Production of antibodies and development of an enzyme-linked immunosorbent assay for 17β-estradiol in milk

Abstract

The residue of 17β-estradiol (E 2) in milk could potentially lead to the occurrence of various reproductive diseases; therefore, a rapid and sensitive method for monitoring E 2 residues in milk was highly necessary. In this study, we produced new polyclonal and monoclonal antibodies using E 2-3-O-carboxymethyl ether as a hapten and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of E 2 in milk. The results showed that the sensitivity of polyclonal antibody was higher than that of monoclonal antibody, providing a half maximum inhibition concentration (IC 50) against E 2 of 0.17 ng/mL, high cross-reactivity (CR) to E 2 benzoate (150%) and oestriol (18.02%), and negligible CR with other oestrogen compounds. Under optimized conditions, the developed icELISA based on the polyclonal antibody had a limit of detection values of 0.093 μg/L, which was enough sensitive to detect E 2 in milk. In spiked samples (0.5, 1, and 2 μg/L), the recoveries ranged from 83.12% to 94.58% with coefficients of variation <12.8%. These results indicated that the icELISA method we developed was suitable for screening of E 2 residue in milk.

Development of an enzyme-linked immunosorbent assay for quantification of estriol in milk

“…However, in this work, we used the similar strategy for hapten design, but the obtained antibody seemed to be unsatisfactory (Table S1). Therefore, we used another strategy, by introducing a carboxyl group spacer from C3-OH of estriol (Bai et al, 2017). The results indicated that by using this antigen (E3-A-BSA) as immunogen, and E3-B-OVA as heterologous coating antigen, the obtained antibody showed better sensitivity in ELISA in comparison with literatures.…”

“…There are extracted with ethyl acetate, thin layer chromatography, high performance liquid chromatography (Gorga, Insa, Petrovic, & Barceló, 2014;Piwowarska, Radowicki, & Pachecka, 2010), gas chromatography analysis-mass spectrometry (Andrási, Helenkár, Vasanits-Zsigrai, Záray, & Molnár-Perl, 2011;Lu, Wu, Stoffella, & Chris Wilson, 2012), electroanalytical determination (Cincotto et al, 2015;Fu et al, 2016;Santos, Braga, Vieira, & Spinelli, 2010), enzyme-linked immunosorbent assay (ELISA) (Bai et al, 2017), fluoroimmunoassay (Tang, Zhao, Wu, Zhou, & Li, 2013), and immunochromatographic assay (Mukunzi et al, 2016;Wang et al, 2017) for estriol and its analogs analysis. Above all of instrument assays, chromatography spectroscopy was generally used to detect estriol in urine of pregnant women and drug.…”

Development of a fluorescent immunoassay strip for the rapid quantitative detection of cadmium in rice

“…Six to nine days after the third immunization, mouse sera were obtained by tail blood collection, and the same step was performed for subsequent immunizations. The titer and inhibition rate of each serum were detected by indirect competitive ELISA (ic-ELISA; Bai et al, 2017). For ic-ELISA, we used 10 μM ITCBE instead of PBS buffer.…”

A self-powered electrochemical aptasensor for the detection of 17β-estradiol based on carbon nanocages/gold nanoparticles and DNA bioconjugate mediated biofuel cells

“…27,28 Accordingly, developing efficient and sensitive methods for the monitoring of E2 levels is of great significance. So far, many different techniques have been established for the determination of E2, such as gas chromatography/liquid chromatography–mass spectrometry (GC/LC-MS), 29,30 enzyme-linked immunosorbent assay (ELISA), 31,32 colorimetry, 33–35 fluorescence, 36–39 electrochemistry, 40–46 and surface-enhanced Raman spectroscopy. 47,48 Nevertheless, these methods might suffer from the high cost of devices, the need for high levels of technical skill, low sensitivity, cumbersome operation processes and the need for sample pretreatment.…”