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Development of the Monoclonal Antibodies Against LMG and Application for the Determination KIT of LMG Residues

Author: GongPengFei
Tutor: YuanHui；ChenYanJun；WangQuan
School: Hunan Agricultural University
Course: Clinical Veterinary Medicine
Keywords: ELISA MG Monoclonal Antibody residue
CLC: S948
Type: Master's thesis
Year: 2005
Downloads: 396
Quote: 10
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Abstract


Malachite Green（MG） was introduced originally as an ectoparasiticide , fungicide and antiseptic in aquaculture . After administration, MG is prevalently reduced into Leuco-Malachite green （LMG）in fishes, and deposited in fatty tissue of the fish, so that the ban on its use and its potential adverse effects in humans in some countries. The European minimum required performance limit （MRPL）, a quality parameter for residue of MG laboratories, is set at 2 μg/kg（Commission Decision 2004/25/EC）.This research synthesized the antigen and developed the antibodies, and established the ELISA method for detecting LMG.To prepare antibodies （Abs） against MG, Pararosaniline was bound to BSA or OVA using nitrous acid as the coupling reagent, and using UV spectrophotometer scanning identified the conjugates. To prepare poly-clonal antibody, the BALB/c mice were primed with three injection the BSA-PA. Antibodies specific for the particular hapten. was identified with indirect ELISA .it show that it was a wrong antibody because LMG donnot compete against hapten.NMG which has been compounded was bound to BSA or RSA, and we got the specific poly-clonal antibody by three injection the conjugates. Three days later with a final booster injection （intra-peritoneally）, spleen cells were isolated. The fresh spleen cell was used for fusion with myeloma cell. Using iELISA and icELISA to screen the positive cell and we got a positive cell which called 5E9. The supernatant of the positive well reacted to the NMG-OVA and could be competed by LMG.The 5E9 cells were cloned by limiting dilutions （three times）,mice （BALB/c） were injected intraperitoneally with 5E9 cell to produce ascites and which was distilled. Indirect competitive ELISA （icELISA） was established with this ascites. The limit of detection of this assay was established to be 1.3×10-5 ng /ml, and the limit of determination was 0. 427 ng/ml 1. To test the specialty of antibody, eight sulfonamides were used in the competitive ELISA as the competitive antigen. The result proved that the ascites shows no cross-reaction to the other eight sulfonamides besides MG.IcELISA was optimized for the kit exterminating the LMG. It will cost four hours in the inspecting process of the kit; the range is 0. 427-1000 ng/ml; it can be recoveries 83. 2-122% for the samples which be from fish.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Fisheries Protection > Fisheries pharmacology, pharmacology
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