An overview and comparison of a recombinant antigen-binding fragment and an antigen-binding fragment from a monoclonal antibody against wogonin glucuronide.

Wogonin glucuronide (wogonin 7-O-ß-D-glucuronide, Wgn) is widely recognized as a constituent of Scutellariae radix, which is used in Kampo medicines. Wgn has been used for both pharmacological (antifebrile uses and in detoxification) and research purposes. A recombinant antigen-binding fragment (rFab) and an antigen-binding fragment from a monoclonal antibody (mFab) against Wgn were constructed and used in an indirect competitive enzyme-linked immunosorbent assay (icELISA) in this study. The rFab and mFab against Wgn showed both activity and recognition against Wgn. The developed icELISA was validated as a quantitative analytical method to detect Wgn by testing both its utility and its reliability using multiple concentrations of Wgn from S. radix. This approach provides a more economic method to analyze and purify Kampo medicines.

An immunochromatographic assay for rapid etection of salvinorin A.

We developed an immunochromatographic assay (ICA) that enables rapid analysis of salvinorin A (Sal A) in Salvia divinorum within 10 min. The result shows that no Sal A in other samples of Lamiaceae plants was detected, but it could recognize Sal A among other substances in complex samples. The main advantage of the ICA is its high performance in combination with low cost, simplicity, and speed. Our newly developed combined ICA/indirect competitive ELISA(icELISA) system enables analysis of large numbers of samples over short periods of time without cumbersome pretreatments in complex mixtures. This method can complement other instrumental analyses for salvinorins, and could be used to deter S. divinorum abuse.

The effect of varying the peptide linker length in a single chain variable fragment antibody against wogonin glucuronide.

Peptide linkers of three different lengths were constructed to join the variable regions of the heavy chain (VH) and the light chain (VL) in a single-chain variable fragment antibody (scFv) specific for wogonin glucuronide (Wgn) that has the structure VH-(GGGGS)n-VL (n=3, 5, or 7). The scFv antibodies, which were expressed in Escherichia coli, were derived from an anti-Wgn monoclonal antibody (315A). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to evaluate their reactivity and sensitivity, which is also used for quantitative analysis of Wgn. Our results, showed that the reactivity and specificity of the three different scFvs were, in fact, similar. Subsequently, the scFv having a VH-(GGGGS)3-VL linker which was slightly better that other two scFvs against Wgn, was applied to indirect competitive ELISA (icELISA) to analyze Scutellariae Radix (S. Radix). The utility of the icELISA was demonstrated for quality control and analysis of S. Radix in this report.

Development of an immunoassay using an anti-wogonin glucuronide monoclonal antibody.

Wogonin 7-O-ß-D-glucuronide (Wgn) is a bioactive flavone present in the dried root of Scutellaria baicalensis Georgi. To generate a monoclonal antibody (MAb) against Wgn, BALB/c mice injected with Wgn-bovine serum albumin yielded splenocytes that we fused with SP2/0 myeloma cells using the polyethylene glycol method. We obtained a hybridoma designated 315A that produced a MAb reactive to Wgn. The anti-Wgn MAb 315A was applied to an indirect competitive enzyme-linked immunosorbent assay (icELISA) to quantify Wgn. Subsequent validation revealed that icELISA using the 315A anti-Wgn MAb is an accurate and reliable method for the quantification of Wgn in S. baicalensis.

Simultaneous determination of soy isoflavone glycosides, daidzin and genistin by monoclonal antibody-based highly sensitive indirect competitive enzyme-linked immunosorbent assay.

Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.

Development of an enzyme immunoassay using a monoclonal antibody against the psychoactive diterpenoid salvinorin A.

Salvinorin A (1), the main active constituent in Salvia divinorum, is a highly selective kappa-opioid receptor agonist with hallucinogenic effects, which is regulated in several countries. In the present study, a monoclonal antibody (mAb) against 1 was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) system was developed for the detection of salvinorins. To raise mAbs against 1, salvinorin B (2) hemisuccinate was synthesized and used to prepare the immunogen 2-bovine serum albumin conjugate. This technique was used to prepare a hybridoma cell line, 3D5, which secreted a mAb that recognized 1. The mAb was shown to have specificity for 1 and other salvinorins in cross-reactivity tests. The intra-assay calibration range by icELISA using the mAb against 1 was 0.0195-0.625 µg/mL. After validating the icELISA using intra- and interassays, a recovery experiment and analysis of several plants in the family Lamiaceae, including S. divinorum, confirmed that the analytical method based on ELISA is not only simple but also precise, accurate, sensitive, and sufficiently reliable. The results indicate that icELISA is a useful tool in the identification of S. divinorum.

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

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 combined technique using a rapid one-step immunochromatographic assay and indirect competitive ELISA for the rapid detection of baicalin.

A colloidal gold conjugated anti-baicalin monoclonal antibody (anti-BA MAb) was prepared and used in an immunochromatographic assay (ICA) for BA in Scutellariae Radix and Kampo medicines. This competitive ICA uses an anti-BA MAb which shows a high specificity for BA and baicalein. Its advantages include a short assay time (15 min), no dependence on any instrumental systems, and it can detect BA in plant materials and Kampo medicines. The limit of detection for the ICA was found to be around 0.6 µg mL(-1)of baicalin. Moreover, the usefulness of the combination of indirect competitive ELISA and the ICA using anti-BA MAb as a quality control method was confirmed for analysis of BA in Scutellariae Radix and Kampo medicines with a sufficient sensitivity (200 ng mL(-1) to 2 µg mL(-1)), obtainable in an easy and timely manner.