Development and Characterization of Anti-Estradiol Polyclonal Antibody

Abstract

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

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

“…Additionally, inhibition curves without interference were obtained after a six-fold dilution, indicating that skimmed milk could be measured following dilution. Furthermore, the LOD of the icELISA was 0.093 ng/mL, which was much lower than that reported previously (Caron et al, 2010;Li & Jiang, 2012). The accuracy of the ELISA developed for E 2 detection in milk was then assessed by measuring spiked raw milk samples and estimating the overall recovery.…”

“…Antibody represents the key reagent in immunoassays, and some E 2 -specific antibodies have been reported (Caron, Sheedy, & Farenhorst, 2010;Li & Jiang, 2012;Yang et al, 2015). However, the sensitivities of some of these antibodies are too low [half maximum inhibition concentration (IC 50 ) = 243 ng/mL (Caron et al, 2010) or 3.76 ng/mL (Li & Jiang, 2012)] to be used for establishing a highly sensitive icELISA to detect traces of E 2 residue in milk. Therefore, this study prepared polyclonal and monoclonal antibodies against E 2 and established a sensitive icELISA using the antibodies exhibiting the highest sensitivities for detecting E 2 in milk.…”

Antibody production and application for immunoassay development of environmental hormones: a review

“…Spier et al [31] also noted that the hydrophobic haptens would limit accessibility of the antibody, because hydrophobicity can cause conformational changes in the protein carrier and result in internalization of the hapten within hydrophobic pockets. Hence, they improved the concentration of DMF to 25% to increase the conjugation efficiency Medroxyprogesterone acetate [99] Estradiol [38] Estriol [100] [101]…”

“…Therefore, characteristic peaks would be changed: for example, the UV spectra of BSA, E2, and E2-BSA ( Fig. 2, [38]).…”

“…As can be seen, the three curves do not coincide, and they have different peaks. The E2-BSA conjugate has a shift compared with the unconjugated E2 hapten (from 272 to 276 nm [38]). The typical UV absorption peak of protein (BSA) was found at 280 nm.…”