Manual
Description
Complement is a major defense system of innate immunity and aimed to destroy microbes. 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 protected against it. One of the central complement regulators of the alternative pathway is complement factor H (CFH). CFH is the major soluble inhibitor of the alternative pathway of the complement system and plays a key role in controlling complement activation
in vivo. CFH is a 150-kDa plasma protein that is mainly produced in the liver and is present in plasma at a concentration of approximately 500 µg/ml. The molecule is made up entirely of a string of 20 folded globular domains known as short consensus repeats (SCRs).
Alternative pathway activation results from a failure to appropriately regulate the constant low level of the abundant complement complement C3 to C3(H2O) due to spontaneous turnover. Complement non-activating cells and other host surfaces are protected from alternative pathway complement attack through binding of CFH. CFH inhibits the alternative pathway by binding C3b and reducing the formation and activation of the alternative pathway C3-convertase (C3bBb). It also accelerates the decay of this convertase and works as a cofactor for the serine proteinase factor I in the degradation of C3b into inactive C3b.
CFH is associated with several diseases like atypical hemolytic syndrome (aHUS), membranoproliferative glomerulonephritis type ll (MNGN) and age-related macular degeneration (AMD). In the western world, AMD is the leading cause of natural irreversible blindness in the elderly, affecting 50 million individuals worldwide. A common polymorphism in the
CFH gene on chromosome 1, has been determined to be strongly associated with a person's risk for developing AMD. The
CFH gene polymorphism is characterized by a T-to-C single nucleotide substitution resulting in a tyrosine-histidine change at amino acid position 402 . The sequence change is in a region of CFH that binds heparin and C-reactive protein. Individuals homozygous for H402 (genotype CC; CFH-HH402) have up to 12-fold increased risk for developing AMD, while heterozygotes (genotype CT; CFH-HY402) have a 2.5-fold greater risk for developing AMD than individuals homozygous for 402Y (genotype TT; CFH-YY402). The CFH-HH402 and CFH-HY402 variants, likely explain about 43% of AMD in older adults.
Application
The Complement factor H, H402 and Y402 variant detection ELISA has been developed for the detection of complement factor H variant in serum and plasma. In serum or plasma samples, the H402/Y402 variant of complement factor H can be determined accurately if serum or plasma samples are diluted at least 250 times.
Features
- Detection of H402 and Y402 variants of complement factor H.
- Working volume of 50 µl/well.
- Working time of 1.5 hours.
Principle
- The HK353 ELISA is a ready-to-use solid-phase enzyme-linked immunosorbent assay based on the sandwich principle with a working time of 1½ hours.
- The efficient format of 1 plate with 3 (CFH, CFH-H402 and CFH-Y402) x 4 disposable 8-well strips allows free choice of batch size for the assay.
- Samples and controls are incubated together with peroxidase-conjugated secondond antibody (conjugate) in microtiter wells coated with antibodies recognizing all variants of CFH (colorless), CFH H402 (white) and CFH Y402 (black).
- During incubation CFH, CFH H402 or CFH Y402 are captured by the solid bound antibody. The conjugated antibodies will bind to the captured CFH variants.
- The peroxidase conjugated antibody will react with the substrate, tetramethylbenzidine (TMB).
- The enzyme reaction is stopped by the addition of citric acid.
- The absorbance at 450 nm is measured with a spectrophotometer. The polymorphism (Y402H) of complement factor H can be determined from the controls.
Aliases
CFHL1; CFHL3; HF1; HF2; HUS; MGC88246; beta-1-H-globulin
Storage and stability
Product should be stored at 4°C. Under recommended storage conditions, product is stable for at least six months. After reconstitution the reagents are stable for 1 month if stored at 2-8°C. After reconstitution the controls are stable for 4 hours. For longer stability we recommend to store aliquots at -20°C.
References
- Hakobyan, S et al; Measurement of factor H variants in plasma using variant-specific monoclonal antibodies: application to accessing risk of age-related macular degeneration. Invest Ophthalmol Vis Sci 2008, 49: 1983
Precautions
For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result from the use or derivation of this product.
Also available
Scientific info
Fast determination of AMD-associated complement factor H polymorphism
Complement factor H (CFH) is the major fluid-phase regulator of the alternative pathway of complement. It plays a key role in controlling complement activation in vivo. CFH is present in plasma at a concentration of ~500 mg/l and is mainly produced in the liver. Deficiency of CFH results in uncontrolled complement activation, and subsequent susceptibility for pyogenic infections. CFH deficiency leads to an increased susceptibility for type II membranoproliferative glomerulonephritis (MPGN II), implying a unique role for CFH in protecting the kidney from injury. Also, CFH has been shown to fulfill a regulatory role in the development of atherosclerosis.
Common polymorphisms in CFH have been associated with several diseases, including atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). AMD is the leading cause of vision loss in the elderly in Western societies. Several risk factors, including increasing age, cigarette smoking and arterial hypertension, have been identified. Since only a fraction of AMD cases can be fully explained by these risk factors, researchers have searched for genetic factors.
A common polymorphism in CFH (Y402H) was reported to correlate with the risk for developing AMD. Persons homozygous for polymorphic amino acid H402 suffer from an increased risk not only for developing AMD but also for myocardial infarction.
CFH H402 phenotype indicates increased risk for:
• Age-related macular degeneration (AMD)
• Myocardial infarction
• Cardiovascular disease (atherosclerosis)
“Special features of the immuno-assay”:
• Determines presence of CFH as well as Y402/H402 amino acid variants
• Useful for plasma and serum
• Positive controls are included
• Determination within 1.5 hours
Besides the assay for detection of Y402H polymorphism, Hycult Biotech also offers an ELISA for quantitative measurement of CFH. The relevance of CFH to homeostasis is apparent in patients with CFH deficiency. The majority of CFH-deficient individuals develop renal disease, predominantly haemolytic uraemic syndrome (HUS) and membranoproliferative glomerulonephritis (MPGN).