Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method Gennady Rezapkin a , Javier Martin a,b , Konstantin Chumakov a, * a Center for Biologics Evaluation and Research, Food and Drug Administration, 1401 Rockville Pike, HFM 470, Rockville, MD 20852, USA b National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, UK Received 20 February 2004; accepted 9 November 2004 Abstract A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains. Ó 2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. 1. Introduction Poliomyelitis is caused by polioviruses of three distinct serotypes that possess the so-called native or D-antigens. These antigens induce protective antibodies in infected individuals and vaccine recipients. After mild heating and other treatments the antigens are rapidly converted to the H or C form that is not protective [1,2]. Therefore the potency of IPV is determined by the D- antigen content. ELISA is currently the main in vitro method for measuring the D-antigen content of IPV. The antigenic structure of IPV consists of at least four different epitopes [8,16,17,19,22] and a variety of Mabs with virus-neutralizing activity can be produced. The total antigenic activity (D-antigen content) of IPV is traditionally measured in a reaction with polyclonal antiserum and is a result of the combined activity of multiple antigenic sites, conceivably present at varying proportions. There is a wide variety of protocols for ELISA D-antigen potency test, and some of them recently have been adapted for use with monoclonal antibodies [20,27]. Despite the long history of IPV use, little is known about the level of variation and consistency of antigenic composition of IPV, but some reports suggest that these differences could be significant [20]. It is possible that two vaccine batches having identical overall D-antigen * Corresponding author. Tel.: C1 301 594 3720; fax: C1 301 827 4622. E-mail address: chumakov@cber.fda.gov (K. Chumakov). 1045-1056/04/$30.00 Ó 2004 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2004.11.001 Biologicals 33 (2005) 29e39 www.elsevier.com/locate/biologicals