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AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE
Citation:
Kitchin, K T., W L. Anderson, AND M. Suematsu. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE. Oxygen Society, RTP, NC, 11/15-19/2001.
Description:
AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE
Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of glutathione, metal exposure (e.g. arsenic and cadmium), hydrogen peroxide, hyperoxia, hypoxia, heat shock and irradiation. Induction of HO-1 is thought to protect against oxidative damage and to help restore normal redox potential in the cell. The spectrophotometric assay for HO measures bilirubin production and does not distinguish " between the HO-1 and HO-2 isoforms. We developed an enzyme linked immunoadsorbent assay (ELISA) to separately quantitate HO-1 .This ELISA assay utilizes the double antibody capture technique. First, monoclonal antibody to HO-1 is immobilized on microtiter plates. Second, HO-1 containing samples are treated with a detergent (nonident P40) to solubilize the membranes and release the HO-1 protein. Samples are then added directly to the microtiter plates and the binding of the HO-1 protein antigen to the capture antibody occurs. In the third step, the second antibody, polyclonal guinea pig antiserum raised against purified HO-1 protein, is added to the wells. In the fourth step, an anti-guinea pig IgG conjugated to alkaline phosphatase is added. 4-Methylumbelliferylphosphate or p-nitrophenylphosphate can be used as the substrate and product formation is detected by either fluorescence at excitation of 355 nm and emission at 460 nm or by spectrophotometry at 405 nm, respectively. This HO-1 specific ELISA requires 6 hours to complete. The EC50 is about 20 ng/ml for HO-1 , and the minimum detectable level of HO-1 is about 1 ng/ml. Excellent specificity for the HO-1 isoform is shown by the failure of either HO-2 human protein or HO-2 peptide (at concentrations as high as 1000 ng/ml) to generate any signal above background. Preliminary studies show that as little as 1 .0 uL of rat liver microsomes (0.1 g tissue/mi homogenate) gives a strong signal in this ELISA HO-1 assay.
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