Preparation of an anti-diethylstilbestrol monoclonal antibody and development of an indirect competitive ELISA to detect diethylstilbestrol in biological samples

Abstract

Based on the preparation of an anti-diethylstilbestrol (DES) monoclonal antibody, a simple and convenient indirect competitive enzyme-linked immunosorbent assay (ELISA) method for DES detection has been developed. The monoclonal antibody demonstrated high sensitivity to DES with an IC 50 value of 275 pg mL -1 and detection limit (LOD) of 90 pg mL -1 . The specificity of the assay was studied by measuring cross-reactivity of the antibody with structurally related compounds of ethinyl estradiol (<7%), estrone (<0.1%), estriol (<0.1%), and diethylstilbestrol benzoate (<0.1%). Chicken, fish, shrimp, urine and bile spiked with different concentration of DES were detected by the developed method, and the recovery rates were greater than 79.5%. Intra-and inter-assay variations were about 6%. This method exhibited high stability with a coefficient of variation less than 10% in buffer and in real samples. The LODs in fish/shrimp, liver, feed and urine spiked with DES were 600, 600, 4800 and 600 pg mL -1 , respectively. These results confirmed that the antibody to DES was successfully produced and could be used to establish ELISA methods for DES detection in food producing animals. Diethylstilbestrol (DES; Figure 1) is an orally active synthetic non-steroid estrogen. Since the 1960s, DES has been used as a growth-promoting agent in livestock [1,2]. Later, DES was found to cause cancer and its use was "phased out in the 1970s" by the FAO/WHO. In 1971, DES was reported to be a teratogen when given to pregnant women and has been associated with a rare vaginal cancer in female offspring [3]. On February 5, 1975, the US Food and Drug Administration (FDA) ordered 25 and 100 mg tablets of DES withdrawn; however, it was still illegally used in the livestock industry.*Corresponding authors (email: xirimo2000@yahoo.com; jintliu@sdu.edu.cn)More than 30 years of researches have confirmed that DES is a teratogen, which can cause malformations of an embryo or fetus. Prenatal exposure to DES is associated with an increased incidence of fibroids of adulthood [4,5]. Therefore, the Ministry of Agriculture of the People's Republic of China issued a banned list (Announcement No. 235; http://www. agri.gov.cn/ zcfg/bmgz/ t20060123_540865.htm, accessed on October 24, 2002) of veterinary drugs and announced that the use of DES in food and animal feed was prohibited. The European Union (EU) requires that residual levels of DES in edible animal food should not exceed 2 ng mL -1 [6]. Consequently, a rapid screening method with high sensitivity and

Development of an immunochromatographic assay for hexestrol and diethylstilbestrol residues in milk

“…Table 2 shows the CR of anti-HES mAb with different compounds. It was obviously noticed that there was no significant CR with other estrogen used in this study; this finding was in agreement with other research reported on stilbene antibodies (Li et al, 2011;Wang et al, 2008). Therefore, the results revealed that the produced 2E6 mAb had a high specificity against HES and more affinity against DES.…”

“…The synthesis of 4-(4-(4-(4-hydroxyphenyl)hexan-3-yl)phenoxy)butanoic acid (HES-4C) was accomplished by linking ethyl 4 bromobutanoate to one of HES ′ -OH group using the method as described by Li et al (2011) and Wang et al (2008) with slight modification (Figure 1). Briefly, 200 µl of ethyl 4 bromobutanoate dissolved into 1 ml of DMSO was slowly introduced into a solution of 15 ml of DMSO containing 0.5 mg of HES and 0.25 g of K 2 CO 3 ; then, the mixture was heated to 60°C for 6 h. After cooling to room temperature, the mixture was extracted with Diethyl ether and the ether extract was dried in fume hood; the product obtained was saponified to produce HES-4C acid.…”

Pharmacokinetics of Geniposide by Monoclonal Antibody-Based icELISA in Mice after Oral Administration of Huanglian-Jiedu-Tang

“…20) In addition, icELISA methods were developed for detection of okadaic acid and dinophytoxin-1 in seafood, 21) and diethylstilbestrol in biological samples. 22) Pharmacokinetic studies are very important in drug development. Currently, high-performance liquid chromatography (HPLC), 23) HPLC-mass spectrum (MS)/MS, 24) gas chromatography (GC) 25) and GC-MS 26) are the main methods employed in pharmacokinetics.…”

A sensitive, rapid chemiluminescence ELISA for the detection of 1, 4-bisdesoxycyadox residue in chicken muscle and liver

“…The limit of detection was determined as the concentration that caused 10% inhibition 14,20,21 and the linear range was determined as the concentrations that caused 20-80% inhibition in both ic-ELISA and CL-ELISA. Cross-reactivity was determined using the optimized CL-ELISA system and ic-ELISA and was calculated as follows: percentage of cross-reactivity ¼ 100 Â IC 50 (Cy4)/IC 50 (the tested compound).…”