| Both5-Methyl salicylic acid and4-methylphenol belong toPPCPs(pharmaceutical and personal care products), which are widelyconcerned as new emerging pollutants. The two kinds of molecules havemuch resemblance in structure. In this study,5-methylsalicylic acid wascoupled with carrier proteins for producing artificial antigen by differentmethods. Appropriate immunogens were screened out to immunizingNewzealand rabbits to abtain antibody with high titer against5-Methylsalicylic acid. Based on the antibody with high titer, the ic-ELISAmethod was established after optimizing detection factors. Uilizing the highcross-reactivity of antibody against5-Methylsalicylic acid with4-methylphenol when accessing the specificity of the antibody, the ic-ELISAmethod to detect4-methylphenol was also established. Both establishedmethods were used to detect some samples with comparison with HPLC.Comparing the results of the ic-ELISA and HPLC, high consistence wasachieved.1The Synthesis and Screening of Artificial Antigens of5-Methylsalicylic AcidFour kinds of artificial antigen were coupled with carrier proteins by theactivating ester combined with carbodiimide method and the mixedacnhydride mehod. The results of Ultraviolet full wavelength scanningindicated that the synthesis of four artificial antigens were successful. Theprotein concerntration of four artificial antigens were detected by Masiliangblue method, which presented results as7.2mg/mL,5.834mg/mL, 1.789mg/mL and4.8mg/mL. The coupling ratios of four artificial antigenswere14.6、8、19.7and3.4.2The Preparation and Identification of antibody against5-Methylsalicylic AcidTwo antigens with coupling ratios as14.6and3.4were used toimmunizing two Newzealand rabbits. After several times of immunization,the whole bloods were collected. The titer of antibodies were assessed byic-ELISA method with results as256000and64000. The concerntration ofprotein of two antibodies were18.02mg/mL and20.97mg/mL.3The Establishment of ic-ELISA Method to Detect5-MethylsalicylicAcidUsing the prepared artificial antigen and antibody, six factors includingthe concentration of coating antigen and antibody and the type of sealing fluidof ic-ELISA method were considered. Then the ic-ELISA method to detect5-Methylsalicylic Acid was established with optimized factors as128000and4000as the best dilution ratio of coating antigen and antibody,0.5%OVA asthe best sealing fluid,2500as the best dilution ratio of IgG-HRP,2hours asthe best coating duration,1hour as the best incubation duration for thereaction between coating antigen and antibody,1hour as the best incubationduration of HRP-IgG.Utilizing the optimized factors, the standard curve was established withthe regression equation as y=-14.31x+81.10，R2=0.944. The50%inhibitionconcentration and the limitis of detect was figured out from the regressionequation as149.04ng/mL and4.19ng/mL. Considering the high cross-activityof the antibody with4-methylphenol, the standard curve to detect4-methylphenol was created with the regression equation as y=9199x-44776R2=0.991. The50%inhibition concentration and the limitis of detect wasfigured out from this regression equation as112.23ng/mL and17.14ng/mL.The specificity of the two method were also assessed with satisfactory results. 4Detecting the concentrations in samples and comparing the results withHPLCThe cosmetic samples and water samples were collected and detected forthe concentrations of5-methylsalicylic acid and4-methylphenol in them. Thestandard curve of HPLC to detect5-methylsalicylic acid was y=9199x-44776R2=0.991, and the standard curve to detect4-methylphenol wasy=9199x-44776R2=0.991. The results of ic-ELISA and HPLC were highconsistent with each other. The recovery rates of5-methylsalicylic acid byic-ELISA was97.25%~103.90%and the recovery rate of4-methylphenol was97.50%~10.67%. The established methods were proved to be reliable andaccurate from above researches and could be applied in many practicalsample detections. |
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