An ELISA assay for heme oxygenase (HO-1)

A double antibody capture ELISA for the HO-1 protein has been developed to separately quantitate HO-1 protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-1 protein from membranes and/or other cellular entiti es and increased the amount of HO-1 protein found in rat liver whole homoge nates as well as the nuclear, mitochondrial and microsomal fractions. Use o f the detergent NP40 did not substantially change HO-1 protein standard cur ves. The ELISA assay for HO-1 has been shown to be reproducible over (i) a 4-day trial period as well as (ii) almost 1 year of general laboratory use. Excellent specificity for the HO-1 isoform is shown by the failure of eith er the human HO-2 protein or HO-2 peptide (at concentrations as high as 100 0 ng/ml) to generate any signal above background. At least a 300-fold great er signal comes from HO-1 protein as compared to the HO-2 protein. The EC,, , is about 200 ng/ml for HO-1, and the minimum detectable level of the HO-1 protein is about 1 ng/ml. The ELISA assay for the HO-1 protein requires a total of 6 h to complete. Of the total cellular HO-1 protein, 20, 19, 9 and 3% appeared in the nuclear, microsomal, mitochondrial and high speed super natant fractions, respectively. As expected, the highest concentration of H O-1 protein per total protein in a subcellular fraction was found in the mi crosomes. For many research projects utilizing this ELISA assay for HO-1 pr otein concentration, use of the whole homogenate will be an excellent choic e, rather than use of the postmitochondrial or microsomal fractions. Much h igher HO-1 protein levels were found in tissues of rats rather than mice. T his may be because the capture antibody and secondary antibody were both ra ised against the rat and not the mouse forms of the HO-1 protein. In rats t he HO-1 concentrations were 1067, 364, 194, 31, 28 19, 5 and 2 ng/g tissue in whole homogenates from testes, brain, liver, lung, spleen, kidney, small intestines and urinary bladder, respectively. The ELISA assay for HO-1 des cribed here will be useful for HO-1 research studies in tissues and cell cu ltures of rats and mice. This ELISA for HO-1 may also work with human tissu es and cells. (C) 2001 Published by Elsevier Science B.V.