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Production and characterization of pertussis toxin specific monoclonal and polyclonal antibodies: Implication for
Danyal Imani1, Tannaz Bahadori1, Maryam Mobini1
1Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Background:
Pertussis is a pulmonary disease caused by the gram-negative bacteria Bordetella pertussis (BP) with a high fatality rate among newborns and young children. Pertussis toxin (PT) is essential for pertussis pathogenesis as well as production of acellular pertussis vaccines (aPV). Traditional PT purification procedures are laborious and yield low purity and recovery rates. Also, due to the low production levels of PT by BP and the difficulties of purification, an appropriate immunoassay is needed to monitor PT concentrations upstream and downstream of the production process. This study investigates production and application of monoclonal and polyclonal antibodies for efficient PT purification and quantification.
Methods:
Rabbits and mice were immunized with native PT to produce polyclonal and monoclonal antibodies (MAbs). The MAbs were selected based on affinity, isotype and specificity, as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. The native PT antigen was purified using an immunoaffinity column. The purity and recovery rates of native PT were analyzed by ELISA, SDS-PAGE, and immunoblotting. Additionally, monoclonal and polyclonal antibodies were used to establish an ELISA assay for measurement of PT concentration.
Results:
A highly pure PT with recovery rates of around 74 ± 4.9 % was obtained following purification by immunoaffinity column, using polyclonal antibodies. Furthermore, the designed ELISA demonstrated suitable reactivity for measurement of the PT antigen.
Conclusion:
Our results indicate suitability of the produced monoclonal and polyclonal anti-PT antibodies for purification and monitoring of PT by immunoaffinity chromatography and ELISA, respectively. The immunoaffinity method offers an efficient replacement for PT purification in the context of developing aPV.
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