Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma
Section snippets
Materials and reagents
Trastuzumab (Herceptin) was supplied as a lyophilized powder from Roche (Basel, Switzerland). This pharmaceutical formulation also contains l-histidine hydrochloride, l-histidine, α,α-trehalose dihydrate, and polysorbate 20. Ammonium acetate, glycine, sodium chloride, Tris, urea, sodium hydrogen carbonate, dimethyl sulfoxide (DMSO), and sulfuric acid were purchased from Merck (Darmstadt, Germany). Crystalline pepsin and Tween 20 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Sterile
Anti-idiotype antibody production and purification
The complementarity-determining regions (CDRs) of a monoclonal antibody are present in the F(ab)2 portion, whereas the Fc tail is the same as that of human IgG. To prevent the rabbits from producing a majority of antibodies against human IgG, only the F(ab)2 fragments of trastuzumab were injected into the rabbit. F(ab)2 fragments were produced by digestion with pepsin. The F(ab)2 fragments could be separated from the Fc tails and intact antibodies by using protein A. The intact antibodies and
Conclusion
The goal of this study was to develop an ELISA for analysis of trastuzumab in serum and plasma. Because no commercially available coat antigen for trastuzumab is available, anti-idiotype antibodies were produced in-house. Anti-idiotype antibodies were purified from rabbit serum using protein A and IVIG-L sepharose columns. From one rabbit, 10 ml of serum was purified and the obtained anti-idiotype trastuzumab antibodies were sufficient to process approximately 1500 assay plates. In addition, 30
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