Development of Monoclonal Antibodies to 4-Hydroxyestrogen-2-N-Acetylcysteine Conjugates: Immunoaffinity and Spectroscopic Studies

Abstract

Catechol estrogen quinones (CEQ) derived from oxidation of the catechol estrogens 4-hydroxyestrone (4-OHE1) and 4-hydroxyestradiol (4-OHE2) can conjugate with glutathione (GSH), a reaction that prevents damage to DNA and can provide biomarkers of exposure to CEQs. Monoclonal antibodies (MAb) to 4-OHE1(E2)-2-N-acetylcysteine [4-OHE1(E2)-2-NAcCys] were developed and characterized by immunological and spectroscopic studies. The NAcCys conjugate is the hydrolytic product of the corresponding conjugate with GSH, followed by N-acetylation of cysteine. MAbs were produced by immunizing mice with 4-OHE1(E2)-2-NAcCys attached to an appropriate linker that was conjugated to keyhole limpet hemocyanin (KLH). Hybridoma cell lines were screened using 4-OHE1(E2)-2-NAcCys conjugated to ovalbumin (OA). There is no immunological cross-reactivity between KLH and OA. Hence, positive hybridoma cell lines secreting antibody against 4-OHE1(E2)-2-NAcCys could be rapidly identified using OA-4-OHE1(E2)-2-NAcCys. An affinity column was developed and used to purify MAb against 4-OHE1(E2)-2-NAcCys. The purified MAb was immobilized on an agarose bead column. This column was used to capture and preconcentrate the hapten of interest out of urine samples. A number of structurally related standards were used to estimate the selectivity and specificity of the chosen MAb. Capillary electrophoresis (CE) with field-amplified sample stacking in absorbance detection mode and laser-induced low temperature luminescence measurements were used to identify and quantitate the 4-OHE1(E2)-2-NAcCys conjugates and related compounds released from the affinity column. Femtomole detection limits have been demonstrated. Future prospects in clinical diagnostics for testing human exposure to CEQ by urine analysis are briefly addressed.