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Haptoglobin (Hp) Typing ELISA Assay Kit

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 Product Description

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Haptoglobin (Hp) Typing ELISA Assay Kit:
For Research Use Only
Size:  1x96 wells
Sensitivity:  Cut-Off Control
Incubation Time:  <90 min 
Sample Type:  Diabetic Serum/Plasma
Sample Size: Variable. 1:10 Sample Dilution 

Product Developed and Manufactured by Savyon Diagnostics

Intended Use

This Eagle Biosciences Haptoglobin (Hp) Typing ELISA Assay Kit is intended for use for diabetic patients only, for the qualitative determination of Hp phenotypes (Hp 1-1, Hp 2-1, or Hp 2-2) in human serum/plasma, to be used in conjunction with clinical evaluation and patient assessment as an aid in predicting risk of coronary arterial and cardiovascular disease. This Eagle Biosciences Haptoglobin (Hp) Typing ELISA Assay Kit is for research use only and not for use in diagnostic procedures.   


Haptoglobin (Hp) is a normally occurring acute phase serum protein whose primary physiological role is a scavenge free hemoglobin (Hb), a potent oxidizing agent, from the circulation (1). Free Hb, released during hemolysis of red blood cells, promotes the accumulation of hydroxyl free radicals which can cause oxidation damage to tissues. Hp acts as an antioxidant by first forming complexes with Hb and then clearing the complexes from the circulation by uptake via the CD163 macrophage receptor (2). 

Hp is polymorphic in man and occurs as either one of three phenotypes, Hp 1-1, Hp 2-1, or Hp 2-2. The prevalence of the three phenotypes of Hp is 16% Hp 1-1, 48% Hp 2-1, and 36% Hp 2-2 (1). Substantial evidence supports the pathogenetic role for the Hp 2-2 phenotype (3). First, the clearance of the Hb/Hp complex is Hp phenotype dependent with Hp 1-1/Hb complexes being cleared more efficiently than Hp 2-2/Hb complexes (4). Second, the Hp 2-2/Hb complex is an inferior antioxidant compared to the Hb 1-1/Hb complex in studies measuring conjugated diene formation of linolenic acid or TBARS formation by oxidized LDL (5). Third, Hp 1-1 is more efficient in preventing heme release from Hp/Hb complexes than Hp 2-2 (6), a finding that may help explain differences in antioxidant capabilities between the different Hp types. Finally, recent studies show impaired reverse cholesterol transport in diabetics carrying the Hp 2-2 genotype, presumably due to the binding of Hp 2-2/Hb complexes to HDL followed by subsequent iron-mediated oxidative damage (7). The presence of the Hp 2-2 phenotype in diabetic individuals predicts cardiovascular risk. Several longitudinal studies have established that the Hp 2-2 phenotype is an independent risk factor for cardiovascular disease in type 1 and type 2 diabetics (3, 8-11). Although the distribution of Hp phenotypes is not different in individuals with or without diabetes (1), the Hp 2-2 phenotype was shown to be a risk factor only in patients with diabetes. This may occur because in a diabetic patient, glycosylation of hemoglobin and the reduction of macrophages expressing the CD163 receptor may contribute to the increase in oxidative stress and tissue damage (4, 12). It has been shown that the oxidation of LDL by gycosylated hemoglobin is not completely blocked by binding to Hp (4) and the impaired removal of the complexes results in their localization in HDL particles (13). This increased oxidation of lipoproteins by the Hp 2-2/Hb complexes like contributes to the development of vascular complications in diabetics.  

Assay Principle

Haptoglobin (Hp) typing ELISA pates are supplied coated with purified monoclonal antibody (mAb) directed against Hp. The serum to be tested in diluted and incubated in the Hp ELISA plate. In this step Hp in the serum binds to the immobilized antibody. The non-bound Hp is removed by washing. Horseradish Peroxidase (HRP) conjugated monoclonal antibody to Hp is added. Note: the same mAb in the unconjugated form was used to coat the microtiter wells. Since Hp 1-1 is dimeric, at most only one HRP conjugated mAb can bind per dimer attached to the well. However, Hp 2-1 and Hp 2-2 are polymeric and can potentially nind 2-8 HRP conjugated mAbs. Unbound conjugate is removed by washing. TMB substrate reagent is added resulting in development of a blue color. The blue color development is stopped with the addition of stop solution changing the color to yellow.  Absorbance is measured using a spectrophotometer at 450 nm. The absorbance of each sample is compared to a user-calculated cut-off to determine its Hp phenotype.


1. Langlois, M.R. and Delanghe, J.R. Biological significance of haptoglobin polymorphism in humans. Clinical Chemistry 42:1589-1600 (1996).
2. Kristiansen, M., Graversen, J.H., Jacobson, C., et al. Identification of the hemoglobin scavenger receptor. Nature 409:198-201 (2001).
3. Costecou, T., Ferrell, R.E., Orchard, T.J. Haptoglobin genotype: a determinant of cardiovascular complication risk in type 1 diabetes. Diabetes 57:1702-1706 (2008).
4. Asleh, R., Marsh, S., Shilkrut, M. et al. Genetically determined heterogeneity in hemoglobin scavenging and susceptibility to diabetic cardiovascular disease. Circulation Research 96:435-441 (2005).
5. Melamud-Frank, M., Lache, O., Enav, B.I., et al. Structure/function analysis of the anti-oxidant properties of haptoglobin. Blood 98:3693-3698 (2001).
6. Bamm, V.V., Tsemakhovich, V.A., Shaklai, M., et al. Haptoglobin phenotypes differ in their ability to inhibit heme transfer from hemoglobin to LDL. Biochemistry. 2004 43:3899-906.
7. Asleh, R., Miller-Lotan, R., Hayek, A.M., et al. Haptoglobin genotype is a regulator of reverse cholesterol transport in diabetes in vitro and in vivo. Circulation Research 99:1419-1425 (2006).
8. Levy, A.P., Hochber, I. Jablonski, K., et al. Haptoglobin phenotype is an independent risk factor for cardiovascular disease in individuals with diabetes: the strong heart study. Journal of the American College of Cardiology 40:1984-1990 (2002).
9. Roguin, A., Koch, W., Kastrati, A. et al. Haptoglobiin genotype is predictive of major adverse cardiac events in the 1 year period after percutaneous transluminal coronary angioplasty in individuals with diabetes. Diabetes Care 26:2628-2631 (2003).
10. Suleiman, M., Aronson, D., Asleh, R., et al. Haptoglobin polymorphism predicts 30-day mortality and heart failure in patients with diabetes and acute myocardial infarction. Diabetes 54:2802-2806 (2005).
11. Milman, U., Blum, S., Shapira, C., et al. Vitamin E Supplementation reduces cardiovascular events in a subgroup of middle-aged individuals with both type 2 diabetes mellitus and the haptoglobin 2-2 genotype. Atherosclerosis, Thrombosis, and Vascular Biology 28:341-347 (2007).
12. Asleh, R. and Levy, A.P. In vivo and in vitro studies establishing haptoglobin as a major susceptibility gene for diabetic vascular disease. Vascular Health and Risk Management 1:19-28 (2005).
13. Asleh, R., Blum, S., Kalet-Litman, S. et al. Correction of HDL dysfunction in individuals with diabetes and the haptoglobin 2-2 genotype. Diabetes 57:2794-2800 (2008).
14. Levy, N.S., Vardi, M., Blum, S., et al. An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype. Clinical Chemistry and Laboratory Medicine 51: 1615–1622 (2013).
15. USA Center for Disease Control/National Institute of Health Manual, "Biosafety in Microbiological and Biomedical Laboratories", (1984).
16. Wassell, J., Keevil, B. A new method for haptoglobin phenotyping. Annals of Clinical Biochemistry 36: 609-612 (1999).
17. Interference testing in Clinical Chemistry – Approved Guideline-2nd addition. CLSI document EP07-A2. Wayne, PA. Clinical and Laboratory Standards Institute, 2005.


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