross-reactivities to heterologous CPs, or carrier proteins like tetanus toxoid (TT) and CRM197 (Reyes et al., 2013). The aim of the present work was to develop four sandwich ELISAs using these MAbs to quantitate CPs from MenA, MenC, MenW and MenY in meningococcal CP-based vaccines.
8 mg of each MAb was coupled to 4 mg of HRP and was purified by gel filtration chromatography. Conjugates containing 1% BSA, 0.05% thiomersal and 50% glycerol were stored in small aliquots at −20 °C. 2.4. Calibrators, quality controls and calibration curve construction
2. Materials and methods
In the absence of International Standards, calibrators consisted of purified CPs from MenA, MenC, MenW and MenY produced at Finlay Institute (Havana, Cuba) and used as internal controls (IC). MenC, MenW and MenY ICs were quantitated using resorcinol colorimetric assay (Chen et al., 1956) with N-acetylneuraminic as the standard while MenA IC was quantitated using Chen colorimetric assay (Svennerholm, 1957) using KH2PO4 as the standard. These ICs are supplied as lyophilized powders with 100 μg of each CP. A two-fold serial dilution of each IC was used to generate a six-point calibration curve with a range from 0.3125 to 10 ng/mL. GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) was used for plotting the optical density (OD492) against the CP concentrations and four-parameter logistic equation (4PL) was applied to obtain the function describing a sigmoid model. The quality controls (QCs) consisted of meningococcal CP powders (Finlay Institute, Havana, Cuba) which were weighted, dissolved in PBS and adjusted to a final concentration of 1.25 ng/mL (w/v). Calibrators and QCs were stored as single ready-to-use aliquots at −20 °C.
2.1. Reagents and buffers
2.5. Description and preparation of vaccines
Unless otherwise stated, reagents were obtained from Sigma-Aldrich (USA). The following buffers were used: Coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), blocking buffer (PBS, 3% non-fat dried milk), washing buffer (PBS, 0.05% (v/v) Tween 20, pH 7.4), and substrate buffer (35 mM citric acid, 67 mM Na2HPO4, 0.012% (w/v) H2O2, pH 5.0).
Three registered vaccines and two lots of two experimental vaccines were analyzed.
2.2. MAbs Murine MAbs against MenA, MenC, MenW and MenY CPs were produced and characterized as described previously (Reyes et al., 2013). Briefly, hybridomas were produced by fusion of SP2/ O myeloma cell line with splenocytes of BALB/c mice that were immunized with subcutaneous injections of meningococcal polysaccharide group A conjugated to TT (MenAfriVac®, Serum Institute of India, Ltd.) and meningococcal polysaccharide groups A, C, W135 and Y conjugated to CRM197 (MENVEO®, Novartis, Switzerland) vaccines. Four hybridomas, coded as 7E1F7 (anti MenA CP), 7E12B3 (anti MenC CP), 5C11F1 (anti MenW CP) and 5H10D9 (anti MenY), were selected and grown as mouse ascites in the peritoneal cavity of pristane-primed BALB/c mice. The MAbs were purified from the ascites fluid by affinity chromatography using HiTrap Protein G (GE Healthcare, Germany) following manufacturer's instructions. 2.3. Peroxidase labeling of MAbs Purified MAbs were conjugated in-house to horseradish peroxidase type VI (HRP) as described by Wilson (1978). Briefly,
– MENVEO® vaccine (Novartis, Switzerland): Meningococcal polysaccharide groups A, C, W135 and Y conjugated to CRM197 (10 μg/dose of MenA CP and 5 μg/dose of each MenC, MenW and MenY CPs). – MENCEVAX®ACWY vaccine (GlaxoSmithKline): Meningococcal polysaccharide groups A, C, W and Y (50 μg/dose of each CP). – vax-MEN-ACW135® (Finlay Institute, Cuba): Meningococcal polysaccharide groups A, C and W (50 μg/dose of each CP). – ACW135Y (lots 105, 106) and ACW135XY (lots 101, 102) vaccines: Experimental vaccines (Finlay Institute, Cuba), meningococcal polysaccharide groups A, C, W, Y or A, C, W, X and Y (50 μg/dose of each CP). All the vaccines were reconstituted as described by the manufacturers and were prepared to a final concentration of 1.25 ng/mL taking into account the standard curve range. 2.6. ELISA sandwich for serogroup quantitation Polystyrene microwell plates (Maxisorp, Nunc, USA) were coated with 10 μg/mL of MAb 7E1F7, 7E12B3, 5C11F1 or 5H10D9 in coating buffer (100 μL/well). After overnight incubation at 4 °C, the coated wells were washed three times and blocked with blocking buffer for 1 h at 37 °C. The calibrators, QCs and samples were added (100 μL/well) and incubated 1 h at 37 °C. Subsequently, the wells were washed three times and peroxidase conjugated MAbs diluted to 1:15000 in washing buffer containing 1% non-fat dried milk were added to the wells
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
F. Reyes et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
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Fig. 1. Calibration curves obtained from sandwich ELISAs using the 4PL equation. A, B, C and D represent the standard curves using MenA, MenC, MenW and MenY CPs respectively. Error bars represent standard deviations of quadruplicate samples.
(100 μL/well) and incubated for 30 min at room temperature. After four washes, 0.4 mg of OPD per mL substrate buffer was added to the wells (100 μL/well) and following 15 min the color development was stopped with 1 M H2SO4. OD was measured at 492 nm using a Microplate Reader (Thermo Electron Corporation, USA) and unknown concentrations were calculated using CDC Software (Plikaytis et al., 1996). The calibrator, QCs and samples were run in triplicates unless otherwise stated. Three independent experiments were performed and results were expressed as mean of CP content. Optimal dilutions of MAbs and HRP conjugated MAbs were determined by standard checkerboard titrations according to the procedures described by Crowther and Walker (2009).
3. Results 3.1. Calibration curve construction Calibration curves are shown in Fig. 1. A four-parameter fit model was applied to obtain the function describing a sigmoid model. Coefficients of determination (R2) ≥0.99 were obtained in all cases.
3.2. Limits of detection and quantitation The LOD and LOQ were found below 1 ng/mL for all CP in each ELISA assay as shown in Table 1.
2.7. Limits of detection and quantitation Lower limits of detection (LOD) and quantitation (LOQ) were defined as the mean of blank values (n = 20) plus either 3 or 10 standard deviation (SD) respectively. 2.8. Intra- and inter-assay precision Intra-assay precision was determined by interplate and intraplate precision as calculated by the coefficient of variation (CV). The interplate precision was evaluated by measuring triplicates of standard curve on four distinct ELISA plates on the same day in parallel experiments. The intraplate precision was evaluated by measuring the standard curve in quadruplicate on a single ELISA plate. The inter-assay precision was determined as the CV on five separate occasions.
3.3. Intra- and inter-assay precision The four ELISA assays showed inter-assay CVs of 3–14%, while intra-assay CVs varied between 1–7% and 1.5–9% for the intraplate and interplate assay respectively (Tables 2–5).
Table 1 Lower limits of detection and quantitation for each capsular polysaccharide.
LOD LOQ
MenA ng/mL
MenC ng/mL
MenW ng/mL
MenY ng/mL
0.382 0.855
0.19 0.56
0.15 0.35
0.08 0.24
LOD was calculated as reagent blank + 3 × SD of reagent blank, whereas LOQ was determined as reagent blank + 10 × SD of reagent blank.
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
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Table 2 Inter- and intra-assay coefficient of variation (%) of MenA capsular polysaccharide standards. MenA CP (ng/mL)
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
3.24 10.58 12.64 11.55 10.42 11.63
Intra-assay
Table 4 Inter- and intra-assay coefficient of variation (%) of MenW capsular polysaccharide standards. MenW CP (ng/mL)
Intraplate n=4
Interplate n = 12
0.91 2.54 3.18 6.89 2.16 4.27
2.62 4.41 9.21 8.28 4.67 4.36
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
6.50 7.66 8.39 10.58 10.63 11.24
Intra-assay Intraplate n=4
Interplate n = 12
2.99 4.39 4.82 2.81 2.43 3.57
2.55 1.64 2.82 3.05 3.20 3.52
Polysaccharide content must be checked on the final product to determine that it contains the stated amount of polysaccharide. In this regard, four in-house quantitative sandwich ELISAs were developed using four specific MAbs for measurement each CP content in multivalent meningococcal CP-based vaccines. Several procedures for the quantitation of meningococcal CP such as phosphorus and sialic acid content measurement, capillary zone electrophoresis (CZE) and HPAEC-PAD have been used (Chen et al., 1956; Svennerholm, 1957; Cook et al., 2013b; Lamb et al., 2005; Ricci et al., 2001; Lei et al., 2000). However, these methods have some limitations: the first can't be used in multivalent meningococcal vaccines (trivalent, tetravalent, pentavalent) because phosphorus is present in MenA and MenX CP (Berti et al., 2012), while N-acetylneuraminic acid is common to MenC, MenW and MenY CP (Cook et al., 2013b). On the other hand, it has been reported that CZE has been used to quantitate native polysaccharides from a mixture of meningococcal polysaccharide serogroups A, C, W and Y (Lamb et al., 2005). Unfortunately, quantitation of polysaccharides in meningococcal multivalent vaccines using this method has not been
fully evaluated. Finally, HPAEC-PAD requires prior conversion to monosaccharides by acid digestion and the presence of saccharide excipients such as sucrose or lactose may interfere with the determination of some polysaccharide serogroups (Cook et al., 2013a) and add considerable time and expense to the procedure. Using the proposed ELISAs the time for analysis could considerably be shortened and the analysis of 22 vaccine samples (in three replicates) can be performed without excipient removal. The developed sandwich ELISAs showed LOD and LOQ below 1 ng/mL for the respective CPs. These results agree with the affinity constant values calculated for each MAb in a previous work (Reyes et al., 2013). Therefore, these assays can be used successfully for the quantitation of CP in meningococcal multivalent vaccines. In addition, using the proposed ELISAs and taking into account their high sensitivity, these assays could be a useful tool to test for free polysaccharide in conjugated vaccines prior separation of the unbound CP from conjugated-CP in such vaccines. Free polysaccharide must be tested to ensure that the amount present is within the limits agreed by the regulatory authorities based on lots shown to be clinically safe and efficacious. On the other hand, the inter-assay and intra-assay CVs were calculated for each point of the standard curve (10–0.312 ng/mL). Reproducibility and repeatability were in accordance with international recommendations, which define the acceptance criterion of precision for standard curves as inter-assay CV ≤ 15% (DeSilva et al., 2003). Five multivalent meningococcal CP-based vaccines were evaluated using the developed sandwich ELISAs. The vaccines have an expected nominal label amount of 50 μg/dose of each CP except for MENVEO®, which has 10 μg/dose of MenA CP and 5 μg/dose of each MenC, MenW and MenY CPs. The common specified range is ±30% of the expected amount per
Table 3 Inter- and intra-assay coefficient of variation (%) of MenC capsular polysaccharide standards.
Table 5 Inter- and intra-assay coefficient of variation (%) of MenY capsular polysaccharide standards.
In general the CV increased with decreasing concentration of CP, and assays for MenA, MenW and MenY were found to have lower inter-assay variation than MenC assays. 3.4. Serogroup quantitation in vaccines Total serogroup polysaccharide content using the four ELISA sandwich are shown in Table 6. All of the five meningococcal CP-based vaccines were found into the accepted range (±30%) of CP content. The CVs were b 9% compared to the acceptance criterion of b15% CV for immunoassays. 4. Discussion
MenC CP (ng/mL)
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
9.27 10.66 11.83 12.44 13.93 14.15
Intra-assay
MenY CP (ng/mL)
Intraplate n=4
Interplate n = 12
1.13 2.20 4.03 3.61 5.05 5.11
3.88 5.68 4.87 4.40 5.71 4.86
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
5.04 4.18 8.28 9.87 9.89 10.10
Intra-assay Intraplate n=4
Interplate n = 12
3.01 3.21 2.98 3.41 2.38 4.75
5.2 4.25 5.11 5.29 5.73 5.04
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
F. Reyes et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
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Table 6 Serogroup polysaccharide content of meningococcal CP-based vaccines. Vaccines
MenA CP Average
ACYW 105 ACYW 106 ACYWX 101 ACYWX 102 vax-MEN-ACW135® MENCEVAX®ACWY MENVEO® a b
50.76 43.05 47.71 48.65 45.83 46.47 8.78
a
MenC CP b
CV
4.19 1.08 1.67 1.35 0.79 2.16 1.91
Average 56.49 57.36 43.68 50.68 51.60 47.47 5.26
MenW CP CV 2.34 1.95 2.06 1.37 4.62 3.96 4.98
Average 48.59 50.31 46.63 57.74 53.65 54.62 4.73
MenY CP CV 2.02 2.47 1.42 4.44 1.43 1.70 2.08
Average 45.62 44.76 46.03 48.68 – 45.34 6.40
CV 3.71 4.38 0.99 8.19 – 4.99 1.94
Mean of triplicate determinations from three independent experiments for each serogroup expressed in μg/dose. Coefficient of variation expressed in %.
dose (0.5 mL) (European Pharmacopoeia). Considering this guideline, MENVEO® vaccine should have between 7 and 13 μg of MenA CP and between 3.5 and 6.5 μg of MenC, MenW and MenY CPs. The remaining vaccines should have between 35 and 65 μg of MenA, MenC, MenW or MenY CPs. Values from Table 6 indicate that all the vaccines screened for CP content were within general specifications for each CP taking into general consideration the European Pharmacopoeia recommendations. 5. Conclusions Four sandwich ELISAs were established for quantitation of CP from N. meningitidis in both conjugated and plain meningococcal CP-based vaccines. The ELISAs showed a very good precision and high sensitivity. These assays are suitable for screening multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability analyses. Acknowledgments The authors thank MsC. M. Ileana Delgado, BsC. Luis A. Izquierdo and Dr. Rolando F. Ochoa for their excellent technical assistance with the ELISAs. We also like to thank Dr. Barbara Bolgiano (National Institute for Biological Standards and Controls, UK) for her kindness in reviewing the draft of the manuscript. References Berti, F., Romano, M.R., Micoli, F., Pinto, V., Cappelletti, E., Gavini, M., Proietti, D., Pluschke, G., MacLennan, C.A., Costantino, P., 2012. Relative stability of meningococcal serogroup A and X polysaccharides. Vaccine 30, 6409. Chang, Q., Tzeng, Y.L., Stephens, D.S., 2012. Meningococcal disease: changes in epidemiology and prevention. Clin. Epidemiol. 4, 237. Chen, P.S., Toribara, T.Y., Warner, H., 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756. Cook, M.C., Bliu, A., Kunkel, J.P., 2013a. Quantitation of serogroups in multivalent polysaccharide-based meningococcal vaccines: optimisation of hydrolysis conditions and chromatographic methods. Vaccine 31, 3702. Cook, M.C., Gibeault, S., Filippenko, V., Ye, Q., Wang, J., Kunkel, J.P., 2013b. Serogroup quantitation of multivalent polysaccharide and polysaccharideconjugate meningococcal vaccines from China. Biologicals 41, 261.
Crowther, J.R., Walker, J.M., 2009. The ELISA Guidebook. Springer. DeSilva, B., Smith, W., Weiner, R., Kelley, M., Smolec, J., Lee, B., Khan, M., Tacey, R., Hill, H., Celniker, A., 2003. Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm. Res. 20, 1885. European Pharmacopoeia, 2012. Meningococcal Polysaccharide Vaccine, 01/ 2008:0250; Meningococcal Group C Conjugate Vaccine, 01/2008:2112 corrected 6.0; Typhoid Polysaccharide Vaccine, 01/2008:1160; Pneumococcal Polysaccharide Conjugate Vaccine (Adsorbed), 01/2008:2150; Haemophilus Tybe b Conjugate Vaccine, 07/2012:1219. Harrison, L.H., Trotter, C.L., Ramsay, M.E., 2009. Global epidemiology of meningococcal disease. Vaccine 27 (Suppl. 2), B51. Lamb, D.H., Lei, Q.P., Hakim, N., Rizzo, S., Cash, P., 2005. Determination of meningococcal polysaccharides by capillary zone electrophoresis. Anal. Biochem. 338, 263. Lei, Q., Shannon, A., Heller, R., Lamb, D., 2000. Quantification of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid conjugate vaccines. Dev. Biol. 103, 259. Ma, S., Mao, Q., Liang, Z., Zhang, C., Yang, W., Sun, Z., Zhang, H., Shen, X., Bi, S., Sun, L., 2014. Development of a sandwich ELISA for the quantification of enterovirus 71. Cytotechnology 66, 413–418. Plikaytis, B., Holder, P., Carlone, G., 1996. Program ELISA for Windows User's Manual, Version 1.00. Centers for Disease Control and Prevention, Atlanta, Ga. Reyes, F., Amin, N., Otero, O., Aguilar, A., Cuello, M., Valdes, Y., Garcia, L.G., Cardoso, D., Camacho, F., 2013. Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests. Biologicals 41, 275. Ricci, S., Bardotti, A., D'Ascenzi, S., Ravenscroft, N., 2001. Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection. Vaccine 19, 1989. Stephens, D.S., Greenwood, B., Brandtzaeg, P., 2007. Epidemic meningitis, meningococcaemia, and Neisseria meningitidis. Lancet 369, 2196. Suzaki, Y., Ozawa, Y., Kobori, H., 2006. Quantification of human angiotensinogen by a novel sandwich ELISA. Peptides 27, 3000. Svennerholm, L., 1957. Quantitative estimation of sialic acids. II. A colorimetric resorcinol-hydrochloric acid method. Biochim. Biophys. Acta 24, 604. Tan, L.K., Carlone, G.M., Borrow, R., 2010. Advances in the development of vaccines against Neisseria meningitidis. N. Engl. J. Med. 362, 1511. Trad, A., Hedemann, N., Shomali, M., Pawlak, V., Grotzinger, J., Lorenzen, I., 2011. Development of sandwich ELISA for detection and quantification of human and murine a disintegrin and metalloproteinase17. J. Immunol. Methods 371, 91. WHO Expert Committee on Biological Standardization, 2006. Annex 2, guidelines for assuring the quality, safety and efficacy of meningococcocal A conjugate vaccines. Fifty-seventh Report. Technical report series no. 962. World Health Organization, Geneva, p. 115. Wilson, M., 1978. Recent development in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. Immunofluorescence and related staining techniques p. 215.
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Research paper
Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroups A, C, W and Y in multivalent vaccines Fátima Reyes ⁎, Oscar Otero, Maribel Cuello, Nevis Amin, Luis García, Daniel Cardoso, Frank Camacho Research and Development Vice Presidency, Finlay Institute, A.P. 16017, La Habana Cod. 11600, Cuba
a r t i c l e
i n f o
Article history: Received 31 January 2014 Received in revised form 21 March 2014 Accepted 25 March 2014 Available online xxxx Keywords: Quantitation ELISA Capsular polysaccharide Meningococcal vaccines
a b s t r a c t Neisseria meningitidis is a Gram negative bacterium that has been classified in 13 serogroups according to the biochemical composition of the capsular polysaccharide (CP). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY). Individual CP quantitation in multivalent meningococcal CP-based vaccines is required for quality control testing of these products. In this regard, four sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the quantitation of CP. The quantitation and detection limits of the four ELISAs were below 1 ng/mL. The assays showed good reproducibility and repeatability as calculated for each point of the standard curve (CV b 15%). In addition, five multivalent meningococcal CP-based vaccines were evaluated and the proposed ELISAs showed that these vaccines were found into the accepted range (±30%) of CP content. These assays are suitable for screening multiple plain or conjugated meningococcal CP-based vaccines and could be useful for monitoring lot-to-lot consistency and stability analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Meningococcal disease, caused by Neisseria meningitidis, occurs mainly as either septicemia or meningitis, and is a worldwide health problem (Chang et al., 2012). N. meningitidis strains have been classified into at least 13 serogroups on the basis of the structure of the capsular polysaccharide (CP)
Abbreviations: CP, capsular polysaccharide; MenA, N. meningitidis serogroup A; MenC, N. meningitidis serogroup C; MenW, N. meningitidis serogroup W; MenY, N. meningitidis serogroup Y; ELISA, enzyme-linked immunosorbent assay; CV, coefficient of variation; HPAEC-PAD, high performance anion exchange chromatography with pulsed amperometric detection; MAbs, monoclonal antibodies; TT, tetanus toxoid; HRP, horseradish peroxidase; IC, internal controls; OD, optical density; 4PL, four-parameter logistic equation; QCs, quality control; LOD, limit of detection; LOQ, limit of quantification; SD, standard deviation; CZE, capillary zone electrophoresis. ⁎ Corresponding author at: Finlay Institute, Calle 27, No. 19805, La Lisa, A.P. 16017, Cod. 11600 Havana, Cuba. Tel.: +53 7 2716911; fax: +53 7 2731218. E-mail addresses: [email protected], freyes@finlay.edu.cu (F. Reyes).
(Harrison et al., 2009). However, invasive infections are most frequently caused by six of these serogroups: A, B, C, W, X and Y (MenA, MenB, MenC, MenW, MenX, MenY) (Stephens et al., 2007). Most meningococcal vaccines are monovalent or multivalent mixtures of either polysaccharides or polysaccharides conjugated to carrier proteins (Tan et al., 2010). Quantitative determination of the individual polysaccharide components in multivalent meningococcal vaccines is an important step in manufacturing and regulatory control (Cook et al., 2013a). Several methods for individual quantitation of meningococcal polysaccharides have been described. Determination of total phosphorus content (Chen et al., 1956) has been employed for measurement of MenA and MenX, while sialic acid content (Svennerholm, 1957) can be used for MenC, MenW, and MenY. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been used for the quantitation of polysaccharides in multivalent meningococcal CP-based vaccines (Cook et al., 2013b). These methods can provide accurate and sensitive measurement of meningococcal
http://dx.doi.org/10.1016/j.jim.2014.03.020 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
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F. Reyes et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
polysaccharides but require prior conversion to monosaccharides by acid digestion. The use of strong acids, often at elevated temperature, represents a potential safety concern for laboratory staff and has a cost associated with material disposal (Lamb et al., 2005). Simple and affordable techniques to quantitate polysaccharides in meningococcal vaccines are essential for the evaluation of antigen content and lot-to-lot consistency of manufacture. Immunological assays such as rate nephelometry or inhibition enzyme-linked immunosorbent assay (ELISA) may be used (WHO Expert Committee on Biological Standardization, 2006). In addition, sandwich ELISA is a common tool for antigen quantitation (Trad et al., 2011; Suzaki et al., 2006; Ma et al., 2013). Either monoclonal or polyclonal antibodies can be used as the capture and detection antibodies in sandwich ELISA systems. However, monoclonal antibodies (MAbs) recognize a single epitope that allows fine detection and quantitation of small differences in antigen. In a previous study, our group obtained MAbs for MenA, MenC, MenW and MenY CPs. The selectivity of these MAbs was confirmed for each CP, with no cross-reactivities to heterologous CPs, or carrier proteins like tetanus toxoid (TT) and CRM197 (Reyes et al., 2013). The aim of the present work was to develop four sandwich ELISAs using these MAbs to quantitate CPs from MenA, MenC, MenW and MenY in meningococcal CP-based vaccines.
8 mg of each MAb was coupled to 4 mg of HRP and was purified by gel filtration chromatography. Conjugates containing 1% BSA, 0.05% thiomersal and 50% glycerol were stored in small aliquots at −20 °C. 2.4. Calibrators, quality controls and calibration curve construction
2. Materials and methods
In the absence of International Standards, calibrators consisted of purified CPs from MenA, MenC, MenW and MenY produced at Finlay Institute (Havana, Cuba) and used as internal controls (IC). MenC, MenW and MenY ICs were quantitated using resorcinol colorimetric assay (Chen et al., 1956) with N-acetylneuraminic as the standard while MenA IC was quantitated using Chen colorimetric assay (Svennerholm, 1957) using KH2PO4 as the standard. These ICs are supplied as lyophilized powders with 100 μg of each CP. A two-fold serial dilution of each IC was used to generate a six-point calibration curve with a range from 0.3125 to 10 ng/mL. GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA) was used for plotting the optical density (OD492) against the CP concentrations and four-parameter logistic equation (4PL) was applied to obtain the function describing a sigmoid model. The quality controls (QCs) consisted of meningococcal CP powders (Finlay Institute, Havana, Cuba) which were weighted, dissolved in PBS and adjusted to a final concentration of 1.25 ng/mL (w/v). Calibrators and QCs were stored as single ready-to-use aliquots at −20 °C.
2.1. Reagents and buffers
2.5. Description and preparation of vaccines
Unless otherwise stated, reagents were obtained from Sigma-Aldrich (USA). The following buffers were used: Coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6), PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4), blocking buffer (PBS, 3% non-fat dried milk), washing buffer (PBS, 0.05% (v/v) Tween 20, pH 7.4), and substrate buffer (35 mM citric acid, 67 mM Na2HPO4, 0.012% (w/v) H2O2, pH 5.0).
Three registered vaccines and two lots of two experimental vaccines were analyzed.
2.2. MAbs Murine MAbs against MenA, MenC, MenW and MenY CPs were produced and characterized as described previously (Reyes et al., 2013). Briefly, hybridomas were produced by fusion of SP2/ O myeloma cell line with splenocytes of BALB/c mice that were immunized with subcutaneous injections of meningococcal polysaccharide group A conjugated to TT (MenAfriVac®, Serum Institute of India, Ltd.) and meningococcal polysaccharide groups A, C, W135 and Y conjugated to CRM197 (MENVEO®, Novartis, Switzerland) vaccines. Four hybridomas, coded as 7E1F7 (anti MenA CP), 7E12B3 (anti MenC CP), 5C11F1 (anti MenW CP) and 5H10D9 (anti MenY), were selected and grown as mouse ascites in the peritoneal cavity of pristane-primed BALB/c mice. The MAbs were purified from the ascites fluid by affinity chromatography using HiTrap Protein G (GE Healthcare, Germany) following manufacturer's instructions. 2.3. Peroxidase labeling of MAbs Purified MAbs were conjugated in-house to horseradish peroxidase type VI (HRP) as described by Wilson (1978). Briefly,
– MENVEO® vaccine (Novartis, Switzerland): Meningococcal polysaccharide groups A, C, W135 and Y conjugated to CRM197 (10 μg/dose of MenA CP and 5 μg/dose of each MenC, MenW and MenY CPs). – MENCEVAX®ACWY vaccine (GlaxoSmithKline): Meningococcal polysaccharide groups A, C, W and Y (50 μg/dose of each CP). – vax-MEN-ACW135® (Finlay Institute, Cuba): Meningococcal polysaccharide groups A, C and W (50 μg/dose of each CP). – ACW135Y (lots 105, 106) and ACW135XY (lots 101, 102) vaccines: Experimental vaccines (Finlay Institute, Cuba), meningococcal polysaccharide groups A, C, W, Y or A, C, W, X and Y (50 μg/dose of each CP). All the vaccines were reconstituted as described by the manufacturers and were prepared to a final concentration of 1.25 ng/mL taking into account the standard curve range. 2.6. ELISA sandwich for serogroup quantitation Polystyrene microwell plates (Maxisorp, Nunc, USA) were coated with 10 μg/mL of MAb 7E1F7, 7E12B3, 5C11F1 or 5H10D9 in coating buffer (100 μL/well). After overnight incubation at 4 °C, the coated wells were washed three times and blocked with blocking buffer for 1 h at 37 °C. The calibrators, QCs and samples were added (100 μL/well) and incubated 1 h at 37 °C. Subsequently, the wells were washed three times and peroxidase conjugated MAbs diluted to 1:15000 in washing buffer containing 1% non-fat dried milk were added to the wells
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
F. Reyes et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
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Fig. 1. Calibration curves obtained from sandwich ELISAs using the 4PL equation. A, B, C and D represent the standard curves using MenA, MenC, MenW and MenY CPs respectively. Error bars represent standard deviations of quadruplicate samples.
(100 μL/well) and incubated for 30 min at room temperature. After four washes, 0.4 mg of OPD per mL substrate buffer was added to the wells (100 μL/well) and following 15 min the color development was stopped with 1 M H2SO4. OD was measured at 492 nm using a Microplate Reader (Thermo Electron Corporation, USA) and unknown concentrations were calculated using CDC Software (Plikaytis et al., 1996). The calibrator, QCs and samples were run in triplicates unless otherwise stated. Three independent experiments were performed and results were expressed as mean of CP content. Optimal dilutions of MAbs and HRP conjugated MAbs were determined by standard checkerboard titrations according to the procedures described by Crowther and Walker (2009).
3. Results 3.1. Calibration curve construction Calibration curves are shown in Fig. 1. A four-parameter fit model was applied to obtain the function describing a sigmoid model. Coefficients of determination (R2) ≥0.99 were obtained in all cases.
3.2. Limits of detection and quantitation The LOD and LOQ were found below 1 ng/mL for all CP in each ELISA assay as shown in Table 1.
2.7. Limits of detection and quantitation Lower limits of detection (LOD) and quantitation (LOQ) were defined as the mean of blank values (n = 20) plus either 3 or 10 standard deviation (SD) respectively. 2.8. Intra- and inter-assay precision Intra-assay precision was determined by interplate and intraplate precision as calculated by the coefficient of variation (CV). The interplate precision was evaluated by measuring triplicates of standard curve on four distinct ELISA plates on the same day in parallel experiments. The intraplate precision was evaluated by measuring the standard curve in quadruplicate on a single ELISA plate. The inter-assay precision was determined as the CV on five separate occasions.
3.3. Intra- and inter-assay precision The four ELISA assays showed inter-assay CVs of 3–14%, while intra-assay CVs varied between 1–7% and 1.5–9% for the intraplate and interplate assay respectively (Tables 2–5).
Table 1 Lower limits of detection and quantitation for each capsular polysaccharide.
LOD LOQ
MenA ng/mL
MenC ng/mL
MenW ng/mL
MenY ng/mL
0.382 0.855
0.19 0.56
0.15 0.35
0.08 0.24
LOD was calculated as reagent blank + 3 × SD of reagent blank, whereas LOQ was determined as reagent blank + 10 × SD of reagent blank.
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
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Table 2 Inter- and intra-assay coefficient of variation (%) of MenA capsular polysaccharide standards. MenA CP (ng/mL)
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
3.24 10.58 12.64 11.55 10.42 11.63
Intra-assay
Table 4 Inter- and intra-assay coefficient of variation (%) of MenW capsular polysaccharide standards. MenW CP (ng/mL)
Intraplate n=4
Interplate n = 12
0.91 2.54 3.18 6.89 2.16 4.27
2.62 4.41 9.21 8.28 4.67 4.36
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
6.50 7.66 8.39 10.58 10.63 11.24
Intra-assay Intraplate n=4
Interplate n = 12
2.99 4.39 4.82 2.81 2.43 3.57
2.55 1.64 2.82 3.05 3.20 3.52
Polysaccharide content must be checked on the final product to determine that it contains the stated amount of polysaccharide. In this regard, four in-house quantitative sandwich ELISAs were developed using four specific MAbs for measurement each CP content in multivalent meningococcal CP-based vaccines. Several procedures for the quantitation of meningococcal CP such as phosphorus and sialic acid content measurement, capillary zone electrophoresis (CZE) and HPAEC-PAD have been used (Chen et al., 1956; Svennerholm, 1957; Cook et al., 2013b; Lamb et al., 2005; Ricci et al., 2001; Lei et al., 2000). However, these methods have some limitations: the first can't be used in multivalent meningococcal vaccines (trivalent, tetravalent, pentavalent) because phosphorus is present in MenA and MenX CP (Berti et al., 2012), while N-acetylneuraminic acid is common to MenC, MenW and MenY CP (Cook et al., 2013b). On the other hand, it has been reported that CZE has been used to quantitate native polysaccharides from a mixture of meningococcal polysaccharide serogroups A, C, W and Y (Lamb et al., 2005). Unfortunately, quantitation of polysaccharides in meningococcal multivalent vaccines using this method has not been
fully evaluated. Finally, HPAEC-PAD requires prior conversion to monosaccharides by acid digestion and the presence of saccharide excipients such as sucrose or lactose may interfere with the determination of some polysaccharide serogroups (Cook et al., 2013a) and add considerable time and expense to the procedure. Using the proposed ELISAs the time for analysis could considerably be shortened and the analysis of 22 vaccine samples (in three replicates) can be performed without excipient removal. The developed sandwich ELISAs showed LOD and LOQ below 1 ng/mL for the respective CPs. These results agree with the affinity constant values calculated for each MAb in a previous work (Reyes et al., 2013). Therefore, these assays can be used successfully for the quantitation of CP in meningococcal multivalent vaccines. In addition, using the proposed ELISAs and taking into account their high sensitivity, these assays could be a useful tool to test for free polysaccharide in conjugated vaccines prior separation of the unbound CP from conjugated-CP in such vaccines. Free polysaccharide must be tested to ensure that the amount present is within the limits agreed by the regulatory authorities based on lots shown to be clinically safe and efficacious. On the other hand, the inter-assay and intra-assay CVs were calculated for each point of the standard curve (10–0.312 ng/mL). Reproducibility and repeatability were in accordance with international recommendations, which define the acceptance criterion of precision for standard curves as inter-assay CV ≤ 15% (DeSilva et al., 2003). Five multivalent meningococcal CP-based vaccines were evaluated using the developed sandwich ELISAs. The vaccines have an expected nominal label amount of 50 μg/dose of each CP except for MENVEO®, which has 10 μg/dose of MenA CP and 5 μg/dose of each MenC, MenW and MenY CPs. The common specified range is ±30% of the expected amount per
Table 3 Inter- and intra-assay coefficient of variation (%) of MenC capsular polysaccharide standards.
Table 5 Inter- and intra-assay coefficient of variation (%) of MenY capsular polysaccharide standards.
In general the CV increased with decreasing concentration of CP, and assays for MenA, MenW and MenY were found to have lower inter-assay variation than MenC assays. 3.4. Serogroup quantitation in vaccines Total serogroup polysaccharide content using the four ELISA sandwich are shown in Table 6. All of the five meningococcal CP-based vaccines were found into the accepted range (±30%) of CP content. The CVs were b 9% compared to the acceptance criterion of b15% CV for immunoassays. 4. Discussion
MenC CP (ng/mL)
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
9.27 10.66 11.83 12.44 13.93 14.15
Intra-assay
MenY CP (ng/mL)
Intraplate n=4
Interplate n = 12
1.13 2.20 4.03 3.61 5.05 5.11
3.88 5.68 4.87 4.40 5.71 4.86
10 5 2.5 1.25 0.625 0.312
Inter-assay n = 50
5.04 4.18 8.28 9.87 9.89 10.10
Intra-assay Intraplate n=4
Interplate n = 12
3.01 3.21 2.98 3.41 2.38 4.75
5.2 4.25 5.11 5.29 5.73 5.04
Please cite this article as: Reyes, F., et al., Development of four sandwich ELISAs for quantitation of capsular polysaccharides from Neisseria meningitidis serogroup..., J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.03.020
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Table 6 Serogroup polysaccharide content of meningococcal CP-based vaccines. Vaccines
MenA CP Average
ACYW 105 ACYW 106 ACYWX 101 ACYWX 102 vax-MEN-ACW135® MENCEVAX®ACWY MENVEO® a b
50.76 43.05 47.71 48.65 45.83 46.47 8.78
a
MenC CP b
CV
4.19 1.08 1.67 1.35 0.79 2.16 1.91
Average 56.49 57.36 43.68 50.68 51.60 47.47 5.26
MenW CP CV 2.34 1.95 2.06 1.37 4.62 3.96 4.98
Average 48.59 50.31 46.63 57.74 53.65 54.62 4.73
MenY CP CV 2.02 2.47 1.42 4.44 1.43 1.70 2.08
Average 45.62 44.76 46.03 48.68 – 45.34 6.40
CV 3.71 4.38 0.99 8.19 – 4.99 1.94
Mean of triplicate determinations from three independent experiments for each serogroup expressed in μg/dose. Coefficient of variation expressed in %.
dose (0.5 mL) (European Pharmacopoeia). Considering this guideline, MENVEO® vaccine should have between 7 and 13 μg of MenA CP and between 3.5 and 6.5 μg of MenC, MenW and MenY CPs. The remaining vaccines should have between 35 and 65 μg of MenA, MenC, MenW or MenY CPs. Values from Table 6 indicate that all the vaccines screened for CP content were within general specifications for each CP taking into general consideration the European Pharmacopoeia recommendations. 5. Conclusions Four sandwich ELISAs were established for quantitation of CP from N. meningitidis in both conjugated and plain meningococcal CP-based vaccines. The ELISAs showed a very good precision and high sensitivity. These assays are suitable for screening multiple vaccine samples and could be useful for monitoring lot-to-lot consistency and stability analyses. Acknowledgments The authors thank MsC. M. Ileana Delgado, BsC. Luis A. Izquierdo and Dr. Rolando F. Ochoa for their excellent technical assistance with the ELISAs. We also like to thank Dr. Barbara Bolgiano (National Institute for Biological Standards and Controls, UK) for her kindness in reviewing the draft of the manuscript. References Berti, F., Romano, M.R., Micoli, F., Pinto, V., Cappelletti, E., Gavini, M., Proietti, D., Pluschke, G., MacLennan, C.A., Costantino, P., 2012. Relative stability of meningococcal serogroup A and X polysaccharides. Vaccine 30, 6409. Chang, Q., Tzeng, Y.L., Stephens, D.S., 2012. Meningococcal disease: changes in epidemiology and prevention. Clin. Epidemiol. 4, 237. Chen, P.S., Toribara, T.Y., Warner, H., 1956. Microdetermination of phosphorus. Anal. Chem. 28, 1756. Cook, M.C., Bliu, A., Kunkel, J.P., 2013a. Quantitation of serogroups in multivalent polysaccharide-based meningococcal vaccines: optimisation of hydrolysis conditions and chromatographic methods. Vaccine 31, 3702. Cook, M.C., Gibeault, S., Filippenko, V., Ye, Q., Wang, J., Kunkel, J.P., 2013b. Serogroup quantitation of multivalent polysaccharide and polysaccharideconjugate meningococcal vaccines from China. Biologicals 41, 261.
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