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The preparation of high specificity and affinity antibodies is challenging due to limited information on characteristic groups of haptens in traditional design strategy. In this study, we first predicted characteristic groups of flurogestone acetate (FGA) using quantitative analysis of molecular surface combined with atomic charge distribution. Subsequently, FGA haptens were rationally designed to expose these identified characteristic groups fully. As a result, seven monoclonal antibodies were obtained with satisfactory performance, exhibiting IC 50 values from 0.17 to 0.45 μg/L and negligible cross-reactivities below 1% to other 18 hormones. The antibody recognition mechanism further confirmed hydrogen bonds and hydrophobic interactions involving predicted FGA characteristic groups and specific amino acids in the antibodies contributed to their high specificity and affinity. Finally, one selective and sensitive ic-ELISA was developed for FGA determination with a detection limit as low as 0.12 μg/L, providing an efficient tool for timely monitoring of FGA in goat milk samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC 50 values for 23 SAs below 100 μg L -1 and 5 ASGs below 50 μg L -1 . The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.

Multiplex lateral flow immunoassay (mLFIA) has attracted great attention due to the increasing need for rapid detection of multiple analytes. However, it has a number of disadvantages with regard to accuracy and interference because of difficulties in simplifying the process of preparing nanomaterial-based probes. In this work, inspired by protein self-assembly, for the first time, a facile natural antibody network (NAN)-based mLFIA for multiple chloramphenicol (CAP) and streptomycin (STR) determination was designed. The NAN structure was constructed by introducing a second antibody (Ab 2 ) as a scaffold to noncovalently combine with various monoclonal antibodies (mAbs), thus permitting each mAb to act as an independent functional unit to maintain bioactivity. Furthermore, the NAN was colored by simple one-step staining using coomassie brilliant blue R-250 (CBBR) to form a chromogenic probe, eliminating the need for complex nanomaterials to improve reproducibility and precision. Under optimal conditions, a satisfactory detection performance (the visual limit of detection (v-LOD) of 3 ng mL -1 for CAP and 20 ng mL -1 for STR) was obtained for whole milk analysis, which met the basic requirement of detection and had good specificity, reproducibility (relative standard deviation (RSD) < 15%), and robustness. In addition, the precision of the detection results was improved usefully since the test procedure was simplified. Overall, the developed system enables fast, simple, and reliable point-of-care assays of multiple analytes.

A biosensor that could simultaneously detect multi-analyte as many as in a single test is more favored when facing lots of samples for screening purpose. In such a biosensor, the recognition element with broad specificity and highly affinity is a key reagent for detection spectrum. Here we report a dihydropteroate synthase (DHPS) based biosensor for detecting 29 sulfonamides (SAs) in milk. The biosensor was established in a competitive format where HRP-labeled tracer and free SAs competitively binding to the limited amount of DHPS-DHPPP (6-hydroxymethyl-7,8-dihydropterin diphosphate) binary complex. Compared to other biosensors based on antibodies with the same objective, the current sensor employing DHPS provided not only highly sensitivity but also preponderant uniform affinities for all individual sulfonamide, which has never been achieved before in any immunosensor. However, the difference of recognition mechanism between DHPS and antibody remains unknown. To address this question, we deconstructed the variable region of two monoclonal antibodies produced by our group and then compared the intermolecular forces and key contacting amino acid residues of both DHPS and antibodies to several typical SAs with help of molecular modeling analysis. Results showed the orientation of the ligands in the binding site played a significant role during the recognition with proteins. The optimized assay provided a limit of detection (LOD) of 1.15-14.91 ng mL -1 and dynamic range was ranging from 1.54 to 126.85 ng mL -1 for 29 SAs. The developed biosensor finally was validated for five SAs in spiked milk with recovery values ranging from 72.7 to 129.3% and coefficients of variation less than 16.0%. The study showed that the biosensor based on DHPS make it a suitable screening method for the simultaneous determination of total SAs residues in food matrices., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The development of multianalyte immunoassays with an emphasis on food safety has attracted increasing interest, due to its high target throughput, short detection time, reduced sample consumption, and low overall cost. In this study, a superior polyclonal antibody (pAb) against sulfonamides (SAs) was raised by using a bioconjugate of bovine serum albumin with a rationally designed hapten 4-[(4-aminophenyl) sulfonyl-amino]-2-methoxybenzoic acid (SA10-X). The results showed that the pAb could recognize 19 SAs with 50% inhibition (IC 50 ) below 100 µg L -1 and a recognition profile for SAs containing, either a five-atom ring or a six-atom ring, with highly uniform affinity. A three-dimensional quantitative structure-activity relationship analysis indicated that the electrostatic features of SAs play a considerably important role, during recognition with pAb than stereochemical effects. Skimmed milk samples were directly diluted five times before analysis. After optimization, the limit of detection for sulfamonomethoxine, sulfamethoxazole, sulfaquinoxaline, sulfadimethoxine, and sulfamethazine were 1.00, 1.25, 2.95, 3.35, and 6.10 µg L -1 , respectively. The average recoveries for these 5 SAs were 72.0⁻107.5% with coefficients of variation less than 14.1%. The established method, based on pAb, with broad specificity and uniform affinity, offered a simple, sensitive, and high-throughput screening tool for the detection of multi-SAs in milk samples., Competing Interests: The authors declare no conflict of interest.

An antibody with broad specificity and principally depending on hapten structure and size is a key reagent for developing a class-selective immunoassay. In the present study, three new generic haptens of antibacterial synergists (ASGs) were proposed using trimethoprim as the starting molecule. These haptens contained carboxyl groups on the meta position of trimethoxybenzene for conjugating to protein, while, the common moiety of ASGs, i.e., diaminopyrimidine, was intentionally and maximally exposed to the immune system in animals in order to induce antibodies with broad specificity against ASGs. Five monoclonal antibodies (mAbs) were finally obtained, and 5C4 from the hapten with a short spacer arm, named Hapten A, showed not only uniform broad specificity but also high affinity to all five ASGs. We further determined the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. An indirect competitive ELISA (icELISA)-based 5C4 was established and exhibited IC 50 values of 0.067-0.139 μg L -1 with cross-reactivity of 48.2%-418.7% for the five ASGs in buffer under optimal conditions. The calculated limits of detection of the icELISA for chicken and milk were 0.06-0.8 μg kg -1 and 0.05-0.6 μg L -1 , respectively. The recoveries in spiked chicken and milk samples were 75.2%-101.4% with a coefficient of variation less than 14.3%. In summary, we have developed, for the first time, a rapid and reliable icELISA for ASGs with significantly improved sensitivity and class selectivity.

The recognition spectrum of immunoassays developed on the basis of class-specific antibodies can include the several nearest analytes but rarely all of the desired representatives of the group. The situation may be sufficiently improved using a hybrid assay combining two antibodies with specificities that complement each other. Two monoclonal antibodies (mAb) with broad but different specificities toward sulfonamides were examined for their binding to a panel of hapten conjugates. mAb-hapten pairs without mutual cross-reactions were identified, and classical direct antigen-coated and mAb-coated ELISAs were developed as formats with referent specificities. Both interactions were combined in a single hybrid assay, which was designed as a one-step double-competitive sandwich-ELISA. For this assay, the intermediate bifunctional reagent mAb(1)-hapten(2) conjugate was synthesized and able to simultaneously bind to hapten(1) and be bound by mAb(2). Formation of a two-mAbs sandwich complex was inhibited by competitors of interaction(1) as well as by competitors of interaction(2). Thus, due to the summation effect, simultaneous determination of analytes recognized by both mAbs was achieved. The hybrid assay can be performed in two reversed arrangements using a coating antigen or coating antibody, the characteristics of which were compared and found to be similar in sensitivity and extended specificity. The suitability of the developed test for the determination of 14 sulfonamides at their maximum residue limit (MRL) concentration was demonstrated using the examples of turkey muscle and milk samples.

The enzyme-linked immunosorbent assay (ELISA) has become the most important and widely used rapid detection technology for food safety because of its simple operation, fast speed and high sensitivity. Multiplex synchronous detection is the goal of ELISA that is always pursuing for. However, the reported multiplex ELISAs have not truly realized synchronous detection because of the complex signal generation and collection procedures. Here, we developed a dual-luciferases competitive direct bioluminescent immunoassay (DBL-cdELISA) with only one substrate addition step followed immediately by simultaneous signal acquisition. It is the first report of simultaneous multiplex analysis of small molecules based on microtiter plates and enzymes without any additional steps. The IC 50 values for norfloxacin (NOR) and sulfamethazine (SMZ) were 0.051 ng mL -1 and 0.211 ng mL -1 , respectively. The results demonstrated that the application of different luciferases and substrates simplified the signal generation and collection procedures and enabled simultaneous detection of small molecules with a simple procedure, high throughput and fast speed, that will be of great significance for the development of multiple assays., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS) and a fluorescence probe for multi-sulphonamide detection. Dihydropteridine pyrophosphate (DHPPP) was synthesised and acts as the first substrate for DHPS. Under optimised conditions, the half-maximal inhibitory concentrations (IC50) of the assay were less than 100 ng mL(-1) for at least 29 sulphonamides and the time needed for the detection was less than 20 min. More importantly, the assay revealed quite uniform affinities for all of the individual sulphonamides tested, which has never before been achieved in an antibody-based assay., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this paper, a rapid and sensitive multiplex flow-through immunoaffinity chromatography test (FTIACT) was developed for the on-site screening of 14 sulfonamide and 13 quinolone residues in milk. The developed FTIACT method combines the purification, preconcentration and immunochemical detection of multiple antibiotics on the sepharose gel test layers. The use of liposome-encapsulated quantum dots (LQDs) with the FTIACT method exhibited the best results, with limits of detection (LODs) of 1 and 0.5ng/mL for the sulfonamides (SAs) and quinolones (QNs), respectively, through qualitative analysis (visual detection by the naked eye). In order to achieve low detection limit, the color intensity of the images were converted into relative optical density values to enable a quantitative evaluation. Quantitative analysis of the samples enabled the detection of SAs (0.13ng/mL) and QNs (0.062ng/mL) in spiked milk samples. The FTIACT described in this work shows promise as a multiplex immunoassay for the qualitative and quantitative screening of multiple chemical residues in milk., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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 IC50 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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The residue of lincomycin (LIN) in edible animal foodstuffs caused by the widespread use of veterinary drugs is in need of rapid, simple, and sensitive detection methods. The present work introduces a fluorescent microsphere immunoassay (FMIA) for detecting LIN in different samples based on the competitive immunoreaction on the chromatography test strip. The residues of LIN in different samples compete with bovine serum albumin (BSA) labeled LIN conjugates on the T-line to bind to the anti-LIN monoclonal antibody labeled fluorescent microspheres (FM-mAbs). Captured FM-mAbs on the T-line represent the fluorescent intensity, which is detected under UV light and quantified by a fluorescent reader. Under optimized conditions, the dynamic range is from 1.35 to 3.57 ng/mL, and the 50% inhibition concentration (IC50) is 2.20 ng/mL. This method has 4.4% cross-reactivity with clindamycin and negligible cross-reactivity (<0.1%) with other analogues. To reduce the matrix effects, a dilution method is used to pretreat the samples, and the recoveries range from 73.92 to 120.50% with coefficient of variations <21.76%. In comparison with the results of ELISA and colloidal gold immunoassay, FMIA has obvious advantages such as easy operation, time savings, high sensitivity and specificity, and broader prospect.

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented., (Copyright © 2013 John Wiley & Sons, Ltd.)

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 μg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 μg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

The soluble form of penicillin-binding protein 3 (sPBP3(∗)) from Streptococcus pneumoniae was expressed in Escherichia coli as a six-histidine fusion protein. The protein was purified and used to develop a microplate assay in direct competitive format for the detection of penicillins and cephalosporins in milk. The assay was based on competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-Amp) to the sPBP3(∗) by free β-lactam antibiotics in milk. Under optimized conditions, most of the β-lactam antibiotics (11 penicillins and 16 cephalosporins) could be detected at concentrations corresponding to the maximum residue limits (MRLs) set by the European Union. Analysis of spiked milk samples showed that acceptable recoveries ranged from 74.06 to 106.31% in skimmed milk and from 63.97 to 107.26% in whole milk, with coefficients of variation (CVs) less than 16%. With the high sensitivity and wide-range affinities to penicillins and cephalosporins, the developed assay based on sPBP3(∗) exhibited the potential to be a screening assay for fast detection of β-lactam antibiotics in milk., (Copyright © 2013 Elsevier Inc. All rights reserved.)

This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.

The fluorescent microsphere has been increasingly used as detecting label in immunoassay because of its stable configuration, high fluorescence intensity, and photostability. In this paper, we developed a novel lateral flow fluorescent microsphere immunoassay (FMIA) for the determination of sulfamethazine (SMZ) in milk in a quantitative manner with high sensitivity, selectivity, and rapidity. A monoclonal antibody to SMZ was covalently conjugated with the carboxylate-modified fluorescent microsphere, which is polystyrene with a diameter of 200 nm. Quantitative detection of SMZ in milk was accomplished by recording the fluorescence intensity of microspheres captured on the test line after the milk samples were diluted five times. Under optimal conditions, the FMIA displays a rapid response for SMZ with a limit of detection of as low as 0.025 ng mL(-1) in buffer and 0.11 μg L(-1) in milk samples. The FMIA was then successfully applied on spiked milk samples and the recoveries ranged from 101.1 to 113.6% in the inter-batch assay with coefficient of variations of 6.0 to 14.3%. We demonstrate here that the fluorescent microsphere-based lateral flow immunoassay (LFIA) is capable of rapid, sensitive, and quantitative detection of SMZ in milk.

A competitive, direct, chemiluminescent immunoassay based on a magnetic beads (MBs) separation and gold nanoparticles (AuNPs) labelling technique to detect chloramphenicol (CAP) has been developed. Horseradish peroxidase (HRP)-labelled anti-CAP monoclonal antibody conjugated with AuNPs and antigen-immobilized MBs were prepared. After optimization parameters of immunocomplex MBs, the IC50 values of chemiluminescence magnetic nanoparticles immunoassay (CL-MBs-nano-immunoassay) were 0.017 µg L(-1) for extract method I and 0.17 µg L(-1) for extract method II. The immunoassay with two extract methods was applied to detect CAP in milk. Comparison of these two extract methods showed that extract method I was advantageous in better sensitivity, in which the sensitivity was 10 times compared to that of extract method II, while extract method II was superior in simple operation, suitable for high throughout screen. The recoveries were 86.7-98.0% (extract method I) and 80.0-103.0% (extract method II), and the coefficients of variation (CVs) were all <15%. The satisfactory recovery with both extract methods and high correlation with traditional ELISA kit in milk system confirmed that the immunomagnetic assay based on AuNPs exhibited promising potential in rapid field screening for trace CAP analysis., (Copyright © 2013 John Wiley & Sons, Ltd.)

In this research, a mixed immunoassay design for multiple chemical residues detection based on combined reverse competitive enzyme-linked immunosorbent assay (ELISA) procedure was developed. This method integrated two reverse ELISA reactions in one assay by labeling horseradish peroxidase to deoxynivalenol (DON) and orbifloxacin. Within this method, IC50 of the two mAbs for each analyte we produced ranged from 23~68 ng mL(-1) for DONs and 4.1~49 ng mL(-1) for quinolones (QNs). The limit of detection measured by IC10 was achieved at 0.45-1.3 ng mL(-1) for DONs and 0.59-6.9 ng mL(-1) for QNs, which was lower than the maximum residue levels. Recoveries in negative samples spiked at concentrations of 100, 200, and 500 ng mL(-1) ranged from 91.3 to 102.2 % for DONs and 88.7-98.05 % for QNs with relative standard deviation less than 9.88 and 12.67 %. The results demonstrated that this developed immunoassay was suitable for screening of low molecular weight contaminants.

Due to the widespread use and potential toxicity of avermectins (AVMs), multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab) was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC₅₀ values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCβ) of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV) were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL(-1)), sulfonamides (0.17 ng mL(-1)) and melamine (7.5 ng mL(-1)), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot-MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen-antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC50) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10-100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1-9.2%.

Erythromycin (ERY) is one of the most common macrolides applied in veterinary medicine to treat diseases or as a feed additive for animal growth promotion. Long-term irrational use of ERY could lead to residues in animal-derived food and the emergence of drug-resistant strains, posing potential threats to human health. In this study, a highly sensitive, specific, robust, and rapid fluorescence polarization immunoassay (FPIA) for the determination of ERY in milk has been described. Herein, to achieve high sensitivity, five tracers of ERY with different fluorescein structures were synthesized and paired with three monoclonal antibodies (mAbs). Under the optimized conditions, the combination of mAb 5B2 and tracer ERM-FITC achieved the lowest IC50 value in the FPIA with 7.39 μg/L for ERM. The established FPIA was used to detect ERY in milk, revealing a limit of detection (LOD) of 14.08 μg/L with recoveries of 96.08–107.77% and coefficients of variations (CVs) of 3.41–10.97%. The total detection time of the developed FPIA was less than 5 min from the addition of samples to the result readout. All the above results showed that the proposed FPIA in this study was a rapid, accurate, and simple method for the screening of ERY in milk samples. [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]

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]

A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (icFLISA) method was developed for detection of melamine in milk based on secondary antibody (Ab2)-conjugated quantum dots (QDs). The sensitivity of icFLISA with a limit of detection (LOD) of melamine (3.88 ng mL−1) and 50% inhibition value (IC50) (31.58 ng mL−1), was much higher than that of icELISA. As a proof of principle, recovery tests involving four fortification levels (150, 80, 40, and 20 ngmL−1) in blank samples were also performed. The recoveries ranged from 80.85 to 110.54% with coefficient of variations (CVs) of 2.82–8.82%. When challenged with authentic samples, these results of icFLISA were consistent with that of icELISA, immuno-chromatographic assay and gas chromatography-single quadruple mass spectrometry (GC-MS)method, indicating that icFLISA can be considered as a new screening method for detection of melamine in milk. [ABSTRACT FROM PUBLISHER]

Zeranol (α-Zearalanol, α-ZAL), well-known as an anabolic promoter, was officially banned in Europe due to its potential carcinogenic and endocrine-disrupting biological activities. In this study, a method for the determination of zeranol residues in bovine milk and urine was developed based on micro-plate chemiluminescence enzyme immunoassay (CLEIA). The limit of detection (LOD) was 50 ng/L, and the linear range was between 100 ng/L and 4510 ng/L, with a correlation coefficient (R2)0.9982. The recoveries of zeranol in bovine milk and urine were between 84.7% and 123.6%. This study showed that CLEIA was a reliable, convenient, and sensitive method for screening zeranol residues in bovine milk and urine. [ABSTRACT FROM AUTHOR]

A competitive indirect chemiluminescent enzyme-linked immunoassay (CL-ELISA) for chloramphenicol (CAP) residues in milk and chicken muscle has been developed. Due to the unique characteristic of the polyclonal antibody, special reaction system and modified extract method, then after optimization (concentration of Tween-20, concentration of PB and pH, incubation time, and temperature), the method gave a detection limit of 0.92 ng/L and a detection range of 3.16–3035 ng/L, with the IC50 of 17.29 ng/L in optimum condition and real sample matrix. When CAP was spiked in milk and chicken muscle at levels of 5–100 ng/L, recoveries ranged from 104.9%–114.8% and 101.0%–118.8%, with coefficients of variation of 3.0%–14.6% and 9.5%–14.4%, respectively. In an actual chicken muscle residue study, although the extract of samples diluted 10-fold, or even 100-fold, which represents extremely lower concentration of CAP, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The developed method is therefore suitable for screening of ultratrace CAP residues in milk and chicken muscle samples. [ABSTRACT FROM AUTHOR]

Considering processing concerns regarding emerging food safety incidents and large-scale food testing, efficient and straightforward screening tools are urgently needed. The requirements for this technology include effective sample pretreatment, sensitive assay, and sample-to-answer solution that enable the detection of trace amounts of contaminants in complex food matrices. Here, we developed a chemiluminescence- and optical fiber-based fully automated immunosensing (COFFAI) system that conducts chemical contaminant food screening in a truly "sample-to-result" manner. Instead of utilizing laborious benchtop sample purification techniques followed by a time-consuming manual ELISA procedure, the user simply injects the sample of interest, and the whole procedure is automatically processed. We demonstrated that the utility of our COFFAI system can be used to detect trace amounts of chemical contamination in various milk samples within approximately 75 min. This method showed an ultrahigh sensitivity with a quinolone detection limit in the 0.022–0.065 µg/L range for skim milk, whole milk, and raw milk samples. The integration of pretreatment and assay into one automated system is an effective method for food testing. We believe that this platform provides a general method for efficient contaminant screening in foodstuffs. [Display omitted] • We designed a fully automated system that enables "sample-to-result" contaminant screening. • A novel immunoaffinity-based sample pretreatment method that does not require the use of an organic solvent was designed. • An optical fiber and charge-coupled device sensor was used for highly sensitive quantification. [ABSTRACT FROM AUTHOR]