Single-chain fragment variable produced by phage display technology: Construction, selection, mutation, expression, and recent applications in food safety.

Immunoassays are reliable, efficient, and accurate methods for the analysis of small-molecule harmful substances (such as pesticides, veterinary drugs, and biological toxins) that may be present in food. However, traditional polyclonal and monoclonal antibodies are limited by animal hosts and hinder further development of immunoassays. With the gradual application of phage display technology as an efficient in vitro selection technology, the single-chain fragment variable (scFv) now provides an exciting alternative to traditional antibodies. Efficiently constructed scFv source libraries and specifically designed biopanning schemes can now yield scFvs possessing specific recognition capabilities. A rational mutation strategy further enhances the affinity of scFv, and allows it to reach a level that cannot be achieved by immunization. Finally, appropriate prokaryotic expression measures ensure stable and efficient production of scFv. Therefore, when developing excellent scFvs, it is necessary to focus on three key aspects of this process that include screening, mutation, and expression. In this review, we analyze in detail the preparation and affinity improvement process for scFv and provide insights into the research progress and development trend of scFv-based immunoassay methods for monitoring small-molecule harmful substances.

Discovery of a potent and long-acting Xenopus GLP-1-based GLP-1/glucagon/Y2 receptor triple agonist.

The combination of incretin-based therapies and PYY analogue has shown great potential for the treatment of type 2 diabetes (T2DM) and obesity. In this study we developed the first example of a unimolecular triple agonist peptide to simultaneously target GLP-1, glucagon and Y2 receptors, aiming for superior weight loss and better glycemic control. The strategy for constructing such a unimolecular triple agonist peptide is the conjugation of the GLP-1R/GCGR dual-agonistic moiety and PYY moiety via maleimide-thiol specific reaction. A novel triple agonist peptide, 3b, was identified via stepwise structure optimization, long-acting modification and in vitro receptor screens. Peptide 3b exhibited potent and balanced GCGR and GLP-1R activities as well as potent and highly selective Y2R activity. Peptide 3b potently reduced food intake without triggering nausea associated behavior in kaolin consumption and conditioned taste aversion assays. In diet induced obesity (DIO) mice, a lower dose of 3b achieved significantly better effects on lipid metabolism, body weight, and glycemic control than higher dose of GLP-1R mono-agonist, GLP-1R/GCGR dual agonist and GLP-1R/Y2R dual agonist counterparts. Collectively, these data support the therapeutic potential of our GLP-1R/GCGR/Y2R triple agonist 3b as a novel anti-obesity and anti-diabetic agent. Targeting GLP-1R, GCGR and Y2R with unimolecular triple agonist peptide offers a route to develop new obesity and T2DM treatments.

A sensitive and specific enzyme-linked immunosorbent assay for the detection of pymetrozine in vegetable, cereal, and meat.

Pymetrozine is a neonicotinoid insecticide with high efficacy against aphids and planthoppers, and has been used worldwide. To monitor its residue in food, a highly specific and sensitive monoclonal antibody (McAb) was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to detect pymetrozine, with a 50% inhibition value (IC50) of 7.70 µg/L. The McAb showed little affinity for acetamiprid, hexazinone, metamitron, nitenpyram, metribuzin, and imidacloprid. The limits of detection (LOD) calculated from the analysis of broccoli, cabbage, wheat, maize, rice, chicken, fish, and crayfish samples were from 1.56 to 2.72 µg/kg and the average recoveries were from 81.25 to 103.19%. icELISA was confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that the optimised icELISA is a convenient and effective analytical tool for monitoring pymetrozine residues in food.

Preparation of Monoclonal Antibody against Pyrene and Benzo [a]pyrene and Development of Enzyme-Linked Immunosorbent Assay for Fish, Shrimp and Crab Samples.

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental and food pollutants that can cause cancer. In this work, a specific monoclonal antibody (mAb) to identify pyrene (PYR) and benzo [a]pyrene (BaP) was prepared, and an indirect competitive enzyme-linked immunoassay (ic-ELISA) was established to detect PYR and BaP residues in living aquatic products for the first time. The effects of complete antigens with different coupling ratios on the production of high-sensitivity mAb was explored. Under the optimal conditions, the IC50 value was 3.73 ± 0.43 µg/L (n = 5). The limits of detection (LODs) for PYR and BaP in fish, shrimp, and crab ranged from 0.43 to 0.98 µg/L. The average recoveries of the spiked samples ranged from 81.5-101.9%, and the coefficient of variation (CV) was less than 11.7%. The validation of the HPLC-FLD method indicated that the ELISA method set up in this experiment provided a trustworthy tool for PAHs residues detection in aquatic products.

The Simultaneous Detection of Multiple Antibiotics in Milk and Pork Based on an Antibody Chip Biosensor.

In the modern farming industry, the irrational or illegal use of veterinary drugs leads to residues in animal-derived food, which can seriously threaten human health. Efficient detection of low concentrations of drug residues in animal products in a short time is a key challenge for analytical methods. This study proposes to use an antibody chip biosensor for rapid and automated analysis of cephalosporins, aminoglycosides, and sulfonamide antibiotics in pork and milk. 3D polymer slides were applied for the preparation of antibody chips. Ovalbumin (OVA) or bovine serum albumin (BSA) conjugates of the haptens were immobilized as spots on disposable chips. Monoclonal antibodies (mAbs) against cefalexin, ceftiofur, gentamicin, neomycin, and sulfonamides allowed the simultaneous detection of the respective analytes. Antibody binding was detected by a second antibody labeled with Cy3-generating fluorescence, which was scanned a with chip scanner. The limits of detection (LOD) for all the analytes were far below the respective maximum residue limits (MRLs) and ranged from 0.51 to 4.3 µg/kg. The average recoveries of all the analytes in each sample were in the range of 81.6-113.6%. The intra- and inter-assay CV was less than 12.9% and showed good accuracy and precision for all the antibiotics at the MRL level. The sample pretreatment method is simple, and the results are confirmed to be accurate by LC-MS/MS; therefore, this method is valuable for the quality control of animal-derived food.

Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism.

To monitor the residue of kitasamycin (KIT), a monoclonal antibody against KIT was prepared, and a 50% inhibition concentration (IC50) of 5.7 ± 1.4 µg/L was achieved with the most sensitive antibody, KA/2A9, by optimizing ELISA conditions. The LODs for KIT in different animal tissues ranged from 22.47 µg/kg to 29.32 µg/kg, and the recoveries of the fortified tissues were 70% ~ 120% with coefficients of variation below 20%. Then, KIT-specific scFv KA/2A9/3 was prepared for the first time. Homologous modeling and molecular docking results indicated that the key amino acids of KA/2A9/3 scFv are TYR-92 (CDRL3), SER-93 (CDRL3), ASP-155 (CDRH1) and GLY-226 (CDRH3), and the hydrogen bond is the main force. And then, virtual mutation provides a method to evolve KA/2A9/3 scFv antibodies. These results contribute to comprehending the antigen-antibody binding mechanism and provide effective information for in vitro affinity maturation of anti-KIT scFv.

Preparation of monoclonal antibody and development of an enzyme-linked immunosorbent assay for the detection of ceftiofur in animal-derived foods.

Ceftiofur (CEF) residues in animal-derived foods are of great concern to farmers, regulatory agencies and consumers. In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was established to quickly monitor CEF residues in edible animal tissues using an easy sample preparation procedure. A monoclonal antibody, 4D5, against CEF has been produced at first, which had IC50 values for CEF, ceftriaxone, cefquinome, cefotaxime and desfuroylceftiofur of 0.78 µg/L, 0.73 µg/L, 13.6 µg/L, 8.99 µg/L and 8.89 µg/L, respectively. The limit of detection (LOD) and limit of quantitation (LOQ) in artificially contaminated animal-derived foods were 0.12-0.19 µg/L and 0.20-0.30 µg/L. The recovery rates were in the range of 89.7-109.0%. The CVs were less than 6.7%. A good correlation (R= 0.9994) between the ic-ELISA and UPLC-MS/MS showed the reliability of the developed ic-ELISA. The ic-ELISA produces a sensitive, accurate and low-cost tool for the screening of residues of CEF in animal-derived foods.

[Simultaneous determination of four Alternaria toxins in apple juice concentrate by ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry].

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed for the determination of altenuene (ALT), alternariol (AOH), tentoxin and alternariol monomethyl ether (AME) in apple juice concentrate (AJC). The sample was diluted with water, and then cleaned up with a PS DVB column. The quantification was carried out using an external standard method. The UPLC was performed on a BEH C18 column (50 mm x 2.1 mm, 1.7 microm) using a gradient elution of acetonitrile and water. The MS/MS was performed with multiple reaction monitoring (MRM) mode. The limits of quantification of the four Alternaria toxins were between 1.0 and 5.0 microg/L. The recoveries were 77.8%-117.2% with the relative standard deviations less than 9.7%. The method is sensitive, stable and reliable. It's suitable for the quantitative and qualitative analyses of the four Alternaria toxins in AJC.

[Determination of arbutin in apple juice concentrate by ultra performance liquid chromatography with electrospray ionization tandem mass spectrometry].

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) method was developed for the determination of arbutin in apple juice concentrate. Samples were diluted with water, then cleaned-up with a PS-DVB column. Quantitation was carried out using an external standard method. UPLC was performed on an Eclipse Plus C, column (100 mm x 2.1 mm, 1.8 microm) using a gradient solvent system (methanol-water). MS/MS was performed with multiple reaction monitoring (MRM) mode. The detection limit of arbutin was 0.02 mg/L. The method showed good linear relationship at the range of 0.04-2.0 mg/L. The recoveries ranged from 75.2% to 102.7% with relative standard deviations (RSDs) less than 8.9%. The method is simple, fast and sensitive. It's suitable for quantitative and qualitative analysis of arbutin in apple juice concentrate.