Cross-reactivity of an automated human haptoglobin immunoturbidimetric assay for detection of haptoglobin in swine serum.

Abstract

The acute phase response has been previously characterized in the pig.2,9 Haptoglobin (Hpt), an a-2 glycoprotein, is a normal serum protein that increases 5–7 fold within 24– 48 hours upon the advent of an acute inflammatory response.9 Serum Hpt increases as a result of natural infections and experimentally induced inflammation in pigs as in other species.1,2,6,9,11,12 Besides serum Hpt as a marker for inflammatory disease, it has been used as an indicator of weight gain4 and stress from environmental conditions.5 The serum Hpt concentration may therefore be useful as a monitor of overall health of pigs in a commercial production unit. Automated assays for measuring serum Hpt have been validated for use in dogs, horses, and sheep.7,11,13 A commercially available immunoturbidimetric assay for human Hpt has recently been validated for use in dogs and horses.13 The purpose of this study is to report on the cross-reactivity of this same assaya for use in determining serum Hpt concentrations in swine. Serum samples were obtained from pigs housed at the Veterinary Medicine Research Farm at the University of Illinois and from samples submitted to the Purina Mills Research Center for unrelated testing. Samples from the University of Illinois were collected by venipuncture into serum clot tubes, and after centrifugation, the serum was withdrawn and stored at 270 C until assayed. Those samples submitted to Purina Mills Research Center were treated in a similar fashion. Serum used for immunoelectrophoresis, western blot, and correlation studies were from the University of Illinois, and serum for all other experiments was from the Purina Mills Research Center. A cyanmethemoglobin (CHB) assay for serum Hpt was performed as previously described.3,4 Hpt was reported in milligrams of CHB binding capacity (HBC) per deciliter of serum. Samples with relatively low, medium, and high HBC were pooled and labeled respectively. The pooled samples were used for further experiments. Immunoelectrophoresis was performed as previously described13 using 1-ml aliquots of pooled swine serum containing various Hpt concentrations as determined by the CHB assay. Serum from a healthy human donor served as a control. Following standard electrophoresis on a 1.0% agarose gel film,b anti-human Hpt antiserum (anti-Hpt antibody) was added to diffusion wells on either side of each electrophoretic lane. The antiserum was allowed to diffuse for 24 hours at room temperature, forming immunoprecipitates. The film