Detection of Two Isotypically Different Antibodies Produced by a Murine Hybridoma

Abstract

A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized. This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells. The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains. Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality. Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted. PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa. A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products. These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging

“…The study proved that indeed SPRi was capable of following in real time the excretion of the expected antibodies and that it was a much easier and quicker method to confirm antibody production compared to more traditional label-based techniques [9]. Traditionally detection and quantification of single hybridoma cell excretion is a complicated procedure [10][11][12]. Recently a new fluorescence-based method was introduced, which uses metal-enhanced fluorescence.…”

Generation of Monoclonal Antibodies Against Human Recombinant Interferon Beta Using Genetic Immunization with Simultaneous Expression of IgM and IgG Isotypes

“…Only a few authors have reported simultaneous secretion of IgM and IgG molecules from the same hybridoma clone. (17)(18)(19)(20) Isotype switching by B cells is highly regulated by a group of cytokines, including IL-4, IFN-g, and TGF-b. A lymphocyte B cell can only express one isotype at a time; however, during an immune response it may be exposed to combinations of stimuli that provide it with conflicting switching instructions.…”

SPRi cytometry: sensitively sensing the sensitive side of cells

“…The study proved that indeed SPRi was capable of following in real time the excretion of the expected antibodies and that it was a much easier and quicker method to confirm antibody production compared to more traditional label-based techniques [98]. Traditionally detection and quantification of single hybridoma cell excretion is a complicated procedure [99][100][101]. Recently a new fluorescence-based method was introduced, which uses metal-enhanced fluorescence.…”