Study On Immunoassay And Chemiluminescence Analysis For Some Environmental Hormone

There has lately been a growing interest in chemicals that might be disrupting theendocrine system of humans and wildlife. A variety of chemicals are known to disruptthe endocrine systems of animals in laboratory studies. The endocrine systems ofcertain wildlife have been affected by chemical contaminants. The female hormone(estrogen) such as 17β-estradiol, estriol, diethylstilbestrol (DES), and bisphenol A(BPA) are considered to be one of the endocrine disrupting chemicals. Estrogen in theenvironment is derived from the excreta of humans and livestock, medicines, and soon. So it is important to establish sensitive methods for the detection of hormone inthe environment.Immunochemical techniques have gained an increasing importance for screeningand quantification of hormone due to their sensitivity, speed, simplicity and low cost.Flow injection chemiluminescence analysis is commonly used for the determinationof trace and ultratrace concentrations of inorganic and organic species. It is becomingincreasingly important in various fields for its high sensitivity, rapidity, handlingfacility and feasibility. The purpose of this dissertation is to develop newimmunoassays and flow injection chemiluminescence methods for detectingenvironmental hormones. For this aim, some researches as follows:1. 17β-estradiol (E₂) was bound to bovine serum albumin (BSA) to form acomplete antigen, after the hydroxy at the position of C3 was activated. A rabbit wasimmunized with the mixture of E₂-BSA and Freund adjuvant. The polyclonal antibodyshowed specific recognition of the 17β-estradiol, and the titer of the antibody by ELISA method were above 1: 25000.2. A simple, sensitive electrochemical enzyme-linked immunosorbent assay(ELISA) for the determination of 17β-estradiol (E₂) was proposed in this paper. Thecomplex of biotinylated anti-E₂ antibody and horseradish peroxidase-labeled avidin(HRP-avidin) were regarded as a probe in this system. The activity of labeled enzymewas measured with electrochemical methods using o-phenylenediamine as substrate.The enzymatic reaction product is 2,3-diaminophenazine, which can be easily reducedon the hanging mercury electrode with improved sensitivity. Coupled with the platecoated antigen indirect ELISA format using E₂-ovalbumin, the electrochemicaldetection was performed for E₂ with the detection limit of 21.0 pg.mL^-1, and the linearrange of determination of E₂ was 50.0-500.0 pg. mL^-1. The proposed method has beenused for the determination of E₂ in river water with satisfactory results.For comparison the spectrophotometric ELISA method using OPD as substratewas also performed with the following result: the detection limit is 2 ng.mL^-1 and thelinear range is 6.0-120.0 ng.mL^-1. So this electrochemical method gives about100-fold increase in sensitivity than the traditional spectrophotometric method.3. A new approach of flow injection chemiluminescent immunoassay withenhancement to detect 17β-estradiol (E₂) was developed. Based on solid-phaseimmunoassay formats, an E₂-OVA immobilized immunoaffinity column inserted inthe flow system was used to trap the unbound horseradish peroxidase (HRP)-labelledanti-E₂ antibody after an off-line incubation of E₂ and HRP-labelled anti-E₂ antibody.The trapped enzyme conjugate was detected by injecting substrates to produce anenhanced chemiluminescence (CL). The linear range for E₂ was 10.0-1000.0 ng mL^-1with a correlation coefficient of 0.996 and the detection limit of 3.0 ng mL^-1. Thesampling and chemiluminescence detection time for one sample was 400 s after apreincubation procedure of 30 min. The method has been used to determine E₂ inhen's feces and serum samples with satisfactory results.4. Estriol was bound to bovine serum albumin (BSA) to form a complete antigen, after the hydroxy at the position of C3 was activated. A rabbit was immunized withthe mixture of E₃-BSA and Freund adjuvant. The polyclonal antibody showed specificrecognition of the estriol, and the titer of the antibody by ELISA method were above 1:21000.5. A new electrochemical substrate for horseradish peroxidase, methyl red, isreported. In this reaction system, horseradish peroxidase can catalyze the redoxreaction of methyl red and H₂O₂. Methyl red exhibits a sensitive voltammetric peak at-0.51V vs. Ag/AgC1 reference electrode, the decrease of the peak current of methylred is in proportion to the concentration of horseradish peroxidase (HRP). The linearrange for determination of horseradish peroxidase is 50.0-500.0 ng.mL-1 and thedetection limit is 18.0 ng.mL^-1. The relative standard deviation is 3.3ï¼…when 200.0ng.mL^-1 HRP was 11 times sequentially determined. A voltammetric enzyme-linkedimmunoassay method for the determination of estriol has been developed, based onthis electrochemical system. The linear range for determination of estriol is1.0-1000.0 ng.mL^-1, and the detection limit is 0.33 ng.mL^-1. The relative standarddeviation for eleven parallel determinations with 200.0 ng.mL^-1 estriol is 4.8ï¼…. It hasbeen used for determination of estriol in pregnancy sera and bed mud with satisfactoryresults.6. A novel approach of flow injection chemiluminescent immunoassay withenhancement to detect estriol was developed. Based on noncompetitive immunoassayformats, a conjugated estriol-ovalbumin immobilized immunoaffinity column insertedin the flow system was used to trap the unbound horseradish peroxidase(HRP)-labeled antibody after an off-line incubation of estriol and HRP-labeledanti-estriol antibody. The trapped enzyme conjugate was detected by injectingchemiluminescence substrates to produce an enhanced chemiluminescence. The linearrange for determination of estriol was 10.0-400.0 ng.mL^-1 with a correlationcoefficient of 0.996 and a detection limit of 5.0 ng.mL^-1. The sampling andchemiluminescence detection time for one sample was 400s after a preincubation procedure of 30 min. Some pregnancy urine samples detected by this method were ingood agreement with the results obtained by ELISA.7. The complete antigen, DES-BSA, was synthesized by combiningdiethylstilbestrol (DES) with BSA. The complete antigen was identified by UVspectrametric scanning method, and the ratio of hapten to carrier protein was 33:1 inDES-BSA. Rabbits were immunized with DES-BSA to obtain the polyclonal antibody,and the titer of the antibody was determined by ELISA method above 1: 11000.A voltammetric enzyme-linked immunoassay method for the determination ofDES has been developed, based on OPD-H₂O₂-HRP electrochemical system. Thelinear range for determination of DES is 1.0-500.0 ng mL^-1, and the detection limit is0.4 ng.mL^-1. The relative standard deviation for eleven parallel determinations with100.0 ng.mL^-1 DES is 11.5%. This method has been applied to the determination ofDES in serum samples and feed with satisfactory results.8. In alkaline medium, a novel flow injection chemiluminescent method has beendeveloped for the determination of diethylstilbestrol, based on the inhibition ofdiethylstilbestrol on the chemiluminescence reaction between luminol andN-bromosuccinimide. Conditions of this chemiluminescent reaction have beendiscussed and optimized, the linear range of determination is 15.0-260.0 ng.mL^-1 fordiethylstilbestrol, the detection limit is 5.0 ng.mL^-1, and the relative standard deviation(RSD) (n=11) is 2.6% for 100.0 ng.mL^-1 diethylstilbestrol. The method has beenapplied to determine the content of diethylstilbestrol in feed and medicine withsatisfactory results.9. A specific polyclonal antibody was produced against BPA by immunization ofrabbits with a conjugate of 4,4-bis (4-hydroxyphenyl) valeric acid and bovine serumalbumin (BVA-BSA). The polyclonal antibody showed specific recognition of thebisphenol structure, while the cross reactions of other common hormone such asestradiol, estriol and phenol were all lower than 1%, and the titer of the antibody byELISA method were above 1: 10000. A rapid and sensitive method based on enzyme linked immunosorbent assay(ELISA) for the determination of bisphenol A (BPA) was established. The linearrange of the presented method was from 2.0 to 1000.0 ng.mL^-1, and the limit ofdetection was found to be 0.5 ng.mL^-1. The method has been applied to determinebisphenol A in serum sample and table-water with satisfactory results. The method isvery useful for monitoring bisphenol A in environmental and biological samples.10. A flow injection chemiluminescence(CL) method was presented for thedetermination of bisphenol A, based on the quench effect of bisphenol A on thechemiluminescence reaction of luminol-potassium hexacyanoferrate system. Thedecrease of the CL emission intensity was linear with the concentration of bisphenol Ain the range of 182.4-2736.0 ng.mL^-1, and the detection limit is 70.7 ng.mL^-1. Thismethod has been used for the determination of bisphenol A in water solvent ofpolycarbonate materials with satisfactory results.11. A new chemiluminescence enzyme immunoassay method is established, inwhich the sodium tetraphenylborate enhanced chemiluminescence reaction system ofLuminol-H202-HRP (Horseradish Peroxidase) is adopted for the end-point monitoringof immunoassay for bisphenol A. The linear range for determination of BPA is25.0-1000.0 ng.mL^-1. And the detection limit is 5.0 ng.mL^-1. It has been used fordetermination of bisphenol A in water with satisfactory results.