Медицинская иммунология (Aug 2020)
Development of ELISA test for the quality control of Pseudomonas aeruginosa recombinant vaccine based on the hybrid recombinant protein
Abstract
A hybrid recombinant protein containing the amino acid sequences of the three most significant Pseudomonas aeruginosa antigens (membrane proteins OprF, OprI and toxoid aTox) was incorporated into a vaccine against Pseudomonas infection. Quality control of a hybrid recombinant protein and appropriate vaccine includes determination of authentity and completeness of adsorption upon aluminum hydroxide adjuvant. The aim of our study was to develop techniques of quality control for a vaccine based on the hybrid OprF-aToxOprI recombinant protein specific to P. aeruginosa. Hybridomas secreting specific monoclonal antibodies for OprF-aTox-OprI were derived from the fusion of myeloma cells and murine spleen cells immunized with recombinant proteins P. aeruginosa. To produce sufficient quantities of antibodies, the hybrid cells were in vivo cultured in BALB/c mice. Supernates and ascite liquids were chromatographically purified with immune sorbent. Conjugation of antibodies with horseradish peroxidase was carried out according to P.K.Nakane. The hybrid OprF-aTox-OprI recombinant protein was detected by the solid-phase ELISA, using a panel of monoclonal antibodies and conjugates of monoclonal antibodies with horseradish peroxidase. Monoclonal antibodies were specific for different OprF-aTox-OprI epitopes. Titration assays containing OprF-aTox-OprI protein at 78 ng/ml to 5000 ng/ml were used as quantitative standards for calibration curves.To identify the recombinant protein OprF-aTox-OprI, 55 variants of of MAb pairs were tested. Limits of quantitative detection served for selection of most sensitive and specific ELISA variants. The quantitative detection limit was calculated for all 11 ELISA variants. Two ELISA variants with the highest sensitivity were selected for quality control of the hybrid recombinant protein. The limits of quantitative detection were, respectively, 2.9 and 13.6 ng/ml (0.0058 and 0.027% of the estimated antigen content in the vaccine) for the first and second ELISA variants. The first variant included a pair of monoclonal antibodies specific for the OprF and OprI epitopes, the second variant represented aTox and OprI epitopes. Two variants of ELISA were developed to detect the hybrid recombinant OprF-aTox-OprI protein. The first variant allows to determine the protein amount and to evaluate completeness of its adsorption on aluminum hydroxide. To confirm authenticity of the protein, both methods must be used, since they can detect all three antigens (OprF, aTox and OprI) which are present in the fusion protein.
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