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Paxilline (PAX) is a tremorgenic mycotoxin that is thought to be associated with various syndromes in animals. To facilitate the determination of this toxin, a hapten was chemically synthesized and then respectively conjugated to Bovine Thyroglobulin (BTG) and Bovine serum albumin (BSA) for antibody production. Monoclonal antibody (mAb) 4F8 was finally obtained and used to establish an accurate and sensitive indirect competitive ELISA (icELISA). Various parameters that could impact the performance of icELISA were optimized, including the ion concentration, pH value and organic solvent. Under optimal condition, this assay exhibits an IC50 value as low as 0.84 µg L−1. The limit of detection (LOD) of the established icELISA method for PAX was 0.03–0.3 µg L−1. In conclusion, this study provided a rapid, sensitive, and high-throughput immunoassay for the detection of PAX in grains. [ABSTRACT FROM AUTHOR]

Nitrovin, classified as a nitrofuran drug, has been banned in numerous countries due to its carcinogenic effect on human health. However, existing analytical techniques for its detection are laborious, requiring an overnight derivatization process lasting 16 h. In this study, novel haptens were designed to prepare antibodies against aminogunidine (AGD), the metabolite of nitrovin, and then developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect AGD from fish samples without derivatization for the first time. Computer-assisted molecular modeling predicted the efficiency of novel haptens by conformational studies and electronic analysis. The results were consistent with the predicted results in producing high-affinity monoclonal antibody (mAb) against AGD. The half-inhibition concentration (IC 50) value of icELISA was 0.80 ng mL−1 for AGD, with a limit of detection of 0.27 μg kg−1 and recoveries of 89.8%–97.2%. The results showed that this new hapten design and synthesis provided a great prospect to obtain high-affinity mAb for AGD. This developed icELISA substantially avoided the conventional and lengthy derivatization process and was an effective method for the direct detection of AGD in fish tissue without derivatization. [Display omitted] • Two novel haptens for nitrovin metabolite were designed and synthesized with new derivative agents. • One high affinity monoclonal antibody with IC 50 of 0.80 ng mL−1 was obtained. • The cross reactivity of mAb 4G8 was investigated by computational chemistry. • An icELISA without derivatization pretreatment was developed with a LOD of 0.27 μg kg−1 in fish samples. [ABSTRACT FROM AUTHOR]

To monitor the contamination of a type A trichothecene, diacetoxyscirpenol (DAS), one monoclonal antibody (mAb) 8A9 with high affinity and specificity was prepared in the present study. The mAb 8A9 showed a 50% inhibition concentration (IC50) of 0.31 μg/L, which is of the highest affinity reported to date. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral flow immunoassay (LFIA) based on mAb 8A9 were developed and exhibited limits of detection as low as 0.65 μg/kg and 100 μg/kg in rice samples, respectively. The molecular recognition mechanism of mAb 8A9 to DAS was explored by molecular docking. The results showed that the hydrophobic amino acids of mAb 8A9 interacted with DAS by forming hydrogen bonds and a pi-sigma bond, which lead to a highly specific recognition of DAS. In summary, we produced one mAb, developed ELISA and LFIA for DAS detection in rice with significantly sensitivity, specificity, accuracy, and precision. [ABSTRACT FROM AUTHOR]

Ribavirin (RBV) is an effective antiviral drug, whose use is prohibited in animal husbandry worldwide. In this work, a novel immunizing hapten of RBV, named Hapten 4, was designed by comparing the conformational and electronic properties of RBV and haptens based on computational chemistry. Hapten 4 was synthesized and conjugated with carrier proteins to produce monoclonal antibody (mAb). The obtained mAb 4C3 for RBV exhibited an IC50 value of 6.24 ng/ml in an indirect competitive enzyme‐linked immunosorbent assay (icELISA) and displayed no cross‐reaction with five other antiviral drugs, including amantadine. The applicability of the developed icELISA was verified in chicken, with a calculated limit of detection of 4.23 µg/kg. The recoveries in spiked chicken were 79.2%–107.3% with a coefficient of variation less than 15.9%. The results indicated that the produced antibody from the new hapten was reliable and would be useful for RBV screening in chicken. Practical Application: RBV is a broad‐spectrum antiviral drug, which is commonly used illegally in poultry farms. A high‐affinity mAb 4C3 against RBV was produced and used to develop icELISA with acceptable sensitivity and accuracy. The constructed icELISA has excellent performance for detecting RBV residues in chicken. [ABSTRACT FROM AUTHOR]

Rifaximin (RIF) is an important drug in clinical veterinary, and its residue in milk might pose a potential threat to human health. To monitor RIF residue in milk, a europium nanospheres-based time-resolved fluorescence immunoassay (TRFIA) was developed. The TRFIA is based on the competitive reaction between RIF-ovalbumin (OVA) and the target RIF in milk for binding to the Eu-Nanospheres-mAbs. The limit of detection (LOD) and limit of quantity (LOQ) were 3.05 and 6.63 ng/mL, respectively. The recoveries of the TRFIA to detect RIF in spiked milk samples ranged from 73.7% to 103.9%, and the interday and intraday variation were respectively less than 12.1% and 10.5%. A quantitative comparison of TRFIA and UPLC-MS/MS method exhibited a significant correlation (R2 = 0.9725) at the six various spiked levels. These results indicated that the TRFIA we developed was applicable for the detection of large numbers of milk samples. [ABSTRACT FROM AUTHOR]

Two haptens were synthesized by introducing spacer arms of different lengths and structures on the free amino of amantadine (AMA). 4-(chlorosulfonyl) benzoic acid (CSBA) was reacted with AMA to produce hapten AMA-CSBA, and succinic anhydride was reacted with AMA to produce hapten AMA-HS. And then, the two haptens coupled with bovine serum albumin (BSA) were prepared as immunogens for the production of monoclonal antibody (mAb). High-sensitive mAbs were obtained and characterized by using indirect competitive enzyme-linked immnunosorbent assay (icELISA). The best antibody was obtained in a heterologous icELISA with an IC value of 0.84 and 0.41 ng/mL for AMA and rimantadine, respectively, and negligible cross-reactivity (CR) with memantine and ribavirin. An icELISA was developed to detect AMA residue in chicken muscle. The limit of detection (LOD) was 1 μg/kg in spiked chicken muscle and the recoveries ranged from 93.6 to 116.9% with the coefficient of variations (CVs) below 9.5%. Good correlation between icELISA and HPLC-MS/MS results in the incurred tissues demonstrated the reliability of the developed icELISA. These results indicated that the new antibody was sufficiently sensitive and specific for the detection of AMA in real samples. [ABSTRACT FROM AUTHOR]

The residue of 17β-estradiol (E2) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E2residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E2-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E2in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC50) against E2of 0.17 ng/mL, high cross-reactivity (CR) to E2benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093 μg/L, which was enough sensitive to detect E2in milk. In spiked samples (0.5, 1, and 2 μg/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E2residue in milk. [ABSTRACT FROM PUBLISHER]

Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or ovalbumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC 50 values for erythromycin of 14.40 and 0.94 μg L − 1 , respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis. [ABSTRACT FROM AUTHOR]

A sensitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody was developed for routine screening of florfenicol (FF) in fish feed. FF succinate was synthesised and conjugated to human serum albumin by N-hydroxysuccinimide-activated ester method as immunogen, while it was conjugated to ovalbumin by mixed anhydride reaction as coating antigen. The ELISA for FF showed an IC50 value of 2.5 ng/mL, and negligible cross-reactivities with other amphenicol family of antibiotics. The inter-assay recoveries of FF from fortified fish feed at levels of 2-20 mg/kg ranged from 98.8 to 117.6% with coefficients of variation (CV) of 9.3-14.2%, intra-assay recoveries ranged from 102.5 to 121.2% with CV of 10.9-13.8%. The developed ELISA were then validated by liquid chromatography method and liquid chromatography-tandem mass spectrometry method, the results indicated this ELISA could be used as convenient method for detection of FF in fish feed. [ABSTRACT FROM AUTHOR]

A new monoclonal antibody (Mab) against sulphamerazine (SMR) was produced and a fluorescence polarisation immunoassay (FPIA) based on the Mab was developed and optimized for the simultaneous qualitative screening of SMR, sulphamethazine (SMZ) and sulphadiazine (SDZ). The Mab, raised from mice immunized with SMR, was bound to bovine serum albumin (BSA) using glutaraldehyde as the coupling reagent. Fluorescein-labelled SMR and SMZ (tracer) were synthesized and purified by thin layer chromatography (TLC). Cross-reactivities below 3.6% were displayed in the optimized FPIA for another 14 sulphonamides when both tracers were employed. The limits of detection (LOD) were 0.9 ng g-1 for SMR, 2 ng g-1 for SMZ and 3.1 ng g-1 for SDZ. Analysis of SMR, SMZ and SDZ fortified chicken muscle and honey samples by the FPIA showed average recoveries of 86-131% with a standard deviation (SD) of 4.6-32. Comparative analyses of a SMZ-treated chicken muscle sample by both FPIA and high performance liquid chromatograph (HPLC) showed a good correlation (r = 0.9991). The study demonstrates the practical application of FPIA in screening chicken muscle and honey samples for sulphonamides residues. [ABSTRACT FROM AUTHOR]

A fluorescence polarisation immunoassay (FPIA) for the screening of sulphamethazine (SMZ) in chicken muscle was developed. Fluorescein-labelled tracers for several sulphonamides were synthesised and monoclonal antibodies (Mabs) and polyclonal antibody (Pab) were produced. The Mabs and Pabs were raised against sulphonamides conjugated to bovine serum albumin by a diazo method. The most sensitive FPIA method for SMZ detection was based on a combination of the Mab against SMZ–bovine serum albumin and tracer SMZ labelled with fluorescein isothiocyanate. The limit of detection and IC50 for SMZ were 1 and 16 ng mL−1. Recoveries of SMZ from fortified chicken muscle samples averaged between 78% and 89%. The proposed FPIA method was applied for determining SMZ in an actual residue study. [ABSTRACT FROM AUTHOR]

The consumption of shellfish contaminated with brevetoxins, a family of ladder-frame polyether toxins formed during blooms of the marine dinoflagellate Karenia brevis, can cause neurotoxic poisoning, leading to gastroenteritis and neurotoxic effects. To rapidly monitor brevetoxin levels in oysters, we generated a broad-spectrum antibody against brevetoxin 2 (PbTx-2), 1 (PbTx-1), and 3 (PbTx-3) and developed a rapid indirect competitive enzyme-linked immunosorbent assay (icELISA). PbTx-2 was reacted with carboxymethoxylamine hemihydrochloride (CMO) to generate a PbTx-2-CMO hapten and reacted with succinic anhydride (HS) to generate the PbTx-2-HS hapten. These haptens were conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen reagents, respectively, using the active ester method. After immunization and cell fusion, a broad-spectrum monoclonal antibody (mAb) termed mAb 1D3 was prepared. The 50% inhibitory concentration (IC50) values of the icELISA for PbTx-2, PbTx-1, and PbTx-3 were 60.71, 52.61, and 51.83 μg/kg, respectively. Based on the broad-spectrum mAb 1D3, an icELISA was developed to determine brevetoxin levels. Using this approach, the limit of detection (LOD) for brevetoxin was 124.22 μg/kg and recoveries ranged between 89.08% and 115.00%, with a coefficient of variation below 4.25% in oyster samples. These results suggest that our icELISA is a useful tool for the rapid monitoring of brevetoxins in oyster samples. [ABSTRACT FROM AUTHOR]

The antigen‐antibody interaction determines the sensitivity and specificity of competitive immunoassay for hapten detection. In this paper, the specificity of a monoclonal antibody against alternariol‐like compounds was evaluated through indirect competitive ELISA. The results showed that the antibody had cross‐reactivity with 33 compounds with the binding affinity (expressed by IC50) ranging from 9.4 ng/mL to 12.0 μg/mL. All the 33 compounds contained a common moiety and similar substituents. To understand how this common moiety and substituents affected the recognition ability of the antibody, a three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) between the antibody and the 33 alternariol‐like compounds was constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The q2 values of the CoMFA and CoMSIA models were 0.785 and 0.782, respectively, and the r2 values were 0.911 and 0.988, respectively, indicating that the models had good predictive ability. The results of 3D‐QSAR showed that the most important factor affecting antibody recognition was the hydrogen bond mainly formed by the hydroxyl group of alternariol, followed by the hydrophobic force mainly formed by the methyl group. This study provides a reference for the design of new hapten and the mechanisms for antibody recognition. [ABSTRACT FROM AUTHOR]

A rapid lateral flow fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay has been developed for the detection of aflatoxin M 1 (AFM 1 ) residues in milk. For this purpose, an ultra-sensitive anti-AFM 1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC 50 value of the MAb 1D3 for AFM 1 was 23 ng/L, and the cross-reactivity (CR) of the MAb with aflatoxin M 2 , aflatoxin B 1 , aflatoxin B 2 , aflatoxin G 1 and aflatoxin G 2 was 18%, 9%, <0.15%, 43% and <0.3%, respectively. The MAb was covalently conjugated with carboxylate-modified FMs, and the FMs were used as the label in a competitive immunochromatography assay. Using a moderate hapten-to-protein coupling ratio (˜2) in the coating antigen resulted in improved immunoassay sensitivity. Under optimal conditions, the IC 50 value of the assay was 36.3 ng/L with the limit of detection (LOD) of 4.4 ng/L in milk samples using a fluorescence reader, and the recoveries of AFM 1 in spiked milk samples ranged from 76.6 to 110.8% with coefficient of variation (CV) of 4–14.7%. The whole procedure could be completed within 30 min. The results demonstrated that the FM-ICTS assay is easy to conduct, rapid, highly sensitive and specific for the detection of AFM 1 residues in milk. [ABSTRACT FROM AUTHOR]

The consecutive growth of antimicrobial resistance and the spread of resistance genes worldwide, especially the emergence of superbugs, have made traditional antibiotic-based treatments inadequate to fight bacterial infections. Therefore, new therapeutic modalities for bacterial infections are urgently needed. Antibodies are considered to be an effective alternative to antibiotics. The emergence and advancement of technologies such as hybridoma, antibody purification, transgenic mice, phage display, and protein engineering have enabled the production of large quantities of humanized antibodies with high purity and affinity. Antibodies has achieved remarkable achievements in the field of medicine in the past decades. Antibody-based therapy is expected to be an effective way to treat drug-resistant bacterial infections in the post-antibiotic era due to its merits of high specificity, which leads to no selective pressure on non-target bacteria and could cooperate with antibiotics to enhance the antimicrobial effect. This review first introduces the mechanism of action of antibodies against bacterial infections, then summarizes the reported antimicrobial antibodies according to different targets, discusses the advantages and limitations of the antibody-based therapy for antimicrobial treatment, and finally, the perspectives of antimicrobial antibodies developing have been prospected, providing a reference for the development of new antimicrobial antibodies. [Display omitted] • Antibodies are effective alternatives to antibiotics for bacterial infection treatment in the post-antibiotic era. • Mechanisms of antimicrobial antibody actions are introduced. • Advances in antimicrobial antibodies for various target antigens in recent years are summarized. • Advantages and limitations of antimicrobial antibodies for clinical treatment are discussed. • The perspectives for antimicrobial antibody development are discussed. [ABSTRACT FROM AUTHOR]

Xylazine (XYL) is an illicit adulterant in opioids and an approved veterinary sedative drug, which has been abused, misused, and residued in food samples, endangering people health, causing drug-facilitated crimes and even death. Immunoassay used antibody as core biomaterial could to achieve highly sensitive and rapid detection screening purpose for XYL in situ. Here, we rationally designed four novel XYL haptens with different spacer arms to produce antibodies with high affinity and specificity. Ten monoclonal antibodies (mAbs) were obtained and mAb 7H5 showed a high affinity with IC 50 of 0.23 ng mL−1 and ignorable cross-reactivity for the other eight analogs. One highly sensitive indirect competitive ELISA (icELISA) and lateral flow immunoassay (LFIA) were established based on heterologous haptens for XYL detection in a series of human urine and food samples. The limit of detections (LODs) of the icELISA were 0.10–2.24 μg L−1 with recovery of 83.5 % to 128.6 % and CV below 15.8 % in different samples, while, the visual limits of detection (vLOD) of LFIA were 0.10–1.80 μg L−1 with the cut-off value of 0.60–4.80 μg L−1. In addition, the molecular recognition mechanism of mAbs was explored. The study provides powerful tools for rapid screening of XYL in human urine and food samples for the first time. [Display omitted] • Four novel XYL haptens with heterologous structures were designed and evaluated. • A high affinity and specificity mAb 7H5 against XYL were obtained. • One highly sensitive icELISA and LFIA were established for XYL detection. • The recognition mechanism of prepared mAbs was investigated by molecular docking. [ABSTRACT FROM AUTHOR]

This study synthesized a new alternariol (AOH) hapten. The AOH had an alkylation reaction with ethyl 4-bromobutyrate, followed by hydrolysis and purification, and then conjugated with keyhole limpet hemocyanin to immunize mice and successfully produced two kinds of monoclonal antibodies (mAbs), namely the antibodies which can recognize both AOH and alternariol monomethyl ether (AME) and the ones that can specifically recognize AOH. Wherein, mAb 13D8 can uniformly recognize AOH and AME with the 50% inhibition concentration (IC 50 ) values of 9.4 ng/mL and 9.7 ng/mL, respectively. An indirect competitive ELISA (icELISA) was established by mAb 13D8 to simultaneously detect AOH and AME, with the limit of detection (LOD) being 0.7 ng/mL and 1.0 ng/mL, respectively. The average recoveries in spiked wheat sample were 84.5–107.6% with the coefficient of variation (CV) lower than 10%. The established icELISA method was used to detect 13 real samples with the same results as HPLC-MS/MS, indicating that the method is suitable for simultaneous detection of AOH and AME in wheat. [ABSTRACT FROM AUTHOR]

To reduce the incidence of false-positive and false-negative results caused by high or low cross-reactivity (CR%) values of the antibodies for total aflatoxins (AFs, AFB 1 +AFB 2 +AFG 1 +AFG 2 ) detection, a new broad-specific monoclonal antibody (MAb) with uniform affinity, named 5H3, was developed. Moreover, magnetic beads (MBs) replaced microplates as immobile phase to improve the sensitivity of the enzymatic immunoassay. Then, a direct competitive enzyme-linked immunosorbent assay (ELISA) based on MBs (MBs-dcELISA) that could simultaneously detect the total AFs with similar sensitivity was developed. Following optimization of conditions, the half maximal inhibitory concentrations (IC 50 ) of the MBs-dcELISA in buffer were 0.05 ng/mL for AFB 1 , 0.04 ng/mL for AFB 2 , 0.05 ng/mL for AFG 1 , 0.06 ng/mL for AFG 2 . The corresponding CR% values were 100%, 125%, 100% and 83.3%, respectively. The limit of detection (LOD) of the MBs-dcELISA for the total AFs was 0.21 ng/g with a working range from 0.22 ng/g to 19.8 ng/g, and the recoveries for the total AFs ranged from 74.5% to 96.5% with coefficients of variation (CV) under 12.1% in spiked maize samples. In addition, the MBs-dcELISA was more sensitive than the conventional dcELISA. Finally, the MBs-dcELISA was applied to screen 9 naturally contaminated maize samples and 6 spiked samples and the results indicated a good agreement with that obtain by HPLC-MS/MS method. [ABSTRACT FROM AUTHOR]

Pollution by the pesticide fipronil (FPN) and three potentially stronger toxicological metabolites (FPNs) threatens ecological balance and human health. Thus, broad-specificity antibodies and immunoassays are required for FPNs determination. Here, we clarified the common skeleton of FPNs by molecular overlay and predicted the effectiveness of haptens by computational chemistry before chemical synthesis; finally, confirmation was obtained by an immunization experiment. One novel monoclonal antibody (mAb), 19F8, with broad specificity to FPNs was obtained based on the predicted hapten H3 that lacks the heterogeneous −SOCF 3 group of FPNs and is stretched by a 6-carbon-long spacer arm at the −NH 2 position. The molecular recognition study showed that the broad specificity of mAb 19F8 to FPNs originated from hydrogen/halogen bonds and pi-pi stacked/pi-alkyl interactions, demonstrating the reliability of the hapten prediction strategy. Finally, a rapid, reliable, and sensitive immunoassay was established based on mAb 19F8 with IC 50 values of 1.85 − 4.89 ng mL−1 and limits of detection of 1.13 − 3.69 μg kg−1 for total FPNs in river water and egg samples. This work not only established a rapid screening method for total FPNs in environmental and food samples but also proposed an effective hapten prediction strategy to improve the efficiency of antibody preparation. [Display omitted] • The effectiveness of hapten structure on antibody response was predicted. • A noval broad-specificity mAb to fipronil and three metabolites was obtained. • The recognition mechanism of mAb 19F8 was investigated by molecular simulation. • An icELISA was developed to detect fipronil and three metabolites in real samples. [ABSTRACT FROM AUTHOR]

Polymyxin B (PMB) and polymyxin E (PME, or named colistin) are old polypeptide antibiotics and recently reintroduced as the last-line therapy for serious infections caused by multidrug-resistant Gram-negative pathogens. A rapid and reliable screening method for PMB and PME in human serum is urgently required due to their narrow therapeutic windows and individual differences. To develop fluorescence polarization immunoassay (FPIA) with high sensitivity and uniform specificity for PMB and PME, one novel fragmentary polymyxins hapten, named H1, was designed only containing the fatty acid acylated tripeptides and intendedly introducing sulfhydryl group for selective conjugation purpose. The H1 induced significantly higher titer and affinity antibody response than the PMB as hapten and produced five monoclonal antibodies (mAbs) with IC 50 of 1.8–6.6 ng/mL for PMB and cross-reactions of 75.0–105.0 % for PME. To investigate the influence of tracer structure on the FPIA sensitivity, 12 tracers differing in spacer arm lengths, fluorophore types and hapten structures were fully paired with five mAbs, showing a safety length of spacer arm ranged from 4 to 17 Å. Under optimal conditions, the IC 50 of FPIA was 3.7 ng/mL in buffer with a limit of detection of 1.4 μg/L in human serum for PMB and PME. The recoveries in spiked human serum were 73.8–91.8 % with CVs less than 12.8 % and whole assay time less than 25 min including sample preparation. These results showed that the FPIA was an efficient, accurate, and sensitive method for rapid screening of polymyxins and suitable for therapeutic drug monitoring. [Display omitted] ● One novel fragmentary hapten of polymyxins ending with sulfhydryl group was designed. ● Five mAbs with high affinity and uniform specificity to PMB and PME were produced. ● The effect of tracer structure on the sensitivity of FPIA was studied using 12 new tracers. ● A FPIA for screening of PMB and PME was developed with comparable sensitivity to ELISA. [ABSTRACT FROM AUTHOR]

Abstract: Fumonisin B1 (FB1) is a mycotoxin, mainly produced by Fusarium fungi and present in food and feed. It causes harmful effects on human and animal health. Therefore, it is necessary to develop sensitive and reliable screening methods. In this study, a highly sensitive monoclonal antibody (MAb) against FB1, clone 2D7, was produced, and the 50% inhibition concentration (IC50) of the MAb was 2.2 ng/mL in buffer. The MAb showed high cross-reactivity with fumonisin B2 (FB2), and negligible cross-reactivity with other mycotoxins. A sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this MAb was developed for the determination of FB1 in maize. In spiked samples (100, 200 and 500 μg/kg), the average recoveries ranged from 78 ± 11 to 107 ± 4%, and the coefficient of variation ranged from 3 to 15%. The limit of detection of the icELISA was 5.4 μg/kg. This method was compared to liquid chromatography tandem mass spectrometry (LC–MS/MS) using naturally contaminated samples, and the correlation coefficient was above 0.82. These results show the reliability of the icELISA method for the determination of FB1 in maize. [Copyright &y& Elsevier]

Monoclonal antibodies (mAbs) are useful biological tools for research, diagnostics, and pharmaceuticals. Here, we proposed a new mAb discovery platform named the two-step cell selection method (TCSM) for mAbs production of some small molecule haptens as antibiotic, toxins, and pesticides. The first step was performed by a fluorescence-activated cell sorter to enrich the hapten-specific B cells, the second step was an image-based precise pick of single hapten-specific hybridoma cells by confocal laser scanning microscopy. In this study, we used tetramethylenedisulfotetramine (TETS) as a model analyte, which is a highly lethal neurotoxic rodenticide. The TETS-specific hybridoma cells selection was completed within 10 days by the TCSM, compared with at least 40 days in the traditional hybridoma method (THM). The half maximal inhibitory concentration (IC 50) of the best mAb 1G6 for TETS in the TCSM was 1.98 ng mL-1, and that of mAb 2B6 in the THM was 11.49 ng mL-1. Antibody-TETS recognition also showed more interactions in mAb 1G6 than in mAb 2B6. Then, the mAb 1G6 was then successfully applied to develop an icELISA for TETS in biological samples with satisfactory sensitivity, accuracy and precision. The results demonstrated that the TCSM was a feasible and efficient method for mAb discovering of poisonous hapten molecules. [Display omitted] • A precise and efficient two-step cell selection method (TCSM) for mAb production was proposed. • A high affinity tetramethylenedisulfotetramine-specific mAb was successfully produced. • MAb has been successfully applied to an immunoassay for detecting TETS in biological samples. • The TCSM is a feasible and efficient method for rare mAb production, especially of toxic molecules. [ABSTRACT FROM AUTHOR]

• New haptens of nifuroxazide metabolite were firstly designed and synthesized. • Effect of hapten structure on affinity and specificity of mAb was studied. • High affinity monoclonal antibody to nifuroxazide metabolite was produced. • An ELISA was developed for nifuroxazide metabolite without derivatization. Derivatization is usually employed in immunoassay for detection of metabolites of nitrofurans and avoiding derivatization could be preferable to achieve an efficient screening. In the study, we designed four haptens of 4-hydroxybenhydrazide (HBH), the nifuroxazide metabolite. The effect of hapten structures on antibody affinity were evaluated and one monoclonal antibody was produced by using the Hapten C with a linear alkalane spacer arm. After optimization, an enzyme linked-immunosorbent assay (ELISA) was established with an 50% inhibition concentration of 0.25 ng mL−1 for HBH, which could ensure the direct detection of HBH without derivatization. The limit of detection of the ELISA for HBH was 0.12 µg kg−1 with the recoveries of 90.1–96.2% and coefficient of variation (CV) values lower than 9.1%. In conclusion, we produced several high affinity antibodies to HBH with new designed hapten and developed an icELISA for the direct detection of HBH without derivatization in chicken. [ABSTRACT FROM AUTHOR]

• Superior monoclonal antibody for BRD is prepared for the first time. • A novel fluorescent immunoassay is developed for diagnostic of BRD poisoning in human. • AuNCs is used as the fluorescent signal probe to enhance the sensitivity of immunoassay via the label-free IFE strategy. • The fluorescent immunoassay is applied for the BRD detection in the clinical real samples. Bromadiolone (BRD) is a widely used second-generation rodenticide and BRD poisoning in humans is relatively common. However, few rapid diagnostic methods for BRD poisoning are currently available. In this study, a highly sensitive and specific fluorescence immunoassay was developed for the rapid detection of BRD in human serum. Briefly, two novel BRD haptens were synthesized, a sensitive and specific mAb for BRD (15C1) was produced. We then incorporated mAb 15C1 as a detection reagent, and gold nanoclusters (AuNCs) as a fluorescent probe into an alkaline phosphatase-based competitive fluorescence immunoassay. The sensitivity of the immunoassay was significantly improved by introducing the fluorescence signal of the AuNCs through the facile inner-filter effect strategy. Our fluorescence immunoassay exhibited high specificity and achieved a limit of detection of 0.047 ng/mL for BRD, which was >10 times better than the corresponding standard enzyme-linked immunosorbent assay using the same immune reagents. The recoveries in spiked human serum were 77.9%–85.6% with coefficients of variation (CVs) of 3.6%–9.8%. There was a good correlation (R2 = 0.987) between the fluorescence immunoassay and ultra-performance liquid chromatography-tandem mass spectrometry. The fluorescence immunoassay could provide a valuable clinical tool for the rapid diagnosis of BRD poisoning. [ABSTRACT FROM AUTHOR]