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.

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.