I am looking for a kit, or well described assay to determine the functionality of complement factor H. I have seen brief protocols involving incubating different amounts of factor H with Facto I and C3b on ice, then incubating at 37*C, then running on SDS-PAGE. Is there a kit that has the Factor I and C3b available? Or do I need to purchase everything separately? If there is no kit for this, do you know of a well cited protocol anywhere?
for the first trials you can possibly perform a standard elisa against Factor H deficient plasma, check the antigenicity first , may be? even i am looking for CFH kits, but so far found nothing really good.
In my experience, fH-deficient serum is very unstable as it auto-activates easily. I suggest testing for cofactor activity of fH with fI and C3/C3b. You can then look for cleavage products. I used purified proteins purchased from CompTech in Tyler, TX (USA).
Hycult has very good kit
http://www.hycultbiotech.com/hk342-01
Complement factor H, Human, ELISA kit
Catalog #: HK342-01
Quantity: 1 x 96 det.
escription: Complement factor H, Human, ELISA kit
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)
Specifications
Catalog number HK342-01
Product type Assays
Quantity 1 x 96 det.
Standard range 3.9 - 250 ng/ml
Detection level 3.9 ng/ml
Working volume 100 µl/well
Species Human
Ishtiaq Qadri, this kit is for quantitating fH, not testing for function. Have you tried making your own fH ELISAs? It's much cheaper and has excellent results.
Thank you for the answers. I am not looking for ELISAs to test serum. My sample will not be serum, but instead fH that I purify myself. I am tagging it for other purposes so I want to make sure the tag is not interfering with functionality. Thus, I want to use my tagged fH with fI and C3b to see if it can still cleave. (I will also purchase a purified wt fH for a control comparison).
Check out my fH functional assay published in PLoS One http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038407 (C3b cleavage to iC3b) using purified proteins. It might help you.
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