We designed the peptide (C3) mimetic carboxyl-terminus (Cterminus) of capsid protein of porcine circovirus type 2b (PCV2b-1A/1B) inducing humoral immunity and generating hybridomas. The positive reactivity of the mAbs to PCV2 capsid protein was demonstrated by Western blot assay. Those mAbs also showed positive signals on PCV2b infected swine lymphocytes by indirect immunofluorescence staining. The mAb 1H3 bound to three minimal linear epitopes (P62, DPPLNP; P67, DPPLNPK; P73, LKDPPLKP), which was located at Cterminus of the capsid protein of PCV2b-1A/1B, PCV2b-1C, and PCV2a-2A respectively. The mAbs 3B2 bound to only one minimal linear epitopes (P59, KDPPLNP). The mAbs 6B8 bound to two minimal linear epitopes (P59 and P67). Our data demonstrate the core motif (P62) within the P59 could be recognized by mAbs (3B2 and 6B8) in the free status by liquid phase blocking immunoassay (LPBI) but not be recognized by these mAbs in the fixed form on the plate by indirect ELISA (iELISA). However, the P73 could be recognized by mAb 1H3 by iELISA but no inhibition of the interactive binding of C3 and mAb 1H3 by LPBI. This study also indicated that IgM mAbs and defective Ig mAb have broad binding, moderate specificity and low affinity. This study confirm that mAbs have pluripotency of binding. It might be a phenomenon of antibody response to Cterminus of capsid protein of PCV2b.
In the context of the carboxyl-terminus (C-terminus) of the capsid protein of porcine circovirus type 2a (PCV2a) and PCV2a vaccines, this study aimed to explore its unrevealing cryptic epitope and its relation to PCV2-infected herd immunity. To discover the C-terminus of the capsid protein of PCV2a, monoclonal antibodies (mAbs) were generated in this work. Two mAbs bound the two minimal linear epitopes (229PPLKP233 and 228DPPLNP233 (or 229PPLNP233)), which were located at the C-terminus of the capsid proteins of PCV2a and PCV2b, respectively. One mAb bound to the minimal linear epitope (220QFREFNLK227, peptide P82), but it neither bound the virus-like particle (VLP) of PCV2a nor produced positive staining in PCV2a-infected cells by immunofluorescence assay. Further, the residues 220–227 were not accessible on the surface of the VLP on the three-dimensional model, but the residues 228–231 extend toward the VLP exterior. Immunoassays were conducted in this study to screen anti-viral peptide-specific IgGs, which could differentiate vaccinated pigs from non-vaccinated ones. The data show two 220QFREFNLKDPPLKP233-containing peptides had a significantly higher binding reactivity with sera from PCV2-infected pigs in the control group than with sera from the VLP-vaccine group, particularly seen in sera from swine aged 15 weeks to 24 weeks. However, the peptide P82 had not this phenomenon in that test. This study confirmed that C-terminal epitopes play an important role in PCV2-induced decoy of swine humoral immunity.
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