CFI is a protein of the complement system (serine protease) , also known as as C3b/C4f inactivator, and is a protein that is encoded by the CFI gene located on chromosome 4. It regulates complement activation by cleaving cellbound or fluid phase C3b and C4b. CFI is synthesised predominantly in the liver, and is initially secreted as a single 88 kDa gene product; this precursor protein is then cleaved by furin to yield the mature CFI protein, which is a disulfide-linked dimer of heavy chain residues (residue 19-335, 51 kDa) and light chain (residues 340-583, 37 kDa). Only the mature protein is active. Genetic polymorphism in CFI has been observed (variants R201S, R406H, R502L). CFI deficiency leads to low levels of complement component 3 (C3) in plasma, due to unregulated activation of the complement alternative pathway, and it has been associated with recurrent bacterial infections in children. More recently, mutations in the CFI gene have been shown to be implicated in development of Haemolytic Uremic Syndrome, a renal disease also caused by unregulated complement activation.
The complement factor I ELISA kit is to be used for the in vitro quantitative determination of human CFI in plasma and serum.
The complement factor I ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 3½ 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 CFI. Biotinylated tracer antibody will bind to the captured human CFI. Streptavidin-peroxidase conjugate will bind to the biotinylated tracer antibody. Streptavidin-peroxidase conjugate 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 CFI standards (log). The human CFI concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
Recovery was determined by mixing CFI depleted serum with different plasma samples with a known CFI concentration. Samples were incubated for one hour at room temperature and measured using the ELISA. The recovery experiment met the requirements and ranged between 87.1% and 109.2%
Precision and reproducibility
The intra-assay precision and reproducibility was tested using four samples with three different dilutions. CV % ranged between 0.5 and 7.1%. To determine the inter-assay variation, four samples were tested by two operators both on three separate days. CV % ranged between 6.7 and 9.6%.