Abstract
Antigen Used for Immunization
Equine influenza virus isolated during the 1974 equine influenza outbreaks in China (A/equine/Beijing/1/1974(H7N7), EQ/BJ). The virus was grown in 10-day-old embryonated chicken eggs (specific-pathogen-free, SPF) and purified by velocity sedimentation through 25 to 70% sucrose gradients.
Method of Immunization
The 5- to 6-week-old female BALB/c mice were injected with immunogen (EQ/BJ) four times over a 2-month period. Each mouse was subcutaneously injected with 100 μg of antigen emulsified with Freund's complete adjuvant (1:1) on days 1, 16, and 31, and intraperitoneally injected on day 46, respectively. Three days after the last injection, the immunized mice were sacrificed, and a single-cell splenocyte suspension was obtained for fusion.
Parental Cell Line Used for Fusion
Mouse Sp2/0.
Selection and Cloning Procedure
Hybrid cells were selected by HAT medium. Hybridoma supernatants were assayed by indirect ELISA based on EQ/XJ and A/equine/Xinjiang/3/2007(H3N8) (EQ/XJ), respectively. The hybridoma cells positive for EQ/BJ and negative for EQ/XJ were sub-cloned using the limiting dilution technique.
Heavy and Light Chains of Immunoglobulin
Not applicable.
Specificity
Indirect immunofluorescence assay
Equine influenza H3N8 viruses A/equine/Qinghai/1/94(H3N8), A/equine/Miami/1/63(H3N8), and EQ/XJ(H3N8), and equine influenza H7N7 virus EQ/BJ were used as coating antigens in IFA, respectively. The culture supernatant of the hybridoma cells was used as primary antibody, and FITC-labeled rabbit anti-mouse IgG was used as secondary antibody in IFA. The MAb H7N7/9 was shown to be reactive with the H7N7 subtype strain (EQ/BJ), but not with all H3N8 subtype strains.
Specific Antigen Identified
The MAb H7N7/9 was shown to direct against equine influenza virus H7N7 subtype.