Preparation Of Anti-AMP Antibody And Development Of Enzyme-Linked Immunosorbent Assay For Detecting AMP Residues In Milk

Ampicillin, a kind ofβ-lactam antibiotics, is a semi-synthetic broad-spectrumpenicillin which has a wider sterilizing range than penicillin G and is active to bothG⁺ and G^-. It is one of the antibiotics commonly used in breeding agricultural animalssuch as birds and livestock. In the process of feeding cows, ampicillin is often addedto the feed and heavy used in the treatment of inflammation and infections of cowssuch as mastitis, urethra and reproductive tract infections, which causes penicillinantibiotics residues in milk. Long-term consumption of the milk containingpenicillin antibiotics residues could lead to body resistance and hypersusceptibilityand do harm to human beings. Therefore, it is very meaningful to establish a rapidmeasurement for detecting penicillin residue in milk. In this paper we have producedanti-ampicillin monoclonal antibody on the basis of preparation of AMP artificialantigen,and then established ELISA detection method. The main results of our studyare as follows:(1) In this paper, ampicillin was coupled with carrier proteins bovine serumalbumin (BSA) and ovalbumin (OVA) by methods of physiology and glutaraldehydeto prepare artificial antigen for immunization and ELISA. Identified by UV scan andSDS-PAGE electrophoresis the couplings were successful.The best preparation conditions were optimized by L9(3⁴) orthogonal test,glutaraldehyde method: the initial molar ratio of AMP and BSA is 40:1, 25%glutaraldehyde 10μ1, reaction time 4 hours; physiological method: the initial molarratio of AMP and BSA is 20:1, reaction time 3 hours. And their coupling rates were10.23 and 17.68 respectively, which were measured by the improved BCA method(2) Each artificial antigens immunised 3 mice, after the fourthimmunization, these mice's antiserum were measured, the titer were reached 1～5×10⁴ by indirect ELISA,and all mice present inhibition to Standard AMP inindirect competitive ELISA with 100ng/mL. The best mouse's number isâ‘¡whoseinhibition rate rezched 93.4 percent. The inhibition rate of the antiserum of mouseimmunized by artificial antigen prepared by method of physiology to standard AMPwas higher than that of glutaraldehyde.Spleen cells of mouseâ‘¡were fused with SP2/0 myeloma cells according toHybridoma Technology. The ratio of fused hole and positive hole were 21% and 5.4% respetively.Limited dilution method was employed to do subclone, threeClones named X.5.2, X.16.3.5 and X.16.4.8 stablely producing anti- ampicillinmonoclonal antibody were finally selected. The clone X.16.3.5 with the highestpositive and the best sensitivity was selected to inject into mice for preparing ascites.The ascites with antibody were purified by caprylic acid-saturated ammoniumsulphate method. Pure antibody's protein concentration was 5.85mg/ml. The titer ofmouse ascites was 3.2×10⁵ determined by indirect ELISA, IC₅₀ value was about12.5ng/ml, had no cross-reaction with the carrier protein OVA, BSA and antibioticssuch as gentamicin, tetracycline, karaoke adriamycin and streptavidin. The selectedmonoclonal antibody had high sensitivity and specificity.(3) The conditions of ELISA experiment were optimized. The standard curve ofdetecting AMP was established by indirect competitive ELISA in Tris-HCL bufferbuffer. The linear range of detection was between 0.5 and 500 ng / ml, the equationwas Y=32.539X+13.651, the correlation coefficient was 0.982, the sensitivity anddetection limit were 13.09ng/ml and 0.77ng/ml respectively, the coefficients ofvariation of ELISA in intra-assay and inter-assay were 4.45% and 5.80%respectively with good reproducibility.The standard curve of detecting AMP residues in milk was successfullyestablished by 5% defatted milk as buffer. The equation was Y=30.427X+13.773,the linear range was between 0.5 and 500ng/ml, the correlation coefficient was0.9904, the sensitivity and detection limit were 15.51ng/ml and 0.75ng/mlrespectively. The precision experiment of this detection method was established. Thecoefficient of variation in intra-assay and inter-assay were 4.35 %＜5% and 5.70%＜10% respectively. The average recoveries of standard addition of the five levels ofmilk was 93.64 %. The detection limit of this method achieved to EU standards withgood reproducibility and accuracy.The anti-AMP monoclonal antibody and the ELISA method can be used todetect AMP residues in milk.