Acute phase proteins are plasma proteins which increase in concentration following infection,inflammation or trauma. The first acute phase proein to be recognized was discovered in humans by Tillet and Frances in 1930 (1). Haptoglobin (Hp) is a heterogeneous plasma protein mostly synthesized by the liver. The haptoglobin monomer consist of two heavy chains, beta chains (40kD) and two light chains, alpha chains, alpha 1 (9kD) and alpha 2 (16kD) that are linked disulfide bonds. The three major haptoglobin types are; Hp1-1 which is monomeric (98kD), Hp1-2 is polymeric at about 200kD, and Hp2-2 at about 400kD. The levels in serum rise quickly following acute tissue damage within 24 to 48 hours and also fall very rapidly once the stimulus is removed. In fact, Hp level are decreased in hemolytic anemia. Hp has a high affinity for hemoglobin (Hb) and its function appears to be to prevent loss of Hb in urine which would lead to loss of iron. Investigations over the past few years have shown that quantification of Hp in plasma or serum can provide valuable diagnostic information in the detection, prognosis, and monitoring of disease not only in humans, but in companion animals and farm herds as well (2).
Sample Type:Serum/Plasma
Intended Use:The Haptoglobin test kits are a highly sensitive twosite enzyme linked immunoassay (ELISA) formeasuring Haptoglobin in serum or plasma of Human. Crossreactivity with Cynomolgus Monkeys has been observed.
Test Principle:The principle of the double antibody sandwich ELISA is represented in Figure 1. In this assay theHaptoglobin present in samples reacts with the anti-Haptoglobin antibodies which have been adsorbed to the surface of polystyrene microtitre wells. After the removal of unbound proteins by washing, anti-Hp antibodies conjugated with horseradish peroxidase (HRP), are added. These enzyme-labeled antibodies form complexes with the previously bound Hp. Following another washing step, the enzyme bound to the immunosorbent is assayed by the addition of a chromogenic substrate, 3,3’,5,5’-tetramethylbenzidine (TMB). The quantity of bound enzyme varies directly with the concentration of Hp in the sample tested; thus, the absorbance, at 450 nm, is a measure of the concentration of Hp in the test sample. The quantity of Hp in the test sample can be interpolated from the standard curve constructed from the standards, andcorrected for sample dilution.
Kit Components:Diluent concentrate (Running Buffer), One bottle containing 1x50ml of a 5X concentrated diluent running buffer.
Wash solution concentrate, One bottle containing 1x50ml of a 20X concentrated wash solution.
Enzyme-antibody conjugate 100X, One vial containing 1x150ul of affinity purified anti-Human Haptoglobin antibody conjugated with horseradish peroxidase in a stabilizing buffer.
Chromogen-substrate solution, One vial containing 1x12ml of 3,3’,5,5’-tetramethybenzidine (TMB) and hydrogen peroxide in citric acid buffer at pH 3.3.
Stop solution, One vial containing 1x12ml 0.3M sulfuric acid.
Anti-human haptoglobulin ELISA micro plate, Twelve removable eight (8) well micro well strips in well holder frame. Each well is coated with affinity purified anti-Human Hp.
Human haptoglobulin calibrator, One vial containing a lyophilized Human Haptoglobin calibrator.
Storage and Stability:Store all components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap.
