A Study Of Fluorescence Immunoassay Methods For α-naphthol And β-naphthol In Environmental Water

Phenolic compounds are important materials in chemical industry. They are important environmental pollutants due to their toxicity to human and difficult to degrade,so it is very necessary to detect the phenolic compounds residues in environmental water samples.α-naphthol andβ-naphthol are widely used in many fields,such as tanning,pharmaceutical industry,rubber industry,pesticide and dyes. They are always mixed in the same industrial product,so developing a new analytical method for determination ofα-naphthol andβ-naphthol is significant for controlling environmental pollution.At present,the main monitoring methods forα-naphthol andβ-naphthol are GC,UV spectrophotometry,HPLC,capillary zone electrophoresis,fluorescence spectra,phosphorescence method and electrochemical method.But the pretreatment of these methods is very complicated,time-consuming and costly.Immunoassay methods have shown to be rapid,sensitive,reliable,cost-effective,and became one of the most competitive and challengeable environmental monitoring methods in the 21st century.The investigation of literature shows that immunoassay is seldom used in china on monitoring environmental pollutants.The work is summarized as follows:(1)The preparation of antigens:α-naphthol andβ-naphthol are small molecules,which can not be directly used for rabbit immunization.They have to combine with carders.Haptens should be synthesized and conjugated to protein for rabbit immunization.Naphthol was synthesized by means of the chloroactic acid method.The haptens were conjugated to the carder proteins(BSA,OVA)with the modified active ester method or with the mixed acid anhydride method to form immune antigens and coating antigens.The conjugates were scanned by UV spectrum to judge whether haptens were linked to the proteins,also the conjugation ratios of artificial antigens were calculated.The results showed that the conjugates were prepared successful.(2)The preparation of antibodies:New Zealand white rabbits were immunized with the mixture of immune antigens and Freund adjuvant. The titers of anti-sera were identificated by tube agglutination reaction test and agar double-diffusion experiment.In tube agglutination reaction test,there were visible precipitation reaction;in agar double-diffusion experiment,there were clear deposit lines.The immune sera were purified by octanoic acid-saturated ammonium sulfate,Sephadex G-25 and DEAE cellulose.Polyclonal antibodies were prepared successful. (3)The establishment of immunoassay methods:Microtiter plates are coated with coating antigens ofα-naphthol orβ-naphthol,and then optimized fluorescence immuno assay conditions.The fluorescence immunoassays(FIA)ofα-naphthol orβ-naphthol were established with the signal of fluorescein isothiocyanate.Microtiter plates are coated with antibodies ofα-naphthol orβ-naphthol.The fluorescence immunoassays (FIA)ofα-naphthol orβ-naphthol were established with the signal of fluorescein isothiocyanate.Both of these methods have great advantages on sensitivity and showed a wider linear range.Satisfactory results were gained when it was used to determine the concentration ofα-naphthol orβ-naphthol in environmental water.Two kinds of new FIA methods of determination ofα-naphthol andβ-naphthol were established.These researches supplied reliable immune antigens and antibodies for establishing new immunoassay forα-naphthol andβ-naphthol residues detection in environmental water.It is significant for environmental monitoring,pollutant controlling,protecting human health.