Fig. 5
Characterization of P18C5 mAb specificity. The vaccinated animals displaying the highest titers based on ELISA were used for the generation of stable productive hybridomas. Supernatants from the resulting hybridoma-producing colonies were screened via standard ELISA to assess the cross-reactivity of hybridoma-secreting mAbs against the isovariants of the Val (V) or Leu (L) residues contained in the C-terminal domain of BSA-conjugated peptides and were used as adsorbed antigens together with BSA-Val-CONH2 and BSA-Val-COO− immunoconjugates in the assay. The P18C5 mAb was tested at increasing dilutions (1:50–1:6400) in ELISAs (a) or at a specific dilution (1:25) in dot-blot assays (b, upper panel). BSA-Val-CONH2, BSA-Val-COO− and BSA-(Val/Leu) peptide immunoconjugates were used as adsorbed antigens in the ELISA or as spotted antigens (10−12–10−16 M) in the dot–blot assay. ELISA (a) was performed in triplicate using serial twofold dilutions of the P18C5 mAb (abscissa). The absorbance of the immunoreactive signals in positive wells (ordinate) was measured at λ = 490 nm (see text for additional details)