Quantification of polysaccharide Vi in the typhoid vaccine VaxTyViTM by sandwich ELISA using a monoclonal antibody Cuantificación del polisacárido Vi en la vacuna contra la fiebre tifoidea VaxTyViTM mediante ELISA en sandwich utilizando un anticuerpo monoclonal

VaxTyVi® is a plain polysaccharide vaccine based on the capsular polysaccharide Vi (PsVi) from Salmonella Typhi, produced at Finlay Institute. Quantification of PsVi in VaxTyViTM, is a mandatory requirement for final product release and has being made by inhibition ELISA. Considering the disadvantages of this type of ELISA and that recently our group obtained a MAb against PsVi, the aim of this work, was to develop a sandwich ELISA to quantify PsVi in VaxTyViTM. M&M. Using MAb 4G3E11 as the capture and detection, a sandwich ELISA was developed. Limits of detection (LOD) and quantitation (LOQ) were calculated, as well as the interand intra-assay for each point of the standard curve. The PsVi content in six batches of VaxTyViTM was also determined. Results. An eight-point calibration curve with a range from 50 to 0.39 ng/mL of PsVi was obtained with a coefficient of determination (R2) ≥0.99. The LOD and LOQ were 0.59 ng/mL and 1.25 ng/mL respectively, representing at least 1000 times more sensitive than inhibition ELISA. The inter-assay CV was 4–15%, and the intra-assay CVs varied between 3-8 % and 4–9% for intraplate and interplate respectively. The PsVi content in all VaxTyViTM lots were found into the accepted range (±30%). In summary, the proposed sandwich shown to be specific, reproducible and highly sensitive. The assay shown to be suitable for use in the quantitation of PsVi in VaxTyViTM, with great potential to replace the inhibition ELISA, implanted in the Finlay Institute since 2006.


Introduction
Typhoid fever, also known as enteric fever, is a potentially fatal multi systemic illness caused primarily by Salmonella Typhi and, to a lesser extent, related serovars paratyphi A, B, and C. Humans are the only source of these bacteria; no animal or environmental reservoirs have been identified.The microorganism is most often acquired through consumption of water or food that has been contaminated by feces of an acutely infected or convalescent person or a chronic, asymptomatic carrier. 1 Typhoid fever is still a health problem, an estimated 22 million cases and 200,000 related deaths occur worldwide each year.Its prevalence is higher in Asia, sub-Saharan Africa and some countries in South America, but 90% of fatal cases occur in Asia, specifically in India, Pakistan and Bangladesh. 2 In Cuba, there has been an increasing tendency to decrease morbidity and mortality from typhoid fever, describing two stages in the epidemiology of the disease: before and after 2002.Before 2002, an average of 178.4 cases reported per year, with a rate of 1.9 per 100 000 inhabitants.Since 2002, we observed a significant decrease in the incidence of the disease, coming from six cases per year to be no reports of the disease in 2013. 3  The change in the behavior of the disease since 2002, is related among other factors, with the introduction since 2001 of the Cuban typhoid vaccine, VaxTyVi TM , in the National Immunization Program.VaxTyVi TM is a plain polysaccharide (Ps) vaccine, based on the capsular polysaccharide Vi (PsVi) from S. Typhi, with proven efficacy and safety for 14 years of implementation and is produced at Finlay Institute. 4ccording to international standards, quantification of PsVi in VaxTyVi TM , is a mandatory requirement for final product release.Several techniques have been used for quantification of PsVi.Immunological assays (IA) such as rate nephelometry or solid phase immunoassay (ELISA) are within them. 5,6In the case of ELISA, different types are described in the scientific literature to quantify antigens. 7In Finlay Institute, Fajardo et al (2006) validated an inhibition ELISA to quantify the PsVi in VaxTyVi™ and until today, it is the method used by the quality control department for the final release of vaccine lots. 8However, this type of ELISA has several disadvantages, among which we can mention: more laborious, consuming more steps including inhibition or neutralization, lower sensitivity and specificity (employs polyclonal serum), and difficult interpretation of the results, because antibody-antigen-enzyme complex bound is inversely related to the concentration of antigen present in the sample. 9Sandwich ELISA nevertheless, is easier to perform, and in the last decade has been more used in the quantification of total and free Ps in polysaccharide vaccines like Haemophilus influenzae b Tetanus toxoid conjugate vaccines and multivalent antimeningococcal polysaccharide vaccines. 10,11ecently our group obtained a MAb against PsVi coded as 4G3E11, with high purity, affinity and specificity confirmed. 12,13he high specificity and affinity constant of the MAb 4G3E11 suggests its use as an analytical tool in enzyme immunoassays.The aim of the present work was to develop a sandwich ELISA to quantify the PsVi in VaxTyVi TM and thus replace the ELISA inhibition assay, considering the disadvantages mentioned above.

MAbs
Murine MAb against PsVi were produced and characterized as described previously. 12,13Briefly, hybridomas were produced by fusion of SP2/O myeloma cell line with splenocytes of BAL-B/c mice that were immunized with three intraperitoneal doses (every 20 days) of PsVi conjugated to diphtheria toxoid (vaccine candidate from Finlay Institute).One hybridoma, coded as 4G3E11 were selected and produced in vivo grown as mouse ascites in the peritoneal cavity of pristane-primed BALB/c mice.The MAb were purified from the ascites fluid (AF) by affinity chromatography using Protein A (GE Healthcare, Germany) following manufacturer's instructions.As part of the characterization was determined: Productivity, Class and Subclass, Purity, Specificity and Affinity constant (Kaff) Table 1 Peroxidase labeling of MAbs Purified MAb were conjugated in-house to horseradish peroxidase type VI (HRP) as described by Wilson (1978).Briefly, 8 mg of 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.

Calibrators, quality controls and calibration curve construction
In the absence of International Standards, calibrators consisted of purified PsVi produced at Finlay Institute (Havana, Cuba) and used as internal controls (IC).PsVi concentration was quantitated using colorimetric Hestrin's assay.The IC was supplied as lyophilized powders with 100 μg of PsVi.A two-fold serial dilution of IC was used to generate an eight-point calibration curve with a range from 50 to 0.39 ng/mL.Ascent software Version 2.6 (Thermo Labsystem Oy, USA) was used for plotting the optical density (OD492) against PsVi concentration and four-parameter logistic equation (4PL) was applied to obtain the function describing a sigmoid model.The quality control (QC) consisted of PsVi powders (Finlay Institute, Havana, Cuba) which were weighted, dissolved in PBS and adjusted to a final concentration of 5 ng/mL (w/v).Calibrators and QC were stored as single ready-to-use aliquots at −20 °C.

ELISA sandwich for PsVi quantitation Samples
Six VaxTyVi TM vaccine lots (9006, 0004, 402, 203, 304 y 405) released by quality control department, with known concentration of 50 µg/mL determined by ELISA inhibition, were evaluated.All VaxTyVi TM lots were provided by Pharmaceutical Deve-lopment Direction of Finlay Institute and were used at dilution of 1:10000, taking into account the standard curve range.

Procedure
Polystyrene microwell plates (Maxisorp, Nunc, USA) were coated with 10 μg/mL of MAb 4G3E11 anti PsVi, in coating buffer (100 μL/well).After overnight incubation at 4°C, the coated plates were washed three times and blocked with blocking buffer for 1 h at 37°C.The calibrators, QC and samples were added (100 μL/ well) and incubated 2 h at 37°C.Subsequently, the wells were washed three times and peroxidase conjugated MAb 4G3E11 diluted 1:2500 in washing buffer containing 1% non-fat dried milk, were added to the wells (100 μL/well) and incubated for 1 h at room temperature.After four washes, 0.4 mg of OPD per mL of substrate buffer was added to the wells (100 μL/well) and following 30 min of incubation in the dark, the reaction was stopped with 1M H 2 SO 4 .OD was measured at 492 nm using a Microplate Reader (Multiskan Ex, Thermo LabSystem, USA).Unknown concentrations were calculated using Ascent Software Version 2.6 (Thermo LabSystem Oy, USA).The calibrator, QC and samples were run in triplicates unless otherwise stated.Three independent experiments were performed and results were expressed as mean of PsVi content.Optimal dilutions of MAb and HRP conjugated MAb were determined by standard checkerboard titrations according to the procedures described by Crowther and Walker  (2009).

Limits of detection and quantitation
Lower limits of detection (LOD) and quantitation (LOQ) were determinated and defined as the mean of blank values (n = 20) plus either 3 or 10 standard deviation (SD) respectively.

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 The assay reproducibility and repeatability was determined by precision assay, calculating the inter and intra-assay expressed in CV% (Table 3).
As can be seen the proposed sandwich ELISA showed an inter-assay CVs of 4-15%, below 20% recommended for this type of assay.The intra-assay CVs varied between 3-8 % and 4-9% for the intraplate and interplate assay respectively, values that are below 10% recommended for intra-assay test in enzyme immunoassays. 14ISA plate.The inter-assay precision was determined as the CV on five separate occasions.

Curve Construction
MAb 4G3E11 was used as the capture and a peroxidase conjugated MAb 4G3E11 was used for detection in a sandwich ELISA assay.An eight-point calibration curve with a range from 50 to 0.39 ng/mL of PsVi was obtained (Fig. 2).A four-parameter fit model was applied to obtain the function describing a sigmoid model.Coefficient of determination (R 2 ) ≥0.99 was obtained.

Sensitivity, Reproducibility and Repeatability
The LOD and LOQ were found to be 0.59 ng/mL and 1.25 ng/mL respectively and could be related with the Kaff value calculated for the MAb in the previous experiment.Table 2 Quantification of polysaccharide Vi in the typhoid vaccine VaxTyViTM by sandwich ELISA using a monoclonal antibody Cuantificación del polisacárido Vi en la vacuna contra la fiebre tifoidea VaxTyViTM mediante ELISA en sandwich utilizando un anticuerpo monoclonal

DISCUSSION
Facile techniques to analyse and quantitate the Ps components of Ps-based vaccines are essential for the evaluation of antigen content and lot-to-lot consistency of manufacture, with the associated implications for efficacy and safety.Content of Ps is directly related to the product dose. 15,16For PsVi quantitation, several methods are available and suitable for use. 17The concentration of the PsVi in its fully O-acetylated, acid form in eluted fractions can be measured using Hestrin's method 18 or another suitable method, such as Nuclear Magnetic Resonance (NMR). 19The acridine orange staining method 20 and high performance

PsVi quantitation in vaccines
The proposed sandwich ELISA was used to measure the PsVi content in six lots of VaxTyVi TM (summarised in Table 4).
As shown in Table 5 all lots of VaxTyVi™ evaluated show concentrations of PsVi determined by the sandwich ELISA within the accepted range (± 30%) according to the product specifications (35 to 65 µg/mL).
All vaccines lots were found into the accepted range (±30%) of PsVi content according to the available European Pharmacopoeia guidelines and the CVs were <4% compared to the acceptance criterion of <15% CV for immunoassays. 15nion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) 21 have been reported to produce comparable results for PsVi in a range of 15-200 μg/ml.In the case of NMR and HPAEC-PAD, these two techniques have a high sensitivity (at least 10 times higher than Hestrin´s assay) but requires the use of expensive reagents and very sophisticated equipment, not accessible for all. 10owever, immunosorbent assays such as ELISAs, have been very useful in quantifying antigens in vaccines, especially for Ps antigens. 10,11ELISAs have a variety of positive qualities that make them great attraction. 9(Figure 3) In Finlay Institute since 2006, an inhibition ELISA was developed and standardized, to quantify PsVi in VaxTyVi TM . 8Although this ELISA has guaranteed over the years, the release of VaxTyVi TM batches efficiently and consistently, it has several disadvantages compared to sandwich ELISA.The main disadvantages are related to the number of steps and the interpretation of results. 9n this manuscript, we describe the use of a simple sandwich ELISA for the quantification of PsVi in VaxTyVi TM .The proposed sandwich ELISA that use the MAb 4G3E11 for coating and the same MAb but conjugated to peroxidase, for detection, shown to be specific, reproducible and highly sensitive.
According to the sensitivity, related with the lowest amount of analyte which can be detected in a sample, in this paper was evaluated by calculating LOD. 22In the proposed sandwich ELISA the LOD was 0.59 ng/mL, value representing at least 1000 fold more sensitive than the value reported for the inhibition ELISA (0.5 μg/mL).This big difference in the detection limit between both ELISAs, could be determined by the use of MAb in sandwich ELISA, regarding the use of polyclonal antibodies (serum) in the ELISA inhibition.Moreover, if we consider that the MAb 4G3E11 used, has a high specificity and affinity constant for the antigen (3.13 x 10 11 M -1 ). 13 The reproducibility and repeatability of the method, was evaluated by assay precision, which takes into account the maximum dispersion of data obtained for a sample processed several times, on different days and in different conditions.The inter-assay and intra-assay CVs were calculated for each point of the standard curve (50-0.39ng/mL) as described Reyes F. et al. 11 Reproducibility and repeatability were in accordance with international recommendations, which define the acceptance criterion of pre-Fig.3. Scheme showing features of ELISA that make it advantageous for a wide range of applications.Four major advantages of ELISA are promoted, all of which add to the reasons that this form of ELISA has been, and will continue to be, successful. 9sion for standard curves with values below 10% and 20 % for intra and inter-assay precision respectively.14 These CV values obtained for inter and intra-assay were lower than those reported by Fajardo et al. 8 for inhibition ELISA (17-19.5% for inter assay and 16-19.8% for intra assay).This result may be because, as mentioned above, in the inhibition ELISA, more steps are employed, including inhibition or neutralization step, compared with the sandwich ELISA. Th use of larger number of steps, resulting in greater manipulation of the sample in the test by staff and therefore greater possibility of error, resulting in reproducibility.23 However, despite the high sensitivity and reproducibility of the proposed sandwich ELISA, was important to demonstrate the application of the assay, in quantify the amount of PsVi present in Vax-TyVi TM , and it was done.Six lots of VaxTyVi TM were evaluated using the developed sandwich ELISA. The CVs were <4% compared to the acceptance criterion of <15% CV for immunoassays.In ELISA inhibition, the CV obtained were between 4.7% and 12.3% for an average of 7.7%, higher than the sandwich ELISA but also within the acceptance criteria.
Therefore, these assays can be used successfully for the quantitation of PsVi in typhoid polysaccharide vaccines, even in conjugate vaccines.In addition, using the proposed sandwich ELISA and taking into account their high sensitivity, these assays could be a useful tool to test free PsVi in conjugated vaccines, prior separation of the unbound Ps from conjugated-Ps 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. 17n summary, the assay described here has been shown to be suitable for use in the quantitation of PsVi in VaxTyVi TM , with great potential to replace the inhibition ELISA, implanted in the Finlay Institute since 2006.

Fig. 1 .
Fig. 1.Behavior of FT in Cuba.The average number of annual cases of FT shown in different periods: before 2002, from 2002 to 2012 and from 2013

Fig. 2 .
Fig. 2. Calibration curve obtained from sandwich ELISA using the Ascent™ Software Version 2.6 (Thermo LabSystem Oy, USA).A four-parameter logistic equation (4PL) was applied to obtain the function describing a sigmoid model.Error bars represent standard deviations of quadruplicate samples.