A secondary antibody format chemiluminescence immunoassay for the determination of estradiol in human serum.

A competitive immunoassay for estradiol (E2) based on secondary antibody format was established. The donkey anti-rabbit IgG was used as the secondary antibody to coat micro-plates, and the horseradish peroxidase (HRP)-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. The addition of sodium trichloroacetate (CCl(3)COONa) in the enzyme buffer as a replaceable packing material can realize directly analysis of E2 in human serum without extraction, which improved reproducibility and resolution of the assay. Additionally, the method showed specific recognition of estrogen, without cross-reaction for the major steroids (estrone (E1), estriol (E3), dihydrotestosterone (DHT), androstenedione, testosterone (T)) commonly found in human serum. The chemiluminescence immunoassay with secondary antibody can be applied to detect E2 with good precision at concentrations as low as 1.48 pg mL(-1). The proposed method has been successfully applied to the determination of E2 in 97 human sera and showed a good correlation compared with the commercially radioimmunoassay (RIA) kit with a correlative coefficient of 0.9881. This method has exhibited great potential in the fabrication of diagnostic kit and can be used in the clinical analysis of E2 in human serum.

Magnetic particle-based chemiluminescence enzyme immunoassay for free thyroxine in human serum.

A magnetic particles-based chemiluminescence enzyme immunoassay with high sensitivity, specificity, rapidity, and reproducibility was developed for the determination of free thyroxine in human serum. A competitive assay has been proposed with horseradish peroxidase labeled thyroxine analog. The immunomagnetic particles coated with anti-fluorescein isothiocyanate antibody was used as dispersed solid phase and separation means for the immunoassay. Experimental conditions, such as temperature, the volume of magnetic particles and substrate, incubation time, dilution ratio and other relevant variables upon the immunoassay have been examined and optimized. The proposed method exhibited high performance which the linear range was 1.59-122 pmol L(-1) and the detection limit was 0.25 pmol L(-1). A coefficient of variance of less than 15% was obtained for both intra-assay and inter-assay precision. The present method has been successfully applied to the analysis of free thyroxine in human serum. The diagnostic accordance rate of the method for normal serum, hyperthyroidism and hypothyroidism are satisfactory. Good correlations were obtained between the results by the proposed method and the commercial radioimmunoassay kit. The present method exhibits good potential in the fabrication of FT4 diagnostic kits which could be used in the clinical analysis and facilitated the development of automated operation systems in the clinical practice.

Development of magnetic particle-based chemiluminescence enzyme immunoassay for the detection of 17beta-estradiol in environmental water.

In the present work, a simple, fast, and highly sensitive chemiluminescence enzyme immunoassay for 17beta-estradiol (E2) in environmental water samples was developed, using magnetic particles (MPs) labeled with secondary antibody as both the immobilization matrix and the separation tools. The specific anti-E2 polyclonal antibody (PcAb) was produced against a conjugate of estradiol-bovine serum albumin. The specificity of the anti-E2 antibody was studied. The results showed that the antibody did not cross-react with the structurally related endocrine-disrupting compounds, including estrone, ethinyl E2, estriol, E2-17-glucuronide, E2-3-sulfate-17-glucuronide, androstenedione, and dihydrotestosterone. The water samples were pretreated with solid-phase extraction using C18 cartridges for the removal of matrix effects. Several physicochemical parameters including the dilution ratios of E2-6-horseradish peroxidase conjugate and anti-E2 PcAb, immunoreaction time, volume of chemiluminescent substrate and MPs, chemiluminescence reaction time, and pH of assay solution were studied and optimized. At optimal experimental conditions, it was found that the proposed method exhibited high performance with detection limit of 2.0 pg/mL, linear range of 20-1,200 pg/mL, and total assay time of 45 min. Both inter- and intra-assay coefficient of variation were less than 10%. The average recoveries of three different spiked concentration samples ranged from 86.3% to 108%. The method was successfully applied to the determination of E2 in river, waste, and tap water, and showed a good correlation with the commercially available radioimmunoassay kit.

Determination of estradiol in human serum using magnetic particles-based chemiluminescence immunoassay.

A magnetic particles (MPs)-based chemiluminescence immunoassay (CLIA) with high sensitivity, specificity, and reproducibility was proposed for the evaluation of estradiol (E(2)) in human sera. The MPs coated with secondary antibody were used as dispersed solid phase for the immunoassay, and the horseradish peroxidase (HRP)-luminol-H(2)O(2) chemiluminescent system with high sensitivity was chosen as the detection system. The method showed specific recognition to E(2), without cross-reaction for the major steroids, including estrone (E(1)), estriol (E(3)), dihydrotestosterone (DHT), androstenedione, and testosterone (T), which was commonly found in human serum. The addition of sodium trichloracetate (Na-TCA) in the enzyme buffer as a blocking agent contributed to the realization of direct analysis of E(2) in human serum without extraction. Besides, the effects of several physicochemical parameters, including the dilution ratios of E(2)-6-HRP conjugate and anti-E(2) polyclonal antibody, immunoreaction time, chemiluminescent (CL) substrate volume, volume of MPs, and CL reaction time, were studied and optimized. The proposed method had a detection limit of 2.51pgmL(-1) with a larger working range of 15-1000pgmL(-1). The inter-assay and intra-assay coefficient of variation (CV) were both less than 15%. The average recoveries of three different spiked concentration samples were 93.3, 106 and 101%, respectively. The method has been successfully applied to the determination of E(2) in 105 human sera and showed a good correlation compared with the commercial radioimmunoassay (RIA) kit with a correlative coefficient of 0.9892. This method has exhibited great potential in the fabrication of diagnostic kit and could be used in the clinical analysis of E(2) in human serum.