Rapid generation of monoclonal antibody-secreting hybridomas against African horse sickness virus by in vitro immunization and the fusion/cloning technique.
Splenocytes from non-immune mice were stimulated in vitro using an equimolar mixture of factors from mixed lymphocyte reaction (MLR) and from phorbol-12-myristate 13-acetate (PMA) stimulated EL-4 cells, and concomitantly immunized with inactivated African horse sickness virus (AHSV) antigen serotype 4 or viral proteins 2 and 5 from AHSV serotype 9. Fusion with NSO myeloma cells was performed five days after primary or secondary stimulation/immunization. The record of hybridoma growth after a standard method of fusion, expansion of cells and subsequent cloning was compared with a fusion/cloning method in which cells were cloned within 2 to 3 days of the fusion event. Detection of antigen specific antibodies in the hybridoma culture supernatants was successful only with cells derived from primary stimulation/immunizations. Antibodies were detected using an indirect ELISA with the immunizing antigen coated on to the surface of the plates. Monoclonal hybridomas were isolated within 2 to 3 weeks using the fusion/cloning method, compared with the standard method, where it took 4 to 5 weeks. Although the total number of clones isolated from the fusion/cloning method was less than that obtained through the standard method, the yield of specific antibody-producing hybridomas as a percentage of the total picked was often more efficient with the fusion/cloning method. With respect to the immunoglobulin isotype produced, not all of the antibodies could be classified by the ELISA system used; 14% of anti-AHSV positive clones were identified as IgG-secreting cells, 25% as IgM-secreting, 18% were cross-reacting with IgG and IgM, and 43% could not be classified. Similar results in all aspects of the work were obtained whether a crude infected cell extract or purified outer capsid polypeptides VP2/5, from serotype 4 and serotype 9 respectively, were used.
Institut für Viruskrankheiten und Immunprophylaxe (IVI), Mittelhäusern, Switzerland.
N
N
Stäuber
U
U
Kihm
K C
KC
McCullough
D000356:African Horse Sickness Virus / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000914:Antibodies, Viral / Q000096:biosynthesis*; D002999:Clone Cells; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006825:Hybridomas / Q000276:immunology*; D007114:Immunization; D007132:Immunoglobulin Isotypes / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007132:Immunoglobulin Isotypes