Rapid and sensitive enzyme-linked immunosorbent assay for the microsomal epoxide hydrolase

Abstract

A rapid and sensitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for microsomal epoxide hydrolase of rat liver. The assay, which is easily and readily performed, is significantly more sensitive than most enzymatic epoxide hydrolase assays routinely used and electroimmunoassays previously developed. The limit of sensitivity of the ELISA is between 2-5 ng of microsomal epoxide hydrolase. Using the ELISA microsomal epoxide hydrolases of mouse and rat liver were shown to be antigenically very similar, while microsomal epoxide hydrolases of guinea pig, monkey and human liver are antigenically distinct from those of rat and mouse. The ELISA developed here is capable of detecting microsomal epoxide hydrolase of rat and mouse liver even when significant enzymatic activity is lost. These results indicate that the antigenic sites recognized by the antibodies used are distinct from the catalytic site of the epoxide hydrolase. Approximately 1.9% of rat microsomal protein was quantified as microsomal epoxide hydrolase by the ELISA. Low levels of microsomal epoxide hydrolase were also detected in rat liver cytosol (approximately 0.02% of the cytosolic protein) demonstrating that microsomal epoxide hydrolase is not totally membrane bound or that an immunologically related protein occurs in the cytosol of normal rat liver. The ELISA developed here will be valuable in investigating further the role of microsomal epoxide hydrolase.

Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase

“…Several assays have been developed to measure the potency of sEH inhibitors since the first assay was reported; however, most of these are substrate based [5,11,21,44,45]. These substrate-based assays are attractive for mechanistic studies and allow one to prioritize the inhibitors with IC 50 values ranging from 0.4 to 100,000 nM.…”

“…Over the years, numerous substrate-based assays have been developed and used [11–20]. Existing sEH assays are able to distinguish among inhibitors of varying potency down to low nanomolar.…”

A methodology for the analysis of the preneoplastic antigen

“…The immunopredpitation assay was run with serum pooled from two animals showing high PNA activity with CSO in the standard assay. Forty pi of solubilized microsomes (diluted to 0.5% original w/v with phosphate-buffered saline -PBS) or rat serum were incubated with 10-100/Jofantiserum (1:1500 dilution in PBS) from a rabbit immunized with rat liver microsomal epoxide hydrolase (17,18). Normal rab-bit serum (1:1500 dilution) was added to bring the total volume to 140 /J.…”

Autoantibody response to microsomal epoxide hydrolase in hepatitis C and A

“…The mEH is a drug-metabolizing enzyme that plays an important role in the metabolism of some mutagenic and carcinogenic epoxides [29]. It is mainly expressed on the ER membrane in the liver [29] and constitutes about 2% of microsomal fraction [30]. It is also expressed on the surface of hepatocytes [31] and may act as a sodium-dependent bile acid transporter [32].…”