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1.  Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein 
Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.
PMCID: PMC3189463  PMID: 22007309
2.  Recombinant Diabody-Based Immunocapture Enzyme-Linked Immunosorbent Assay for Quantification of Rabies Virus Glycoprotein▿  
The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (VH) and light chain (VL) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human × mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r2 value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.
PMCID: PMC2916251  PMID: 20573881

Results 1-2 (2)