A synthetic antibiotic class with a deeply-optimized design for overcoming bacterial resistance.

The lack of new drugs that are effective against antibiotic-resistant bacteria has caused increasing concern in global public health. Based on this study, we report development of a modified antimicrobial drug through structure-based drug design (SBDD) and modular synthesis. The optimal modified compound, F8, was identified, which demonstrated in vitro and in vivo broad-spectrum antibacterial activity against drug-resistant bacteria and effectively mitigated the development of resistance. F8 exhibits significant bactericidal activity against bacteria resistant to antibiotics such as methicillin, polymyxin B, florfenicol (FLO), doxycycline, ampicillin and sulfamethoxazole. In a mouse model of drug-resistant bacteremia, F8 was found to increase survival and significantly reduce bacterial load in infected mice. Multi-omics analysis (transcriptomics, proteomics, and metabolomics) have indicated that ornithine carbamoyl transferase (arcB) is a antimicrobial target of F8. Further molecular docking, Isothermal Titration Calorimetry (ITC), and Differential Scanning Fluorimetry (DSF) studies verified arcB as a effective target for F8. Finally, mechanistic studies suggest that F8 competitively binds to arcB, disrupting the bacterial cell membrane and inducing a certain degree of oxidative damage. Here, we report F8 as a promising candidate drug for the development of antibiotic formulations to combat antibiotic-resistant bacteria-associated infections.

Antiviral activity of flavonol against porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) remains one of the major causative microorganisms of viral diarrhea in piglets worldwide, with no approved drugs for treatment. We identified a natural molecule, flavonol, which is widely found in tea, vegetables and herbs. Subsequently, the antiviral activity of compound flavonol was evaluated in Vero cells and IPEC-J2 cells, and its anti-PEDV mechanism was analyzed by molecular docking and molecular dynamics. The results showed that flavonol could effectively inhibit viral progeny production, RNA synthesis and protein expression of PEDV strains in a dose-dependent manner. When flavonol was added simultaneously with viral infection in Vero cells, it demonstrated potent anti-PEDV activity by affecting the viral attachment and internalization phases. Similarly, in IPEC-J2 cells, flavonol effectively inhibited PEDV infection at different stages of infection, except for the release phase. Moreover, flavonol mainly interacts with PEDV Mpro through hydrogen bonds and hydrophobic forces, and the complex formed by it has high stability. Importantly, flavonol also showed broad-spectrum activity against other porcine enteric coronaviruses such as TGEV and PDCoV in vitro. These findings suggest that flavonol may exert antiviral effects by interacting with viral Mpro, thereby affecting viral replication. This means that flavonol is expected to become a potential drug to prevent or treat porcine enteric coronavirus.

Preparation of high-affinity and high-specificity monoclonal antibody and development of ultra-sensitive immunoassay for the detection of 1-aminohydantoin, the metabolite of nitrofurantoin.

1-Aminohydantoin (AHD), the residual marker of nitrofurantoin, is usually detected after derivatisation using the derivatisation reagent 2-nitrobenzaldehyde. Avoiding the antibody recognition of the derivatisation reagent is essential for the accurate detection of AHD residues. In this paper, a novel hapten called hapten D was designed, and then, a monoclonal antibody that did not recognise 2-nitrobenzaldehyde was prepared based on this novel hapten. An ultra-sensitive indirect competitive enzyme linked-immunosorbent assay (icELISA) was established under optimal conditions. The 50% inhibition concentration and limit of detection of AHD were 0.056 and 0.0060 ng/mL, respectively, which improved the sensitivity by 9-37-fold compared with the previously reported icELISA methods. The average recovery rates were 88.1%-97.3%, and the coefficient of variation was <8.6%. The accuracy and reliability of the icELISA were verified using liquid chromatography-tandem mass spectrometry. These results demonstrated that the developed icELISA is a useful and reliable tool.

Rapid immunoassays for the detection of quinoxalines and their metabolites residues in animal-derived foods: A review.

Quinoxalines are a class of veterinary drugs with antibacterial and growth-promoting functions. They are often widely used to treat and prevent animal diseases and are illegally used as animal growth promoters to increase economic benefits. Quinoxalines could be easily metabolized in animals to various residue markers and remain in animal-derived foods, which would pose a serious threat to human health. Consequently, it is necessary to detect the residues of quinoxalines and their metabolites. This article reviewed and evaluated immunoassays for quinoxalines and their metabolites in animal-derived foods, mainly including enzyme-linked immunosorbent assays, fluorescence immunosorbent assays, immunochromatography, and surface plasmon resonance biosensors. In addition, we deeply explored the design of haptens for quinoxalines and their metabolites and analyzed the effect of haptens on antibody performance. This paper aims to provide guidance and references for their accurate and sensitive detection, thereby ensuring food safety and human public health.

Quantitative Determination of Aflatoxin B1 in Maize and Feed by ELISA and Time-Resolved Fluorescent Immunoassay Based on Monoclonal Antibodies.

In this study, a highly sensitive monoclonal antibody (mAb) was developed for the detection of aflatoxin B1 (AFB1) in maize and feed. Additionally, indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and time-resolved fluorescence immunoassay assay (TRFICA) were established. Firstly, the hapten AFB1-CMO was synthesized and conjugated with carrier proteins to prepare the immunogen for mouse immunization. Subsequently, mAb was generated using the classical hybridoma technique. The lowest half-maximal inhibitory concentration (IC50) of ic-ELISA was 38.6 ng/kg with a linear range of 6.25-100 ng/kg. The limits of detections (LODs) were 6.58 ng/kg and 5.54 ng/kg in maize and feed, respectively, with the recoveries ranging from 72% to 94%. The TRFICA was developed with a significantly reduced detection time of only 21 min, from sample processing to reading. Additionally, the limits of detection (LODs) for maize and feed were determined to be 62.7 ng/kg and 121 ng/kg, respectively. The linear ranges were 100-4000 ng/kg, with the recoveries ranging from 90% to 98%. In conclusion, the development of AFB1 mAb and the establishment of ic-ELISA for high-throughput sample detection, as well as TRFICA for rapid detection presented robust tools for versatile AFB1 detection in different scenarios.

Monospecific and ultrasensitive detection of ofloxacin: A computational chemistry-assisted hapten screening strategy and analysis of molecular recognition mechanism.

Contamination in food and the environment with fluoroquinolones (FQs) has become a serious threat to the global ecological balance and public health safety. Ofloxacin (OFL) is one of the most widely utilized sterilization agents in FQs. In the process of monitoring OFL, broad-spectrum monoclonal antibodies (mAb) cannot meet the demand for monospecific detection. Here, a computational chemistry-assisted hapten screening strategy was proposed in this study. Differences in the properties of antigenic epitopes were precisely extracted through a comprehensive comparative study of 16 common FQs molecules and a monospecific and ultrasensitive mAb-3B4 for OFL was successfully prepared. The screened fleroxacin (FLE) hapten was applied in a heterologous competition strategy resulting in a 20-fold improvement in the half inhibitory concentration (IC50) of mAb-3B4 to 0.0375 µg L-1 and cross-reacted only with marbofloxacin (MAR) in regulated FQs. In addition, a single-chain variable fragment (scFv) for OFL was constructed for the first time with an IC50 of 0.378 µg L-1. Molecular recognition mechanism studies validated the reliability of this strategy and revealed the key amino acid sites responsible for OFL specificity and sensitivity. Finally, ic-ELISA and GICA were established for OFL in real samples. This work provides new ideas for the preparation of monospecific mAb and improves the monitoring system of FQs.

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.

Broad-spectrum portable magnetic relaxation switching immunosensor with gold-functionalized magnetic nanoprobes for the sensitive detection of multiple pyrethroids.

At present, the most available pyrethroid (PYR) detection methods still suffer from a narrow detection spectrum, low sensitivity, and less portability. Herein, a novel magnetic relaxation switching (MRS) sensor was elaboratively designed to detect multiple PYRs, combining a novel broad-spectrum antibody CL-CN/1D2 and synthesized immune gold-functionalized magnetic nanoparticles, with the inherent response of the sensor. A series of antibodies and the immune gold-functionalized magnetic nanoparticles were designed and synthesized. The broad-spectrum antibody CL-CN/1D2 and high-performance gold-functionalized magnetic nanoprobe were further selected. The target analytes were effectively captured by the gold-functionalized magnetic nanoparticles in 20% (v/v) ethanol, resulting in the number increase of the signaling probes in the supernatant after magnetic separation. This sensor can detect multiple PYRs with a detection limit of 2.72 µg/L for cypermethrin, 3.58 µg/L for ß-cypermethrin, 4.07 µg/L for cyfluthrin, 3.66 µg/L for λ-cyhalothrin, 4.42 µg/L for ß-cyhalothrin, 3.51 µg/L for fenpropathrin, 4.41 µg/L for fenvalerate, and 4.12 µg/L for deltamethrin in lake water and milk within 35 min. This study not only achieves broad-spectrum PYRs detection at a trace amount but also provides an effective and universal strategy for enhancing the sensitivity and stability of the portable MRS sensor when detecting hydrophobic analytes in the environment.

Highly Sensitive Magnetic-Nanoparticle-Based Immunochromatography Assay for Rapid Detection of Amantadine in Chicken and Eggs.

Amantadine (AMD) is an antiviral drug that is prohibited for use in livestock and poultry. In this study, carboxyl-modified magnetic nanoparticles (MNPs) were synthesized using the solvothermal method in one step with harmless and inexpensive regents, and they were used to label monoclonal antibodies (mAbs) of AMD in microwells with electrostatic adsorption. Then, a magnetic immunochromatography assay (MICA) method was successfully established. Under optimal conditions, the MICA showed a good performance, with a linear range of 0.2~10.0 µg/L. The limit of detection (LOD) was 0.068 µg/L with the instrument, and the visual LOD (vLOD) was 0.5 µg/L. There was no cross-reaction with rimantadine and ribavirin. The vLOD in real samples was 1.0 µg/kg. The developed MICA has the advantages of convenience, speed, and sensitivity, which make it suitable for the on-site rapid detection of AMD residues in chicken tissues and eggs.

A Sensitive and Specific Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Pretilachlor in Grains and the Environment.

Pretilachlor is a chloroacetamide herbicide mainly used for weed and broadleaf weed control in rice, that is widely utilized in China. In order to detect the residue of pretilachlor in the environment and food, a highly sensitive and specific monoclonal antibody (mAb) against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47 ± 2.35 µg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 µg/L was developed. The specificity of the antibody was explained by computer simulations and experimental validation. The mAb exhibited negligible cross-reactivity towards alachlor, acetochlor, propisochlor, butachlor, and metalaxyl, and the limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 µg/L. The recoveries of all samples were 78.3~91.3%. The reliability of the ic-ELISA method for residue detection of pretilachlor in the environment and grains was confirmed using high performance liquid chromatography.

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.

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.

Study of the effect of intestinal immunity in neonatal piglets coinfected with porcine deltacoronavirus and porcine epidemic diarrhea virus.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhea virus (PEDV) have often been detected simultaneously in piglets with coronavirus diarrhea. However, the intestinal immune response to the interaction between circulating PDCoV and PEDV is unknown. Therefore, this study was conducted to investigate the intestinal immunity of neonatal piglets that were exposed first to PDCoV and then to PEDV. The amounts and distribution of CD3+ T lymphocytes, B lymphocytes, and goblet cells (GCs) in the small intestine were analyzed by immunohistochemistry and periodic acid-Schiff staining, respectively. The expression levels of pattern recognition receptors and downstream mediator cytokines were analyzed by qPCR and ELISA. The results showed that the numbers of GCs, CD3+ T lymphocytes, and B lymphocytes in the duodenum and jejunum of the PDCoV + PEDV coinoculated piglets were increased compared with those of piglets inoculated with PEDV alone. The piglets in the PDCoV + PEDV group had significantly upregulated IFN-α and IFN-λ1 compared with the PEDV single-inoculated piglets. These results suggest that PDCoV + PEDV-coinfected piglets can activate intestinal antiviral immunity more strongly than piglets infected with PEDV alone, which provides new insight into the pathogenesis mechanism of swine enteric coronavirus coinfection that may be used for vaccination in the future.

PK-PD Modeling and Optimal Dosing Regimen of Acetylkitasamycin against Streptococcus suis in Piglets.

Streptococcus suis (S. suis) causes severe respiratory diseases in pigs and is also an important pathogen causing hidden dangers to public health and safety. Acetylkitasamycin is a new macrolide agent that has shown good activity to Gram-positive cocci such as Streptococcus. The purpose of this study was to perform pharmacokinetic-pharmacodynamic (PK-PD) modeling to formulate a dosing regimen of acetylkitasamycin for treatment of S. suis and to decrease the emergence of acetylkitasamycin-resistant S. suis. The minimal inhibitory concentration (MIC) of 110 S. suis isolates was determined by broth micro dilution method. The MIC50 of the 55 sensitive S. suis isolates was 1.21 µg/mL. The strain HB1607 with MIC close to MIC50 and high pathogenicity was used for the PK-PD experiments. The MIC and MBC of HB1607 in both MH broth and pulmonary epithelial lining fluid (PELF) was 1 and 2 µg/mL, respectively. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to determine the concentration change of acetylkitasamycin in piglet plasma and PELF after intragastric administration of a single dose of 50 mg/kg b.w. acetylkitasamycin. The PK parameters were calculated by WinNolin software. The PK data showed that the maximum concentration (Cmax), peak time (Tmax), and area under the concentration-time curve (AUC) were 9.84 ± 0.39 µg/mL, 4.27 ± 0.19 h and 248.58 ± 21.17 h·µg/mL, respectively. Integration of the in vivo PK data and ex vivo PD data, an inhibition sigmoid Emax equation was established. The dosing regimen of acetylkitasamycin for the treatment S. suis infection established as 33.12 mg/kg b.w. every 12 h for 3 days. This study provided a reasonable dosing regimen for a new drug used in clinical treatment, which can effectively be used to treat S. suis infection and slow down the generation of drug resistance.

Clinical Breakpoint of Apramycin to Swine Salmonella and Its Effect on Ileum Flora.

The purpose of this study was to establish the clinical breakpoint (CBP) of apramycin (APR) against Salmonella in swine and evaluate its effect on intestinal microbiota. The CBP was established based on three cutoff values of wild-type cutoff value (COWT), pharmacokinetic-pharmadynamic (PK/PD) cutoff value (COPD) and clinical cutoff value (COCL). The effect of the optimized dose regimen based on ex vivo PK/PD study. The evolution of the ileum flora was determined by the 16rRNA gene sequencing and bioinformatics. This study firstly established the COWT, COPD in ileum, and COCL of APR against swine Salmonella, the value of these cutoffs were 32 µg/mL, 32 µg/mL and 8 µg/mL, respectively. According to the guiding principle of the Clinical Laboratory Standards Institute (CLSI), the final CBP in ileum was 32 µg/mL. Our results revealed the main evolution route in the composition of ileum microbiota of diarrheic piglets treated by APR. The change of the abundances of Bacteroidetes and Euryarchaeota was the most obvious during the evolution process. Methanobrevibacter, Prevotella, S24-7 and Ruminococcaceae were obtained as the highest abundance genus. The abundance of Methanobrevibacter increased significantly when APR treatment carried and decreased in cure and withdrawal period groups. The abundance of Prevotella in the tested groups was significantly lower than that in the healthy group. A decreased of abundance in S24-7 was observed after Salmonella infection and increased slightly after cure. Ruminococcaceae increased significantly after Salmonella infection and decreased significantly after APR treatment. In addition, the genera of Methanobrevibacter and Prevotella were defined as the key node. Valine, leucine and isoleucine biosynthesis, D-Glutamine and D-glutamate metabolism, D-Alanine metabolism, Peptidoglycan and amino acids biosynthesis were the top five Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the ileum microbiota of piglets during the Salmonella infection and APR treatment process. Our study extended the understanding of dynamic shift of gut microbes during diarrheic piglets treated by APR.

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.

A Novel Metabolite as a Hapten to Prepare Monoclonal Antibodies for Rapid Screening of Quinoxaline Drug Residues.

Quinoxalines (Qx) are chemically synthesized antibacterial drugs with strong antibacterial and growth-promoting effects. Qx is heavily abused by farmers, resulting in large residues in animal-derived foods, which pose a serious threat to human health. Desoxyquinoxalines (DQx), which have the highest residue levels, have been identified as the major toxicant and have become a new generation of residue markers. In this study, we prepared monoclonal antibodies (mAb) based on a new generation metabolite (desoxymequindox, DMEQ) and establish an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the rapid determination of Qx residues in food. The mAb exhibited high sensitivity with half maximal inhibitory concentration (IC50) and a linear range of 2.84 µg/L and 0.8-12.8 µg/L, respectively. Additionally, the cross-reactivity (CR) of the mAb showed that it recognized multiple DQx to varying levels. The limits of detection (LOD), limits of quantification (LOQ), and recoveries for the ic-ELISA assay of pork, swine liver, swine kidney, chicken, and chicken liver were 0.48-0.58 µg/kg, 0.61-0.90 µg/kg, and 73.7-107.8%, respectively, and the coefficients of variation (CV) were less than 11%. The results of the ic-ELISA showed a good correlation with LC-MS/MS in animal-derived foods. This suggests that this analytical method can be used for the rapid screening of QX residues.

Metabolic Disposition and Elimination of Tritum-Labeled Sulfamethoxazole in Pigs, Chickens and Rats.

Sulfamethoxazole (SMZ), as a sulfa antibiotic, is often used in the treatment of various infectious diseases in animal husbandry. At present, SMZ still has many unresolved problems in the material balance, metabolic pathways, and residual target tissues in food animals. Therefore, in order to solve these problems, the metabolism, distribution, and elimination of SMZ is investigated in pigs, chickens, and rats by radioactive tracing methods, and the residue marker and target tissue of SMZ in food animals were determined, providing a reliable basis for food safety. After a single administration of [3H]-SMZ (rats and pigs by intramuscular injection and chickens by oral gavage), the total radioactivity was rapidly excreted, with more than 93% of the dose excreted within 14 days in the three species. Pigs and rats had more than 75% of the administered volume recovered by urine. After 7 days of continuous administration, within the first 6 h, radioactivity was found in almost all tissues. The highest radioactivity and longest persistence in pigs was in the liver, while in chickens it was in the liver and kidneys, most of which was removed within 14 days. A total of six, three and three metabolites were found in chickens, rats and pigs, respectively. N4-acetyl-sulfamethoxazole (S1) was the main metabolite of SMZ in rats, pigs and chickens. The radioactive substance with the longest elimination half-life is sulfamethoxazole (S0), so S0 was suggested to be the marker residue in pigs and chickens.

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.

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.