Detection of Haptoglobin from Concentrated Urine Samples by Enzyme Immunoassay

Abstract

The detection of haptoglobin (Hp) from serum and bloodstains is utilized extensively in forensic science laboratories in order to include or exclude possible donors. There is an increasing need to make the same discriminations utilizing genetic markers from urine samples. This paper describes the use of enzyme immunoassay and Western blotting (electrophoretic) techniques to determine Hp phenotypes from concentrated urine samples. Serum and urine specimens were collected from volunteer donors. The serum sample from each donor was typed for Hp. The urine specimens were concentrated 3000-fold from the starting volume of 15 mL to a final volume of 5 microL and applied to the gradient polyacrylamide gels. This procedure allows the separation of Hp samples into the three common phenotypes as well as the other rare variants found in humans. The Western blotting electrophoretic technique was used to achieve the transfer of Hp bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-Hp antiserum and rabbit anti-goat immunoglobulin alkaline phosphatase conjugate were used to identify the Hp bands from the concentrated samples. Specimens stored for six months at -22 degrees C were also concentrated and typed successfully. Recent implementation of drug-screening policies has resulted in an increase in the submission of substituted urine specimens. The above procedure can be used to detect an additional genetic marker from urine samples and thus facilitate the identity of the donor.