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TUTORIAL Capturing the Clone More Attention Should Be Paid to This Critical Step to Success Thomas O. Kohl, Ph.D. 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 com- mitments are entered into for the develop- ment of multiple antibodies displaying superior performance in an assay. All too often, companies initiating antibody devel- opment 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 dis- ease diagnostic detection tests of tomorrow. Without appropriate reagents to capture from Lewis rats, Armenian hamsters, or New Zealand white rabbits. Representation of a well-balanced antimouse-specific antibody reactivity against individual heavy and light chain isotypes. analyte-specific antibodies secreted by paren- tal 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 an- tibody possessing a well-balanced reactivity pattern against all antibody isotypes, as ad- dressed in this article. The development of monoclonal antibodies through hybridoma generation is initialized by the immunization of host animals with the an- tigen of interest. Dependent on the immuniza- tion regimen, together with analyte antigenic- ity 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 2x108 splenocytes, whereas higher B-cell counts are obtained Thomas O. Kohl, Ph.D. (thomas.kohl@ rockland-inc.com), is director, scientific business development at Rockland Immu- nochemicals. Web: www.rockland-inc.com. Implementation of an optimized electro- fusion protocol for the fusion of harvested splenocytes to murine SP2/O-Ag14 my- eloma fusion partner cells at a ratio of 1:1, results in a higher rate of fusion efficiency when compared to the traditional polyeth- ylene glycol-based (PEG) fusion methodol- ogy. 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 fo- cuses on the screening of established, actively growing parental clones. The primary screen- ing 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 to- tal 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 incorpo- rates the screening of supernatant from as little as ten to several hun- dred 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 antibod- ies 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 ca- pable 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 unequiv- ocal detection of a-IgG1, a-IgG2a, a-IgG2b, a-IgG3, µ-IgM and _-IgA heavy chain iso- types. Similarly this holds true for the iden- tification of analyte-specific rat hybridoma- derived antibodies, in which a well-balanced detection of the a-IgG2c isotype among the re- maining a-IgG1, a-IgG2a, a-IgG2b, µ-IgM and _-IgA heavy chain isotypes is required. Application of a secondary detection anti- body not displaying this well-balanced reac- tivity pattern against respective isotypes, will result in failure to identify positive clones—a Genetic Engineering & Biotechnology News | genengnews.com | October 1, 2012 | 23 fact that is often overlooked. This, in turn, presents a problem, especially when the num- ber of visually confirmed hybridomas is lim- ited and the identification of numerous paren- tal clones has been requested. To overcome this limitation and iden- tify each antibody-secreting hybridoma dur- ing the screening process, Rockland (www. rockland-inc.com) has developed antigamma globulin antibodies against mouse and rat heavy (a, µ, and _) and light chain (g and h) isotypes. An affinity purified mixture of heavy and light (H & L) isotype-specific antibodies is conjugated to a reporter prior to the evalu- ation 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 cri- teria, individual antibody levels are adjusted according to reactivity until complete equiva- lent reactivity is obtained (Figure). Use of these versatile broad-spectrum an- tibodies results in consistent detection of ana- lyte-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 im- munohistochemistry or flow cytometry. Not only are these antimouse or rat anti- bodies incorporated into the initial screen- ing 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 mono- clonal antibody development project spans the use of monitoring the stability and antibody ex- pression levels of positive hybridomas through- out the subcloning process and their general incorporation into various assays make them a valuable tool in the laboratory. Drug Discovery