Abstract
Antigen Used for Immunization
PEDV S1 gene (1-789aa) was amplified by PCR and expressed in sf9 insect cells. The recombinant S1 protein was purified from the culture supernatant by immunoaffinity chromatography as immunogen.
Method of Immunization
For the first immunization, equal volumes of antigen and Freund's complete adjuvant (Sigma-Aldrich, St. Louis, MO) were hypodermically injected into mice. Equal doses of antigen in incomplete Freund's adjuvant (Sigma-Aldrich) were injected into mice on days 14 and 28 after primary immunization, respectively. The equal amount of antigen was injected into mice on day 42 for the boost.
Parental Cell Line Used for Fusion
Mouse myeloma Sp2/0.
Selection and Cloning Procedure
The culture supernatants were tested using indirect-ELISA coated by recombinant S1 protein.
Heavy and Light Chains of Immunoglobulin
Specificity
MAb 8A3A10 showed a neutralizing effect to PEDV in cell neutralization test in Vero cells. MAbs against S1 protein of PEDV were identified by indirect immunofluorescence assay in 96-well cell plate and Western blot analysis with recombinant S1 protein.
Specific Antigen Identified
MAbs 8A3A10 and 12C4G12 recognized the recombinant S1 protein expressed in sf9 insect cells, and 8A3A10 showed a neutralizing effect to PEDV.