Hybridoma technology for the generation of rodent mAbs via classical fusion.

Monoclonal antibodies (mAbs) have proven to be instrumental in the advancement of research, diagnostic, industrial vaccine, and therapeutic applications. The use of mAbs in laboratory protocols has been growing in an exponential fashion for the last four decades. Described herein are methods for the development of highly specific mAbs through traditional hybridoma fusion. For ultimate success, a series of simultaneously initiated protocols are to be undertaken with careful attention to cell health of both the myeloma fusion partner and immune splenocytes. Coordination and attention to detail will enable a researcher with basic tissue culture skills to generate mAbs from immunized rodents to a variety of antigens (including proteins, carbohydrates, DNA, and haptens) (see Note 1). Furthermore, in vivo and in vitro methods used for antigen sensitization of splenocytes prior to somatic fusion are described herein.

Screening hybridomas for cell surface antigens by high-throughput homogeneous assay and flow cytometry.

Described herein are methods for the successful screening of monoclonal antibodies (mAbs) of the desired specificities via high-throughput (HTP) homogeneous assay and flow cytometry. We present a combination of screening techniques that allow the scientist to efficiently eliminate nontarget-specific antibody as soon as possible. This compilation of protocols will enable researchers with basic immunology skills to make decisions regarding the design of screening algorithms for the generation of mAbs. Although we have provided an informative overview of both HTP homogeneous assay and flow cytometry, it is imperative for the beginner to acquire fundamental knowledge on how both of these technologies work so as to use these screening strategies effectively.