An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

Abstract

Background Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. Methods A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. Results Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. Conclusions The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies.

Effect of vitamin E supplementation on HDL function by haptoglobin genotype in type 1 diabetes: results from the HapE randomized crossover pilot trial

“…Exclusion criteria included acute coronary syndrome in the prior six months, pancreatic transplant, unwillingness to limit antioxidant vitamin use to trial medications, or a known allergy to vitamin E. Willing and eligible participants were scheduled for a first clinical visit, where study aims were explained in detail, eligibility was re-assessed, and written informed consent and a blood sample for Hp genotype assessment were obtained, if needed (the majority of EDC participants had already had Hp genotype determined). Samples collected were stored in −70 C freezers and batched until Hp genotype was assessed by an ELISA test [21]. All follow-up procedures concluded by December 2013.…”

The Haptoglobin 1 Allele Correlates With White Matter Hyperintensities in Middle-Aged Adults With Type 1 Diabetes

“…Hp was genotyped by an amplification method ( 18 ). When DNA was unavailable, Hp phenotype was assessed ( 19 ).…”

Haptoglobin genotype and cerebrovascular disease incidence in type 1 diabetes

“…Glomerular filtration rate (eGFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI) (27). For 486 participants who provided DNA, Hp was genotyped by an amplification method (28) as previously described (29) while for 129 participants without DNA but with stored blood samples available, Hp phenotype was assessed using an Elisa test (30). Of 615 participants with DNA available, four individuals (one Hp 1-1, one Hp 2-1 and two Hp 2-2) with prevalent cerebrovascular disease and an additional four missing clinical information were excluded from the analyses.…”