Cat No.: 31400 Other Names: Hp
Introduction
Human haptoglobin is an acute-phase glycoprotein produced predominantly by liver. It is composed of two a subunits (M.W. 16-23
kDa) and two b subunits (M.W. 35-40 kDa). Haptoglobin can bind to free hemoglobin released from lysed erythrocytes and prevent
the formation of free radical superoxide that can be formed by the reaction of oxygen and iron from hemoglobin. It is also known to
be involved in immune regulation and anti-inflammation. Elevated amount of haptoglobin is observed during infections and
inflammations, obesity, tissue damage etc. Hence, haptoglobin is used as a biomarker to detect acute allograft rejection, proliferative
diabetic retinopathy (PDR) and diabetic kidney disease (DKD). Additionally, low haptoglobin levels are mainly observed during
hemolytic anemia.
Principle of the Assay
This assay is a quantitative sandwich ELISA.The micro-plate is pre-coated with a rabbit polyclonal antibody against human
haptoglobin. Standards and samples are pipetted into the wells and any human haptoglobin present is bound by the immobilized
antibody. After washing away any unbound substances, a horseradish peroxidase (HRP)-linked polyclonal antibody specific for
human haptoglobin is added to the wells. After the last wash step, an HRP substrate solution is added and colour develops in
proportion to the amount of human haptoglobin bound initially. The assay is stopped, and the optical density of the wells are
determined using a microplate reader. Since the increase in absorbance is directly proportional to the amount of captured human
haptoglobin, the unknown sample concentration can be extrapolated from a reference curve included in each assay.
Assay Performance
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each assay.
Human haptoglobin (ng/mL) | Absorbance (450 nm) | Blanked Absorbance |
0 | 0.087 | 0.000 |
3.12 | 0.135 | 0.048 |
6.25 | 0.194 | 0.107 |
12.5 | 0.347 | 0.260 |
25 | 0.692 | 0.605 |
50 | 1.349 | 1.262 |
100 | 2.650 | 2.563 |
B. Sensitivity
The lowest human haptoglobin level that can be measured by this assay is 1.56 ng/mL.
C. Precision
Intra-assay Precision (Precision within an assay) C.V.<5.63%.
Inter-assay Precision (Precision between assays) C.V.<1%.
D. Spiking
The recovery of human haptoglobin was 104.4%.
E. Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human haptoglobin were serially
diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.
Sample Dilution | Absorbance (450 nm) | Concentration (ng/mL) | Recovery (%) |
1:2 | 0.601 | 5.843 | 95.37 |
1:4 | 0.269 | 6.010 | 98.10 |
1:8 | 0.116 | 6.790 | 110.82 |