There is a need for such quality control as many locally produced FMD vaccines show poor quality (45, 46)

There is a need for such quality control as many locally produced FMD vaccines show poor quality (45, 46). 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did Hoechst 33258 not bind FMDV strains from additional serotypes. Inside a sandwich enzyme-linked immunosorbent assay (ELISA) utilizing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3C4.6?g/l. They can be utilized for FMD vaccine quality control and study and development, such as, to identify virion stabilizing excipients. Keywords: enzyme-linked immunosorbent assay, single-domain antibody, foot-and-mouth disease, virion stability, foot-and-mouth disease virion, vaccine quality control Intro Foot-and-mouth disease (FMD) is an animal disease that is caused by a picornavirus, FMD disease (FMDV), which encompasses seven serotypes: A, Hoechst 33258 O, C, Asia1, SAT1, SAT2, and SAT3. Illness with any one serotype does not create significant humoral immunity against additional serotypes. In FMD endemic areas vaccination is used as a preventive method (1). Due to variations in serotype prevalence in the field most vaccines are used for serotypes O and A. Further vaccines generally are specific for Asia1 or SAT2 serotypes. Standard FMD vaccines (2) are based on chemically inactivated FMDVs that are formulated with an adjuvant. FMD virions consist of an RNA molecule and a capsid composed of 60 copies each of VP1, VP2, VP3, and VP4 proteins (3). Intact virions sediment at 146S in sucrose gradients. Some FMDV strains also create bare capsids Hoechst 33258 that lack the RNA molecule and sediment at 75S. Mild heating or incubation at pH below Gadd45a 6.5 prospects to irreversible dissociation of 146S or 75S particles into stable 12S particles that contain five copies each of VP1, VP2, and VP3. Dissociation into 12S particles results in a strongly reduced immunogenicity (4C7). Several methods have been developed to measure the concentration of 146S particles of the crude FMDV antigen preparation utilized for vaccine preparation. This is traditionally measured by sucrose denseness gradient (SDG) centrifugation (8). Novel methods that are more easy to automate are based on size-exclusion high-performance liquid chromatography (9, 10) or lateral circulation immunoassay (11). All these methods possess the advantage of becoming suitable for all FMDV strains, but the disadvantage of low level of sensitivity, limited sample throughput, and failure to discriminate different vaccine strains in multivalent vaccines. Two times antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISAs) using monoclonal antibodies (mAbs) were also developed for FMDV antigen quantification (7, 12C16). They may be more sensitive, but often not specific for undamaged 146S particles. Only two DAS ELISAs were specific for 146S particles due to use of a mAb showing such specificity. They were suitable for detection of A or Hoechst 33258 O serotype strains (15, 16). We have recently developed two DAS ELISAs using two recombinant llama single-domain antibody fragments (VHHs) that bind specifically to either 146S particles or 12S particles of strain O1 Manisa (17, 18). In each ELISA, the same VHH was utilized for coating as well as for detection of captured antigen using biotinylated VHH. The DAS ELISA utilizing 146S-specific VHH M170 was specific for particular O serotype strains, including strain O1 Manisa. The DAS ELISA utilizing 12S-specific VHH M3 could detect FMDV antigen of several A, O, and Asia 1 strains but not SAT2 strain. We were also able to measure 146S particles of A and Asia 1 serotype strains utilizing the M3 DAS ELISA of heated and untreated samples (17). However, this second option ELISA approach is not suitable for detecting 146S particles in the presence of higher concentrations of 12S particles. An ELISA approach.