Complement factor H (CFH) is the first regulatory protein of the alternative pathway of the complement system. There are three pathways of complement activation. The classical pathway is initiated by Immune complexes; the lectin pathway by surface bound mannan binding lectin; and the alternative pathway by all the surfaces that are not specifically protected against it. Each generates a C3 convertase, a serine protease that cleaves the central complement protein C3, and generates the major cleavage fragment C3b. The complement system mediates a number of essential biological functions that participate in host defense against infection, initiation of the inflammatory reaction, processing and clearance of immune complexes and regulation of the immune response. CFH is a single-chain serum glycoprotein of 150 kD with a modular structure consisting of a tandem of 20 homologous units of about 60 amino acid, called short consensus repeats (SCR). Numerous functional sites have been identified along the 20 SCR domain structure of factor H. Three C3-binding sites have been identified; in the SCR1-4 in SCR6-10 and SCR13-20. Three polyanion binding sites like heparin and several glycoaminoglycans have also been identified in the SCR7, 13 and 20. FH displays anti inflammatory functions and acts as a ligand for CRP. CFH has two important functional domains that are located at the opposite ends of the protein. The N-terminal fragment of the factor H molecule is an essential fluid phase regulator of the alternative pathway. With the C terminal domain and SCR 7 CFH binds to cell and tissue surface and thus mediates its protective role also on host cell surface. CFH is a relatively abundant plasma protein, with a concentration of 400-800 µg/ml, that is essential to maintain complement homeostasis and to restrict the action of complement to activating surfaces. CFH binds to C3b, accerates the decay of the alternative pathway C3-convertase (C3bBb) and act as co-factor for the factor I-mediated proteolytic inactivation of C3b. Factor H regulates complement both in fluid phase and on cellular surfaces. Genetic analyses reveal a clear association of CFH with different human diseases. These incude diseases of the kidney, the atypical form of Hemolytic Uremic Syndrome (aHUS) and membranoproliferative glomerulonephritis (MPGN), and of the eye, age-related macular degeneration (AMD)
The human complement factor H ELISA kit is to be used for the in vitro quantitative determination of human complement factor H in serum, plasma and urine samples.
The human complement factor H ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 2½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are incubated in microtiter wells coated with antibodies recognizing human complement factor H.
Peroxidase conjugate antibody will bind to the captured human complement factor H.
Peroxidase conjugate antibody will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the human complement factor H standards (log).
The human complement factor H concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Normal human blood samples (plasma), containing baseline levels of human complement factor H, were spiked with human complement factor H in concentrations of 62.5 and 15.6 ng/ml. Samples with and without human complement factor H were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Values for human complement factor H ranged between 65% and 134% (mean 102%).