Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours

Wang³

et al. 2022

Molecules

Background: This study aimed to prepare monoclonal antibodies (mAbs) with high immunoreactivity, sensitivity, and specificity for the chelate (Cr(Ш)－EDTA) of trivalent chromium ion (Cr(Ш)) and ethylenediamine tetraacetic acid (EDTA). Further, the study established an indirect competitive enzyme-linked immunosorbent assay (icELISA) for detecting the total chromium content in food, feed, and environmental samples. Methods: Hapten Cr(Ш)－iEDTA was synthesized by chelating Cr(Ш) with isothiocyanatebenzyl－EDTA (iEDTA). Immunogen Cr(Ш)－iEDTA－BSA formed by chelating Cr(Ш)－iEDTA with bovine serum albumin (BSA), and coating antigen Cr(Ш)－iEDTA－OVA formed by chelating Cr(Ш)－iEDTA with ovalbumin (OVA) were prepared using the isothiocyanate method and identified by ultraviolet spectra (UV) and inductively coupled plasma optical emission spectrometry (ICP-OES). Balb/c mice were immunized with the Cr(Ш)－iEDTA－BSA, and the anti Cr(Ш)－EDTA mAb cell lines were screened by cell fusion. The Cr(Ш)－EDTA mAbs were prepared by induced ascites in vivo, and their immunological characteristics were assessed. Results: The immunogen Cr(Ш)－iEDTA－BSA was successfully synthesized, and the molecular binding ratio of Cr(Ш) to BSA was 15.48:1. Three hybridoma cell lines 2A3, 2A11, and 3D9 were screened, among which 2A3 was the best cell line. The 2A3 secreted antibody was stable after six passages, the affinity constant (Ka) was 2.69 × 109 L/mol, its 50% inhibition concentration (IC50) of Cr(Ш)－EDTA was 8.64 μg/L, and it had no cross-reactivity (CR%) with other heavy metal ion chelates except for a slight CR with Fe(Ш)－EDTA (1.12%). An icELISA detection method for Cr(Ш)－EDTA was established, with a limit of detection (LOD) of 1.0 μg/L and a working range of 1.13 to 66.30 μg/L. The average spiked recovery intra-assay rates were 90% to 109.5%, while the average recovery inter-assay rates were 90.4% to 97.2%. The intra-and inter-assay coefficient of variations (CVs) were 11.5% to 12.6% and 11.1% to 12.7%, respectively. The preliminary application of the icELISA and the comparison with ICP-OES showed that the coincidence rate of the two methods was 100%, and the correlation coefficient was 0.987. Conclusions: The study successfully established an icELISA method that meets the requirements for detecting the Cr(Ш)－EDTA chelate content in food, feed, and environmental samples, based on Cr(Ш)－EDTA mAb, and carried out its preliminary practical application.

Section: Methodsmentioning

confidence: 67%

Indirect Competitive ELISA for the Determination of Total Chromium Content in Food, Feed and Environmental Samples

Wang¹,

Wang³

et al. 2022

Molecules

“…Cell fusion and screening of positive hybridoma cell lines were conducted as reported in previous literature [ 33 , 34 ]. Ten to fourteen days after cell fusion, a homologous inELISA and an icELISA were used to screen the positive hybridomas obtained from the supernatants.…”

Section: Methodsmentioning

confidence: 99%

Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples

et al. 2022

Toxins

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 μg/L, 8.69 μg/L, and 0.92–82.24 μg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48–99.48%, 94.18–96.13%, 12.55–12.98%, and 12.53–13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

“…Kiko et al [ 52 ] prepared 3-succinylaconitine by succinylation at the C-3 hydroxyl position of aconitine. The same method has also been used to prepare haptens such as camptothecin [ 53 ], morphine [ 54 ], retrorsine [ 55 ], and tropane [ 56 ]. Wang et al [ 57 ] prepared a hapten of methamphetamine by introducing a carboxyl group on the second amino group.…”

Section: Synthesis and Identification Of Antigenmentioning

confidence: 99%

“…Gandhi et al [ 64 ] synthesized an acidic derivative of MAM and eventually induced antibodies that simultaneously recognize heroin, MAM, and morphine because they have the same common structure. Wang et al [ 56 ] selected the simplest compound with the structure of tropine ring from 12 kinds of atropine alkaloids with similar structures and their structural analogs for hapten design and finally obtained an antibody that can cross-recognize seven kinds of atropine alkaloids.…”

Section: Synthesis and Identification Of Antigenmentioning

confidence: 99%

“…Computer-aided molecular modeling is an effective strategy, which can evaluate and select the designed haptens from the aspects of molecular configuration and electronic properties, and ultimately improve the possibility of obtaining the target antibodies. Now, this method has only been preliminarily applied to atropine [ 56 ] and pyrrolizidine alkaloids [ 55 ], and other related studies have not been reported. The structures of the toxic alkaloids in plant-derived medicines are complex and varied, but the same species has the same parent structure and properties.…”

Section: Synthesis and Identification Of Antigenmentioning

confidence: 99%

See 1 more Smart Citation

Progress in Immunoassays of Toxic Alkaloids in Plant-Derived Medicines: A Review

Ren

Zhang

et al. 2022

Toxins

Self Cite

Plants are the cradle of the traditional medicine system, assuaging human or animal diseases, and promoting health for thousands of years. However, many plant-derived medicines contain toxic alkaloids of varying degrees of toxicity that pose a direct or indirect threat to human and animal health through accidental ingestion, misuse of plant materials, or through the food chain. Thus, rapid, easy, and sensitive methods are needed to effectively screen these toxic alkaloids to guarantee the safety of plant-derived medicines. Antibodies, due to their inherent specificity and high affinity, have been used as a variety of analytical tools and techniques. This review describes the antigen synthesis and antibody preparation of the common toxic alkaloids in plant-derived medicines and discusses the advances of antibody-based immunoassays in the screening and detection of toxic alkaloids in plants or other related matrices. Finally, the limitations and prospects of immunoassays for toxic alkaloids are discussed.