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A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of theAphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Tb-lymphocyte epitopes (4, 5, 7, 8), although a Ti-lymphocyteindependent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter

SUMMARYNeutralizing monoclonal antibodies raised against type 0 foot-and-mouth disease virus have been characterized on the basis of their reactivity with a panel of single site monoclonal antibody-resistant mutants which had defined three antigenic sites. Five antibodies neutralized all these mutants, but by selecting further single site mutants with one of these antibodies it was possible to define a fourth site involved in virus neutralization. Two monoclonal antibodies still neutralized these mutants and all multiple site resistant mutants. One multiple site resistant mutant was resistant to neutralization at each of four antigenic sites but was still efficiently neutralized by type 0 convalescent cattle sera. The relationship between sites recognized by different monoclonal antibodies generated in different laboratories is discussed. INTRODUCTIONStudies on the antigenic structure of two picornaviruses, poliovirus and rhinovirus, have been considerably enhanced by the solving of their three-dimensional crystal structure (Hogle et al., 1985;Rossmann et al., 1985) and the isolation and sequence analysis of monoclonal antibody (MAb) escape mutants (Minor et al., 1986;Sherry et al., 1986). Crystallographic studies on footand-mouth disease virus (FMDV), another picornavirus, are also in progress (Fox et al., 1987) and a number of laboratories have generated MAbs capable of neutralizing type O virus. characterized some 30 MAb-resistant mutants of the O1 Kaufbeuren strain of FMDV isolated using five different neutralizing MAbs. Three distinct antigenic sites were involved in virus neutralization. In this context the term antigenic site is used to describe an area of the virus surface which may contain several MAb epitopes; if one epitope is changed and this affects the ability of a second MAb to neutralize the virus then it is considered that the second MAb recognizes an epitope in the same antigenic site as that changed.Recently Stave et al. (1988) have used different MAbs to define three sites of neutralization. In this communication we now use the panel of mutants generated by Xie et al. (1987) to map the binding sites of MAbs generated in different laboratories and then use these antibodies to show the existence of further sites involved in the neutralization of type O FMDV. We have also generated multiple site mutants which are resistant to neutralization at each of two, three or four sites.

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