Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Development of an enzyme linked immunosorbent assay for the determination of phenothiazine drugs in meat and animal feeds.

In this study, 2-chlorophenothiazine was used to synthesize a hapten for production of monoclonal antibody. The obtained monoclonal antibody showed high crossreactivities to chlorpromazine, promethazine and perphenazine, and showed low crossreactivities to acepromazine and fluphenazine. After evaluation of three coating antigens, a heterologous competitive indirect enzyme linked immunosorbent assay was developed to determine the five phenothiazines in animal feeds and the residues of chlorpromazine, promethazine and perphenazine in meat. The crossreactivities to the five analytes were in a range of 2.4%-98%. The limits of detection for the five drugs in feeds were in a range of 0.1-3.0 µg g-1, and that for chlorpromazine, promethazine and perphenazine in meat were in a range of 0.5-0.8 ng g-1. Their recoveries from standards fortified blank samples (chicken, pork and feeds) were in a range of 74.1%-96.5% with coefficients of variation of 6.4%-15.1%. Therefore, this method could be used as a rapid screen tool to determine phenothiazine drugs in animal feeds and animal derived foods.

Effect of dietary nonphytate phosphorus content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of laying hens that were or were not orally inoculated with Salmonella Typhimurium.

OBJECTIVE: To evaluate the effects of dietary nonphytate phosphorus (NPP) content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of hens that were or were not inoculated with Salmonella Typhimurium. ANIMALS: 64 Salmonella-free hens. PROCEDURES: Hens were fed a diet with 0.22% (control; n = 32) or 0.42% (high-P; 32) NPP for 6 weeks and then orally inoculated with S Typhimurium (5 × 10(7) CFUs) or PBSS. Tissues were obtained from 8 S Typhimurium-inoculated and 8 PBSS-inoculated hens from each group at 2 and 7 days postinoculation (DPI). Percentages of ileal CD4+ and CD8+ lymphocytes were determined by flow cytometry. Cytokine mRNA expression was determined by quantitative real-time PCR assays. RESULTS: For S Typhimurium-inoculated hens, plasma parathyroid hormone concentration was significantly increased and 1,25-dihydroxyvitamin D3 concentration was decreased in hens fed the high-P diet, compared with values in hens fed the control diet. Salmonella Typhimurium inoculation caused an increase in the percentage of ileal CD8+ lymphocytes and the expression of interleukin (IL)-1ß, IL-6, IL-8, interferon-γ, IL-12, and IL-18 in the cecal tonsils and spleen and a decrease in the expression of IL-4 and IL-10 in the cecal tonsils. Hens fed the high-P diet had significantly increased splenic expression of interferon-γ at 2 DPI and IL-1ß, IL-6, IL-12, and IL-18 at 7 DPI, compared with hens fed the control diet. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested there was a T-helper 1 cytokine reaction in the cecal tonsils and spleen of S Typhimurium-inoculated hens, and dietary NPP content altered calcium regulation hormone concentrations and affected splenic cytokine expression.

Immunoassay of red dyes based on the monoclonal antibody of ß-naphthol.

The objective of the present study was to develop a multi-analyte immunoassay for the determination of eight red dyes in food samples. Two dye intermediates (2-hydroxy-1-naphthoic acid and 1-amino-2-naphthol) were used as the haptens to produce the monoclonal antibodies. The obtained monoclonal antibodies recognized Sudan 1-4, Para red, Sudan red G, Sudan red B and Acid orange II simultaneously. After evaluation of different antibody/coating antigen combinations, a heterologous indirect competitive enzyme linked immunosorbent assay was developed to determine the eight red dyes in food samples (chili oil, chili powder, tomato sauce, hotpot seasoning). The crossreactivities to the eight analytes were in the range of 61%-79% (with ß-naphthol as 100%), and the limits of detection were in the range of 1.3-1.9 ng/mL. The recoveries of the eight analytes from the fortified blank samples were in the range of 84.2%-115% with coefficients of variation lower than 18.3%. Therefore, this method could be used as a rapid and simple tool to detect the residues of the eight red dyes in foods.

Production of monoclonal antibody against clonazepam for immunoassay of benzodiazepine drugs in swine tissues.

The objective of the present study was to produce a generic monoclonal antibody for immunoassay of residues of benzodiazepine drugs in swine tissues. Clonazepam was used to synthesize a hapten that was coupled to bovine serum albumin as an immunogen for the production of monoclonal antibody. Results showed that the obtained monoclonal antibody was able to recognize five benzodiazepine drugs simultaneously (clonazepam, flunitrazepam nitrazepam, diazepam, and oxazepam). The cross-reactivities were in the range of 24-100% and the limits of detection were in the range of 0.2-1.5 ng mL(-1) depending on the drug. Then a competitive indirect enzyme-linked immunosorbent assay was developed to determine the residues of five benzodiazepines in swine tissues (muscle, liver and kidney). The recoveries of five analytes from the fortified blank samples were in the range of 74.5-96.5% with coefficients of variation lower than 16.7%. Therefore, this immunoassay could be used as a rapid and simple method for the screening of residues of five benzodiazepine drugs in animal-derived foods.

Alteration in lymphocytes responses, cytokine and chemokine profiles in laying hens infected with Salmonella Typhimurium.

Salmonella Typhimurium has been reported to contaminate egg production across the world, but the exact nature of the immune mechanisms protective against Salmonella infection in laying hens has not been characterized at the molecular level. The experiment was conducted to determine Salmonella colonization and lymphocytes subpopulation in the ileum and spleen, and the mRNA expression of pro-inflammatory cytokines [interleukin (IL)-1ß and IL-6], chemokine IL-8, and T helper (Th)1/Th2 cytokines [Interferon (IFN)-γ, IL-12 and IL-18; IL-4 and IL-10 respectively] in the cecal tonsil and spleen of Salmonella challenged hens. Forty Salmonella-free laying hens were challenged orally with Salmonella Typhimurium or phosphate-buffered saline (PBS; control). The Salmonella challenged or non-challenged hens (n=10) were sacrificed at 2 and 7 days post-infection (DPI). The lymphocyte subpopulation was determined via flow cytometric analysis in the ileum and spleen. The cecal tonsil and spleen samples were collected for mRNA expression through quantitative-RT-PCR. The Salmonella counts were higher (P<0.05) in the ileum than that in the spleen at 2 and 7DPI, and were higher (P<0.05) at 7DPI than that at 2DPI in the spleen. Salmonella challenge increased (P<0.05) ileal CD4+ and CD8α+ cells ratios at 2 and 7DPI, whereas it increased (P<0.05) splenic CD8α+ cells ratio only at 7DPI. The magnitude of increase in ileal CD8α+ cells ratio was higher (P<0.05) than that in CD4+ cells ratio. The mRNA expression of IL-1ß, IL-6, IL-8, IFN-γ, IL-12 and IL-18 were significantly up-regulated in the cecal tonsil of Salmonella challenged hens, and the magnitude of increases in IL-6, IL-8 and IL-12 were significantly higher at 7DPI than that at 2DPI. However, Salmonella challenge increased (P<0.05) the mRNA expression of IL-1ß, IL-10 and IL-18 at 2 and 7DPI, and IL-8 and IFN-γ mRNA only at 7DPI in the spleen. These findings demonstrated that there appeared the induction of cellular immune responses, and a Th1-cytokines reaction in the intestine and spleen of laying hens infected with Salmonella Typhimurium.

Synthesis of novel hapten and production of generic monoclonal antibody for immunoassay of penicillins residues in milk.

The objective of this study was to produce a generic monoclonal antibody for determination of penicillins residues in milk. The compound 6-aminopenicillanic acid was used as the template to synthesize two novel generic haptens that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 11 penicillin drugs (amoxicillin, ampicillin, penicillin G, penicillin V, sulbenicillin, carbencillin, methicillin, cloxacillin, dicloxacillin, oxacillin, and nafcillin). After evaluation of different reagent combinations, a heterologous indirect competitive enzyme immunoassay was developed to multi-determine the 11 drugs in milk. The crossreactivities to the 11 drugs were in a range of 16%-117% and the limits of detection were in a range of 0.7-9.3 ng/mL depending on the drug. The recoveries from the fortified blank milk were in a range of 77.6%-99.4% with coefficients of variation lower than 13.5%. This method could be used as a rapid screen tool for routine monitoring the residues of the 11 penicillin drugs in animal derived foods.

Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk.

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%-102% and the limits of detection were in the range of 1.5-6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%-106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.

Production of the broad specific monoclonal antibody against sarafloxacin for rapid immunoscreening of 12 fluoroquinolones in meat.

The objective of this study was to produce a generic antibody for immunoassay of fluoroquinolone drugs in meat. Two novel haptens of sarafloxacin were synthesized that were used to produce the monoclonal antibodies. The obtained monoclonal antibodies simultaneously recognized 12 fluoroquinolone drugs (sarafloxacin, diflocaxin, marbofloxacin, ofloxacin, ciprofloxacin, enrofloxacin, norfloxacin, pefloxacin, lomefloxacin, amifloxacin, enofloxacin and danofloxacin). After evaluation of different coating antigen/antibody combinations, a heterologous competitive indirect enzyme linked immunosorbent assay (ELISA) was developed to determine the 12 drugs. The crossreactivities to these analytes were in the range of 18%-113% and the limits of detection were in the range of 0.8-6.5 ng/mL depending on the compound. Eight fluoroquinolones licensed as veterinary drugs in China were fortified into blank chicken for ELISA analysis. The recoveries were in the range of 67.6%-94.6% with coefficients of variation lower than 12.4%. Therefore, this method could be used as a screen tool for routine monitoring of the residues of these fluoroquinolone drugs in animal derived foods.

Synthesis of novel haptens against ciprofloxacin and production of generic monoclonal antibodies for immunoscreening of fluoroquinolones in meat.

BACKGROUND: The residues of fluoroquinolone drugs in foods of animal origin are dangerous to the consumers. The objective of this study was to produce a generic monoclonal antibody for determination of fluoroquinolone residues in meat. RESULTS: Two novel haptens of ciprofloxacin containing a free amidogen group on the piperazinyl ring were synthesised that were used to produce the monoclonal antibodies. The antibodies obtained simultaneously recognised 12 fluoroquinolones (ciprofloxacin, enrofloxacin, norfloxacin, sarafloxacin, diflocaxin, danofloxacin, ofloxacin, marbofloxacin, pefloxacin, lomefloxacin, amifloxacin and enofloxacin). After evaluation of different coating antigen-antibody combinations, a heterologous competitive indirect ELISA was used to determine the 12 drugs. The cross-reactivities were in the range of 23-120% and the limits of detection were in the range of 1.0-4.5 ng mL(-1). Eight fluoroquinolone drugs licensed as veterinary drugs in China were fortified into blank chicken for analysis. The recoveries were in the range of 61.5-82.5% with coefficients of variation in the range of 7.5-15.2%. CONCLUSION: This method could be used as a rapid screening tool for routine monitoring the residues of these fluoroquinolone drugs in animal-derived foods.

Synthesis of hapten and production of a monoclonal antibody against a derivative of L-hydroxyproline, a special amino acid in hydrolyzed animal protein.

China's government has prohibited the addition of simply hydrolyzed animal protein from solid leather waste into milk. The objective of this study was to produce a monoclonal antibody against L-hydroxyproline, a special amino acid in hydrolyzed animal protein. L-hydroxyproline was derivatized with N-acetylsulfanilyl chloride and 5-chlorovaleric acid to synthesize the haptens HP1 and HP2. Then, two immunogens from the two haptens were prepared to produce the antibodies. Results showed that only HP1 was able to stimulate the animal immune system and generate the specific antibody to L-hydroxyproline (as the formation of HP1). The obtained monoclonal antibody from HP1 and the heterologous coating hapten HP2 were incorporated into a competitive indirect enzyme linked immunosorbent assay (ELISA) to determine the antibody's specificity and sensitivity. The IC(50) and the limit of detection for HP1 were 0.16 µg/mL and 0.05 µg/mL respectively. The antibody showed low crossreactivity to parental L-hydroxyproline and showed negligible crossreactivity to D-hydroxyproline and other amino acids. The monoclonal antibody was therefore suitable for the development of an immunoassay to monitor the simply hydrolyzed animal protein from solid leather waste in foodstuffs with L-hydroxyproline as the target analyte.

Effects of different fermented soy protein and apparent ileal digestible lysine levels on weaning pigs fed fermented soy protein-amended diets.

Two experiments were conducted to evaluate the effect of different fermented soybean proteins and the apparent ileal digestible lysine levels on weaning pigs fed fermented soy protein (FSP)-amended diets. In Exp. 1, 70 crossed piglets (6.25 ± 0.40 kg) were used in a 5-week trial to evaluate two different FSP. In Exp. 2, 20 weaning barrows (6.15 ± 0.45 kg) were used in a metabolism trial to determine the effects of the apparent ileal digestible (1.2, 1.3, 1.4 and 1.5%) lysine levels in weaning pigs fed FSP (5%) diet. In Exp. 1, pigs fed the diet containing Lactobacillus spp. FSP showed higher nitrogen (N) digestibility (P<0.05), lower blood urea nitrogen and serum creatinine levels (P<0.05) than those fed the Aspergillus oryzae FSP diet. In Exp. 2, increasing dietary lysine levels increased the average daily gain, apparent dry matter, N digestibility, N retention and essential amino acids in the current study (P<0.05), with the 1.5% showing the highest value. In conclusion, pigs fed Lactobacillus spp. FSP had a higher N digestibility than those fed A. oryzae FSP. The optimal apparent ileal digestibility lysine level in fermented soy protein diets (3550 kcal/kg metabolizable energy) for maximizing growth performance and N utilization in the first 7 days (6.25 kg) was 1.5%.