October 1, 2012 (Vol. 32, No. 17)

More Attention Should Be Paid to This Critical Step to Success

Highly specific or functional monoclonal antibodies (mAbs) are sought after by the biopharmaceutical industry for integration into numerous assays specific to a variety of platforms.

Considerable time and monetary commitments are entered into for the development of multiple antibodies displaying superior performance in an assay. All too often, companies initiating antibody development projects are particularly interested in generating the greatest possible number of parental clones from which, upon vigorous performance screening, only a select few will progress to the integration into the early disease diagnostic detection tests of tomorrow.

Without appropriate reagents to capture analyte-specific antibodies secreted by parental hybridomas, the quest for detecting the holy mAb may be delayed or lost. In order to successfully capture all clones, one has to consider the use of a secondary detection antibody possessing a well-balanced reactivity pattern against all antibody isotypes, as addressed in this article.

The development of monoclonal antibodies through hybridoma generation is initialized by the immunization of host animals with the antigen of interest. Dependent on the immunization regimen, together with analyte antigenicity and the host’s antibody-mediated immune response, a candidate demonstrating a positive serum titer can be chosen for the performance of a fusion within 42 to 98 days.

Performance of a fusion begins with the euthanization of the host animal followed by the aseptic removal of the spleen. Dependent on the host, maceration of a murine spleen yields an average of 2×108 splenocytes, whereas higher B-cell counts are obtained from Lewis rats, Armenian hamsters, or New Zealand white rabbits.

Implementation of an optimized electrofusion protocol for the fusion of harvested splenocytes to murine SP2/O-Ag14 myeloma fusion partner cells at a ratio of 1:1, results in a higher rate of fusion efficiency when compared to the traditional polyethylene glycol-based (PEG) fusion methodology. This effectively translates into a greater number of fused hybridomas, which actively grow upon selection in HAT medium while unfused cells die off.

Ten to fourteen days post fusion, the next step in the antibody development process focuses on the screening of established, actively growing parental clones. The primary screening of clones is predominantly conducted by indirect enzyme linked immunosorbant assay (ELISA), in which the hybridoma-derived supernatant is screened for specific antibody reactivity against the immunizing analyte absorbed onto the ELISA plate surface. At this stage and dependent on the total number of 96-well plates used containing cells at a predetermined count per well, 20 or more 96-well plates are visually inspected for cell growth prior to clone selection for analysis. Often this incorporates the screening of supernatant from as little as ten to several hundred individual hybridomas, with screening of entire 96-well plates not being extraordinary.

This hybridoma screening stage includes a crucial step, in that special attention needs to be paid to the reporter-labelled antibody used for the detection of the hybridoma-derived antibody in the indirect ELISA. Established hybridomas express and secrete analyte-specific antibodies at different rates and little is known about the antibody concentration within the 100 µL of collected supernatant used for analysis. Furthermore, the secreted antibody isotype remains undetermined at this stage and use of a reporter-labelled secondary antibody not capable of detecting all species-specific heavy and light chain isotypes will result in the inability to identify a positive clone.

For example, the precise identification of mouse-derived antibodies necessitates that the secondary antibody is capable of unequivocal detection of γ-IgG1, γ-IgG2a, γ-IgG2b, γ-IgG3, µ-IgM and α-IgA heavy chain isotypes. Similarly this holds true for the identification of analyte-specific rat hybridoma-derived antibodies, in which a well-balanced detection of the γ-IgG2c isotype among the remaining γ-IgG1, γ-IgG2a, γ-IgG2b, µ-IgM and α-IgA heavy chain isotypes is required.

Application of a secondary detection antibody not displaying this well-balanced reactivity pattern against respective isotypes, will result in failure to identify positive clones—a fact that is often overlooked. This, in turn, presents a problem, especially when the number of visually confirmed hybridomas is limited and the identification of numerous parental clones has been requested.

To overcome this limitation and identify each antibody-secreting hybridoma during the screening process, Rockland has developed antigamma globulin antibodies against mouse and rat heavy (γ, µ, and α) and light chain (κ and λ) isotypes. An affinity purified mixture of heavy and light (H & L) isotype-specific antibodies is conjugated to a reporter prior to the evaluation of overall reactivity against individual species-specific isotypes in the ELISA. Should the initial reactivity pattern of the antibody not comply with the product acceptance criteria, individual antibody levels are adjusted according to reactivity until complete equivalent reactivity is obtained (Figure).

Use of these versatile broad-spectrum antibodies results in consistent detection of analyte-reactive clones and ensures that all target-specific hybridomas are initially captured in the screening process. Considering that individual clones might lose analyte-specific reactivity throughout their time in culture, the greater number of identified positive clones represents an important facet for customers requiring a large number of clones for evaluation by immunohistochemistry or flow cytometry.

Not only are these antimouse or rat antibodies incorporated into the initial screening process, but their involvement throughout secondary screening procedures incorporating Western blot and/or cell-based ELISA analyses, for example, aid in the further characterization of antibodies secreted by positive clones. Their continued use in the completion of a monoclonal antibody development project spans the use of monitoring the stability and antibody expression levels of positive hybridomas throughout the subcloning process and their general incorporation into various assays make them a valuable tool in the laboratory.

Figure. Representation of a well-balanced antimouse-specific antibody reactivity against individual heavy and light chain isotypes.

Thomas O. Kohl, Ph.D. ([email protected]), is director, scientific business development at Rockland Immunochemicals.

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