Development of an indirect competitive enzyme-linked immunosorbent assay (icELISA) using highly specific monoclonal antibody against paclitaxel

Abstract

Paclitaxel, the major active component of the yew tree, is used as an important anti-cancer agent. To obtain the monoclonal antibody (MAb) against paclitaxel for paclitaxel determination using immunoassay, 7-xylosyltaxol was conjugated to the carrier protein bovine serum albumin (BSA) to construct the immunogen, and the ratio of hapten in XylTax-BSA conjugate was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. After immunization of mice with this conjugate, hybridomas secreting MAbs against paclitaxel were obtained by fusing the splenocytes with the mouse myeloma cell line SP2/0. After hybridoma screening, the anti-paclitaxel MAb 3A3 was obtained, which showed a relatively high specificity to paclitaxel (cross-reactivities against other naturally occurred taxanes: 7-xylosyltaxol, 31.8%; cephalomannine, 6.17%; baccatin III, 10-deacetyl-baccatin III, 1-hydroxybaccatin I, 13-acetyl-9-dihydrobaccatin III and 1-acetoxyl-5-deacetyl-baccatin I, <0.11%). Using the MAb 3A3, we established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for paclitaxel determination with a detection range of 0.098-312.5 μg ml(-1). Determination of paclitaxel contents in various yew tree samples with this icELISA resulted in recovery rates ranging from 92 to 94.8%, and intra- and inter-assay variations of 3.6 and 4.7%, respectively. This icELISA provides a valuable method of paclitaxel determination for various purposes.

Development of a Competitive Time‐Resolved Fluoroimmunoassay for Paclitaxel

“…Rabbit anti‐mouse IgG was purchased from Rockland Immunochemicals (Limerick, PA, USA). Murine monoclonal antibody (MAb) 3A3 against Tax was previously prepared (Chao et al ., ), and is available from Prof. Hiroyuki Tanaka on request. Taxus materials were collected from local botanical gardens and were identified by the author as the shoots and barks of T. wallichiana var.…”

“…Tax contents in Taxus samples were also determined using ELISA as previously described (Chao et al ., ) to validate the established TRFIA. Briefly, an immunoplate, to which 100 μL of 1 μg/mL XylTax–HSA had been adsorbed, was treated with 300 μL SPBS for 1 h to reduce non‐specific adsorption.…”

“…We previously tested the effectiveness of immunoassay in quality control of several natural medicines (Fujii et al ., ; Tung et al ., ; Oiso et al ., ; Li et al ., ; Zhao et al ., ); in fact, the earliest attempt of using immunoassay for Tax detection was reported over 20 years ago (Grothaus et al ., , ; Jaziri et al ., ; Leu et al ., , ; Svojanovsky et al ., ). Recently, we successfully prepared an anti‐Tax monocolonal antibody (MAb) 3A3, based on which we established an indirect competitive enzyme‐linked immunosorbent assay (icELISA) for Tax determination with a wide detection range of 9.8 to 312.5 ng/mL, showing obvious advantages over the previously reported methods (Chao et al ., ).…”

Pharmacokinetics of Geniposide by Monoclonal Antibody-Based icELISA in Mice after Oral Administration of Huanglian-Jiedu-Tang

“…The indirect competitive ELISA (icELISA), an immunological assay based on mAb, was reported to provide a valuable tool for paclitaxel determination. 20) In addition, icELISA methods were developed for detection of okadaic acid and dinophytoxin-1 in seafood, 21) and diethylstilbestrol in biological samples. 22) Pharmacokinetic studies are very important in drug development.…”

Development of Fluorescence-Linked Immunosorbent Assay for Paeoniflorin

“…This method of detection has multiple benefits, including minimal pre-treatment of samples prior to the assay, as well as being inexpensive, fast, highly specific, and sensitive assays [8,9].…”