IVDT_In Vitro Diagnostics Technology

IVD Technology, Spring 2013

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ASSAY DEVELOPMENT antibodies (Figure 1). It is noted that rabbits display less immunodominance and less IgM occurrence when polymers with repeated domains such as lipids or carbohydrates are immunized as immunogens. Although the rabbit immune system generally sets the foundation for better antibody selection, in order to develop the most suitable antibody for a particular application one must also consider the immunogen quality, its structure and format; the conjugation of an immunogen with a carrier, if necessary; the immunization scheme; antibody screening assays; and the screening strategy. Rabbit Monoclonal Antibody Technology Rabbit monoclonal antibodies combine the best properties of monoclonal antibodies with the most desirable attributes of rabbit polyclonal antibodies.3-7 In 1995, Katherine Knight and her colleagues at Loyola University of Chicago developed a plasmacytoma cell line, termed 240E-1, from a double transgenic rabbit overexpressing the oncogenes v-abl and c-myc. Fusion of 240E-1 cells with rabbit lymphocytes produced hybridomas that secreted rabbit monoclonal antibodies.8 However, the stability of 240E-1 derived hybridomas was a concern. Te resulting hybridomas were not genetically stable and antibody activity was often lost after multiple passages of cells. In 1996, Weimin Zhu and Robert Pytela, then at University of California, San Francisco, put forth great efort into improving 240E-1 and developed a better fusion partner cell line, 240E-W, with high fusion efciency and reliable hybridoma stability. A number of characteristics that distinguish 240E-W from its parent 240E-1 were observed.9 Tis new fusion partner cell line has made rabbit hybridoma production a routine lab technique, making largescale development of rabbit monoclonal antibodies a reality. Similar to the original mouse fusion partner cell line, the 240E-W cell line inherited the undesirable traits of endogenous IgG chains.8 To solve this problem, 2 4 IVD TEC HNOLO G Y | SP RIN G 2013 magenta cyan yellow black Weimin Zhu and his colleagues at Epitomics developed new derivative lines of 240E-W, termed 240E-W2, which does not carry an endogenous heavy chain gene, and 240E-W3, which lacks both endogenous heavy and light chain genes. Table I summarizes the features of diferent rabbit fusion partner cell lines. Te rabbit hybridoma generation procedure is similar to that of mouse hybridomas (Fig 2). So far, with the newer fusion partner cell lines (240EW2 and 240E-W3), thousands of hybridoma cell lines have been generated for life science, diagnostic, and therapeutic applications. Among them, several thousand research reagents and more than 150 IVD immunohistochemistry (IHC) products have been used in the market, and three rabbit monoclonal antibodies (RabMAb) have been approved by FDA as Class II or Class III IVD products. Epitomics has developed a humanized RabMAb drug candidate, which was demonstrated to have better in vitro and in vivo efcacies than a commercial benchmark.10 Tis drug candidate is currently in clinical trials. Advantages of Rabbit Monoclonal Antibody High sensitivity and specifcity. Typically, RabMAbs have 10 to 100 times higher afnity than mouse Figure 2. Rabbit hybridoma generation process. B cells are isolated from a rabbit that is immunized with an immunogen and are fused with a rabbit fusion partner cell line to generate hybridomas. Antibodies from hybridomas are screened using various assays. Select hybridoma clones are subcloned to ensure that they are monoclonal. The fnal clone is chosen to produce a larger quantity of RabMAbs in a CELLine Integra fask, or the hydridoma IgG gene is cloned and recombinant antibody is produced in a mammalian expression system. i v d t e c hnol ogy. com ES237160_IV1305_024.pgs 04.25.2013 01:08 UBM

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