| Melamine, with a1,3,5-triazine skeleton, is an important nitrogen heterocyclic organic chemical raw materials and widely used in the production of plastics, dyes, fertilizers, and fabrics. Animal experiments show that melamine may lead to reproductive damage, bladde, chronic kidney inflammation, nephrolithiasis and even bladder carcinoma. So far, the most commonly used method to analyze melamine is HPLC or LC-MS(liquid chromatography-mass spectrometry). These methods requires complex sample pre-trestment processes, sophisticatid detection equipments, good experimental environment and well-trained operators, which limit its application. Compared with HPLC or LC-MS, the advantage of ELISA is simple of sample preparation, sensitivity, high specificity and low cost, it could be uesd to detect the melamines in the scene. Therefore, to develop the ELISA (enzyme-linked immunosorbent assay) method has important practic alvalue.In order to establish a sensitive method to detect melamine, in this study monoclonal antibody against melamine was prepared, the synthesis of HRP-OVA-MEL and the Ci-ELISA and Di-ELISA were developed.First the immunogen (MEL-BSA) and coating antigen (MEL-OVA) were identified by UV and SDS-PAGE methods. BALB/c mice were immunized with MEL-BSA, after a fusion of mouse spleen cells with SP2/0cells, four hybridoma cells secreting monoclonal antibody against MEL were selected, named as A85, Bll, C4and B6. The results of analysis revealed that characteristecs of mAbs. The results of specific test comfirmed that C4and B6in ascites had high specificity becaouse they could only react with melamine but not with carrier protein BSA or OVA.Their subclass belonged IgM. One of them, C4strain, had a titers of1:104at least in ascites by indirect ELISA and a high affinity constant (Ka) with2.92×108L· mol-1To purify the mAb the saturated sulfate precipitation, PEG6000precipitation and optimal globulin precipitation were used, in which the PEG6000precipitation needed shortest time, high yield and affinity of products.Secondly ic ELISA method was developed with the purified C4monoclonal antihbodies. The optimized condition was as follow:the melamine-OVA was added to microtiter plates at a concentration of2.5μg/mL, the optimum block condition was hatching1h at37℃with5%glycine; the optimum labelled antibody (1:4000) reactive condition was hatching1h at37℃and the stopping solution (2mo1/L sulfuric acid) was added after incubated20min in the dark at37℃. Through analysis of ic-ELISA,the standard curve was drawed,and its standard equation was y=-25.093x+62.409(R2=0.9877); IC50was3.12μg/L; detect concentrations ranged from0.05-10μg/L.Finally, melmine was conjugated with HRP, and the conjugates activity were identified by ELISA. And the dc-ELISA was estabished to detect melamine, the standard curve was drawed, anf its standard equation was y=-11.432x+70.072(R2=0.9948); IC50was56.88μg/L; detect concentrations ranged from0.1-1000μg/L.All these results showed that the mAb against melamine could be used for detecting melamine. It will provide foudations for the development of melamine test kit and colloidal gold test. |
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