Elsevier

Analytical Biochemistry

Volume 391, Issue 2, 15 August 2009, Pages 114-120
Analytical Biochemistry

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

https://doi.org/10.1016/j.ab.2009.05.030Get rights and content

Abstract

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to support these pharmacokinetic studies. For this immunoassay, we raised anti-idiotype antibodies in rabbits. After purification of the rabbit material, the anti-idiotype antibodies are used as capturing antibodies on the ELISA plate. After trastuzumab has bound to the catcher antibody, a sandwich ELISA procedure is followed whereby biotinylated anti-idiotype antibodies can bind to trastuzumab. Detection is performed by streptavidin–polyHRP (poly-horseradish peroxidase) conjugate and (3,5,3′,5′)-tetramethylbenzidine (TMB) substrate. The reaction is stopped using sulfuric acid, and the absorbance is measured at 450 nm. The calibration range of the assay is 0.039 to 5 ng/ml in well. Because samples are analyzed in multiple dilutions, the validated range corresponds to 1.6 to 1600 ng/ml in undiluted serum. Samples above the upper limit of quantification (ULOQ) can be diluted before transfer to the assay plates. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum and plasma. The assay is now used to support pharmacokinetic studies with 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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