Use of protein-bearing nitrocellulose as immunogen for in vitro production of monoclonal antibodies: application to myelin basic protein electrophoretically separated from a complex brain protein mixture

doi:10.1016/0165-2478(87)90065-4

Immunology Letters

Volume 16, Issue 1, October 1987, Pages 75-81

https://doi.org/10.1016/0165-2478(87)90065-4 Get rights and content

Abstract

A procedure for producing monoclonal antibody to myelin basic protein (MBP) using in vitro immunization with MBP transferred to nitrocellulose is described. Following the separation of brain proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS—PAGE) and electrophoretic transfer of the electrophoretogram onto nitocellulose, the MBP band located by immunodetection was excised from the nitrocellulose, ground, and used as immunogen for in vitro stimulation of unprimed mouse spleen cells. While in vitro immunization with soluble MBP was able to generate many hybrids, all the wells in the fusions carried out with the immobilized MBP contained hybrids, 33 to 42% of which were positive to MBP. Among these, six were further characterized; all were IgM and all bound to epitopes common to the 18.5K and 21.5K MBP forms of several species. In view of its simplicity, this technique should have a wide application for the rapid production of monoclonal antibodies to selected proteins or their fragments present in small quantity or difficult to purify on a large scale.

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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.
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Use of protein-bearing nitrocellulose as immunogen for in vitro production of monoclonal antibodies: application to myelin basic protein electrophoretically separated from a complex brain protein mixture

Abstract

Anal. Biochem.

Molecular Immunology

J. Immunol. Methods

Anal. Biochem.

Anal. Biochem.

J. Immunol. Methods

J. Neuroimmunol.

J. Neuroimmunol.

Int. J. Biochem.

J. Biol. Chem.

Cell

Trends Biotech.

J. Immunol. Methods

Cell