Micro-plate chemiluminescence enzyme immunoassay for aflatoxin B1 in agricultural products.

In this work, a micro-plate chemiluminescence enzyme immunoassay by antibody-coated for the determination of aflatoxin B1 (AFB1) in agricultural products has been established. Aflatoxin B1 antibody (AFB1-Ab) was adsorbed physically on polystyrene micro-plate hole as solid phase antibody, which took place immunity-reaction between antigen and antibody with AFB1 standard solution or samples by direct competition. Luminol-hydrogen peroxide chemiluminescence system catalyzed by horseradish peroxidase (HRP) with p-iodophenol enhancement was used as signal detecting system. The effects of several factors, including composition and pH of coating solution, dilution ratio and amount of antibody and enzyme labeled antigen, time of antibody-coating, incubation and chemiluminescence reaction, and other relevant variables upon the immunoassay were studied and optimized. The linear range of proposed method for AFB1 was 0.05-10.0 ng g(-1) with a correlative coefficient of -0.9997. The sensitivity of the proposed method was 0.01 ng g(-1). The RSDs of intra- and inter-assay were less than 12.2% and 10.0%, respectively. This method has been successfully applied to the evaluation of AFB1 in agricultural products with recoveries of 79.8%, 101.9% and 115.4% for low, middle and high concentration samples, respectively. It shows a good correlation with the commercial available ELISA kit for AFB1 with correlative coefficient of 0.9098 indicating that the established CLEIA method can be used to determine AFB1 in real samples.

Development of a competitive radioimmunoassay for glypican-3 and the clinical application in diagnosis of hepatocellular carcinoma.

OBJECTIVE: Glypican-3 (GPC3) is a promising specific tumor maker for hepatocellular carcinoma (HCC). The aim of this study is to establish a method to detect serum GPC3 and evaluate the clinical application on clinical diagnosis. DESIGN AND METHODS: A competitive radioimmunoassay for detecting serum GPC3 was developed. Clinical sera were detected by the proposed method and AFP, CA19-9 chemiluminescence immunoassay kit. RESULTS: The proposed method with high sensitivity, specificity and precision had no or little detectable cross-reactivity with relating tumor markers in the dynamic range from 15 to 500 ng/mL, and the detection limit was 0.5 ng/mL. The level of GPC3 in HCC was obviously higher than that in normal liver or other liver diseases. Additionally, our method showed high shows higher sensitivity and specificity for GPC3 than AFP and combined AFP/CA19-9. CONCLUSIONS: This paper provided an applicable competitive radioimmunoassay for GPC3 with high sensitivity, specificity and precision. In addition, using GPC3 for HCC diagnosis was more valuable than AFP.

Development of a sensitive, rapid, biotin-streptavidin based chemiluminescent enzyme immunoassay for human thyroid stimulating hormone.

A highly sensitive "two-site" chemiluminescent immunoassay specific for human thyroid stimulating hormone (TSH) was developed. The signal amplification was achieved via a biotin-streptavidin system (BSAS). The HRP-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. Biotinylated anti-TSH monoclonal antibody (MAb) and HRP-labeled streptavidin were first synthesized. Then the signal amplification was achieved through the interaction between the biotinylated anti-TSH MAb and the HRP-streptavidin conjugate. The light intensity developed was in proportion to the TSH present in the samples. The assay showed little cross-reactivity with three other glycoprotein hormones (human chorionic gonadotropin (HCG), luteinizing hormone (LH), follicle stimulating hormone (FSH)) due to the high specificity of the antibody. The working range for human thyroid stimulating hormone was 0.1-40 mU L(-1). Both the intra-assay and inter-assay coefficients of variation were less than 10% for the BSAS based chemiluminescent enzyme immunoassay (CLEIA). The proposed assay had a sensitivity of 0.01 mU L(-1) which was 10-fold higher than the HRP-MAb conjugate based TSH immunoassay. Thus the higher sensitivity facilitated the clinical testing for thyroid states. The effects of several reaction parameters, such as incubation time, temperature, and reaction volume of the method, were also studied. This method has been successfully applied to the evaluation of TSH in human serum. Compared with the commercial enzyme chemiluminescent immunoassay, the correlation was satisfied.

Development of a high-throughput, indirect antibody immobilization format chemiluminescence enzyme immunoassay (CLEIA) for the determination of progesterone in human serum.

A high-throughput and simple chemiluminescence (CL) enzyme immunoassay (CLEIA) for the determination of progesterone (P) in human serum was developed, with the highly sensitive 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD)-alkaline phosphatase (ALP) system as the CL detection system. The results showed that the indirect immobilization of rabbit anti-progesterone polyclonal antibody (RAPA) through secondary antibody exhibited apparent advantages over direct coating in terms of antibody saving and improvement of the coating stability and uniformity. The direct analysis of P in human serum without extraction was realized by using 8-anilino-1-naphthalenesulphonic acid (ANS) to displace P from its binding proteins. The effect of several relevant parameters of the immunoreaction were examined and optimized. Compared with some commercial progesterone kits, the presented CLEIA has higher sensitivity with detection limitation as low as 0.06 ng/mL. The recoveries were 95.9-101%. The coefficient of variation was <8.4% and 9.9% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of P in human serum.

Development of high-performance magnetic chemiluminescence enzyme immunoassay for alpha-fetoprotein (AFP) in human serum.

BACKGROUND: A high-performance chemiluminescence enzyme immunoassay (CLEIA) for alpha-fetoprotein (AFP), a tumor marker for the diagnosis of hepatocellular carcinoma (HCC), was constructed by using magnetic particles (MPs) as both the immobilization matrix and separation tools. METHODS: A double sandwiched immunocomplex was formed through the reaction among anti-fluorescein isothiocyanate (FITC) antibody coated MPs, FITC-labeled anti-AFP antibody, AFP antigen, and alkaline phosphatase (ALP)-labeled anti-AFP antibody. The subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) gave light intensity that was directly proportional to the amount of analyte present in the samples. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-AFP antibody, the dilution ratio of ALP-labeled anti-AFP antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. RIA and microplate CLEIA were used as comparison methods. RESULTS: The proposed method had a sensitivity of 3.0 ng/ml, low cross reactivities, and an assay time of 1 h. The linear range was 0-1200 ng/ml through using MPs and is useful for samples with extremely high AFP concentrations without dilution while avoiding the hook effect. The intra- and inter-assay precision was <3% and <5%. The present method has been successfully applied to the detection of AFP human serum with recoveries from 90 to 108%, and showed a good correlation with the commercially available AFP RIA kit. CONCLUSIONS: This proposed assay provided apparent advantages over microplate CLEIA and RIA, and facilitated the development of high-throughput screening and automated operation systems in the clinical practice.

Evaluation of carbohydrate antigen 50 in human serum using magnetic particle-based chemiluminescence enzyme immunoassay.

A magnetic particles (MPs)-based chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, specificity, rapidity, and reproducibility was proposed for the evaluation of tumor marker, carbohydrate antigen 50 (CA50) in human serum. The immunomagnetic particles coated with anti-fluorescein isothiocyanate (FITC) antibody was used as dispersed solid phase for the immunoassay, which was based on a sandwich immunoreaction of FITC-labeled anti-CA50 antibody, CA50 antigen, and alkaline phosphatase (ALP)-labeled anti-CA50 antibody, and was based on a subsequent chemiluminescence reaction of ALP with 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) solution. The CL emission intensity was directly proportional to the amount of analyte present in a sample solution. The effects of several physicochemical parameters, including the concentration of FITC-labeled anti-CA50 antibody, the dilution ratio of ALP-labeled anti-CA50 antibody, the volume of MPs and substrate, the immunoreaction time and other relevant variables upon the immunoassay were studied and optimized. The proposed method exhibited advantages in a lower minimum detectable concentration of 1.0 U mL(-1) with comparison to the commercially available immunoradiometric assay (IRMA), and showed a larger linear range of 0 to 300 U mL(-1), as well as less total assay time of only 50 min with comparison to both IRMA and microplate CLEIA. The coefficient of variation was less than 7 and 11% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of CA50 in human serum with recoveries from 82 to 112%, and showed a good correlation with the commercially available CA50 IRMA.

Development of a highly sensitive and selective microplate chemiluminescence enzyme immunoassay for the determination of free thyroxine in human serum.

A microplate chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, selectivity and reproducibility was developed for the determination of free thyroxine (FT4) in human serum. A competitive assay has been utilized with horseradish peroxidase (HRP) labeled thyroxine analog in the chemiluminescence (CL) detection. The CL signal produced by the emission of photons from luminol was directly proportional to the amount of analyte. The linear range was 0.45-7.5 ng dL(-1 )and the detection limit was 0.09 ng dL(-1). Experimental conditions, such as temperature, pH, incubation time, titration level and other relevant variables upon the CL signal have been examined and optimized. A coefficient of variance of less than 16% was obtained for intra- and inter-assay precision. The present method has been successfully applied to the analysis of FT4 in human serum. The positive and negative coincidence ratios are satisfactory. Good correlations were obtained between the results by the proposed method and radioimmunoassay (RIA), as well as a Bayer ACS-180SE detection system.

Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen.

A micro-magnetic chemiluminescence (CL) enzyme immunoassay with high sensitivity, selectivity, and reproducibility was developed for the determination of the tumor marker, carcinoembryonic antigen (CEA) in human serum. A sandwich scheme assay has been utilized with fluorescein isothiocyanate antibody (FITC)-labeled anti-CEA antibody and alkaline phosphate (ALP)-labeled anti-CEA antibody being used in the CL detection. The CL signal produced by the emission of photons from 4-methoxy-4-(3-phosphate-phenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD) was directly proportional to the amount of analyte present in a sample solution. The influences of the reaction time of antigen with antibody, the reaction time of substrate with label, the dilution ratio of ALP-labeled anti-CEA antibody, the concentration of FITC-labeled anti-CEA antibody, and other relevant variables upon the CL signal were examined and optimized. The CL responses depended linearly on the CEA concentration over the range from 2 to 162 ng mL-1 in a logarithmic plot. Assay sensitivity as low as 0.69 ng mL-1 was achieved. A coefficient of variance of less than 13% was obtained for intra- and inter-assay precision. This method has been successfully applied to the analysis of CEA in human serum. According to the procedure based on spiked standards, the recoveries obtained were 80-110%. Comparison experiments were carried out with the commercially available CEA chemiluminescence immunoassay. Satisfactory results were obtained according to a paired t-test method (t value