Monoclonal antibody EP-5C7 (PDL BioPharma, Inc.)

Epidemic diarrhea virus nsp13 gene was amplified from PEDV (KX058033.1) and cloned into pET-28a expression vector.The solubilized and refolded inclusion bodies of his-tagged nsp13 from pET-28a-PEDV nsp13 vectors were chosen for immunization.One hundred micrograms of the recombinant nsp13 mixed with Freund′s adjuvant was s.c. immunized into 5-wk-old female BALB/c mice at 2-wk intervals.The procedure included three addnl. immunizations followed by a final booster injection.Pos. hybridoma cells were screened by using ELISA and subcloned three times by limiting dilutionThen, the stable hybridoma clones were obtained and injected into liquid paraffin-pretreated abdominal cavities of BALB/c mice to obtain ascites (mAbs).PCAGGS-HA-nsp13 vectors transfected into HEK 293T cells grown on microscope coverslips in 24-well plates.At 28 h post transfection, cells were inoculated with primary antibody at a dilution of 1:300 for 1 h at 37°C.After 3 times washing with PBST, cells were inoculated with fluorescein isothiocyanate isomer-labeled goat anti-mouse secondary antibody at a dilution of 1:200 at 37°C for 1 h.Images were acquired after cells were observed under the fluorescence microscope (Carl Zeiss).The results of immunofluorescent assay showed that all mAbs could bind to HA-tagged nsp13 and revealed specific green fluorescence, whereas neg. control cells could not reveal any fluorescence.

The beta8 integrin, which forms alphavbeta8 heterodimers, is being widely investigated because of its specific structure and functions compared with other integrins. In this report, a 12 aa-long peptide of beta8 integrin cytoplasmic domain was synthesized according to a published sequence and covalently coupled to keyhole limpet hemocyanin (KLH). Three stable strains of hybridomas (3G6, 5C7, 5H3) that can secrete high specific monoclonal antibodies against beta8 integrin were successfully established by hybridoma technique. The isotypes of these MAbs were tested to be IgG2a. Their characterizations were shown by enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunocytochemistry (ICC). The affinity constants (Kaff ) of the MAbs 3G6, 5C7, and 5H3 were measured by non-competitive ELISA respectively. Western blot analyses and immunocytochemistry demonstrated that all the MAbs were directed against beta8 integrin with high specificity.