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Complement factor B, Human, ELISA kit-0
Catalog # HK367

Complement factor B, Human, ELISA kit

The auto-activation process of the alternative pathway starts with the spontaneous hydrolysis of C3, which results in the formation of C3(H2O). In turn, C3(H2O) binds complement factor B. Once factor B associates with C3(H2O), factor B itself changes conformation and can then be cleaved by the constitutively active serum protease factor D, generating Ba and Bb.

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Complement is a major defense system of innate immunity and aimed to destroy microbes. There are three pathways of complement activation. The classical and lectin pathways are initiated by the binding of recognition proteins to specific targets. The classical pathway is activated by IgM, isotypes of IgG, and several other proteins such as C-reactive protein and serum amyloid P protein. The lectin pathway is initiated by the binding of mannose binding lectin to carbohydrate moieties found primarily on the surface of pathogens.

In contrast to the other two pathways, the alternative pathway is continuously activated as a result of spontaneous hydrolysis of complement component C3. Because of this spontaneous activating capability, the alternative pathway requires continuous regulation. Endogenous cells and tissues protect themselves from uncontrolled activation by expressing complement regulatory proteins (especially Factor H) that are able control alternative pathway activation.

An “activating” surface is, in large part, one without adequate regulatory protein function to control alternative pathway activation (such as the surface of pathogens). The auto-activation process starts with the spontaneous hydrolysis of C3, which results in the formation of C3(H2O). In turn, C3(H2O) binds complement factor B. Once factor B associates with C3(H2O), factor B itself changes conformation and can then be cleaved by the constitutively active serum protease factor D, generating Ba and Bb. The Bb fragment remains associated with the complex and can then, through its own serine protease domain, cleave additional C3 molecules, generating a form designated C3b. Once C3b is generated, it associates with factor B to generate more C3-convertase, resulting in the activation of the complement cascade. Factor B circulates in the blood as a single chain polypeptide. The human Factor B ELISA is to be used for the quantitative determination of Factor B in plasma samples.

Application
The Human Factor B ELISA kit is to be used for the in vitro quantitative determination of Factor B in plasma and serum samples. This kit is intended for laboratory research use only and is not for use in diagnostic or therapeutic procedures.

Principle
The Human Factor B 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 Factor B. Biotinylated tracer antibody will bind to the captured Human Factor B. 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 Factor B standards (log).  The Human Factor B concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.

Linearity
Linearity of 6 EDTA plasma samples was determined. CV of the samples is between 6.0 and 11.4.

Precision and reproducibility
The intra-assay was tested by testing 3 EDTA plasma samples in quadruple and was between 3.4 and 10.7%. The inter-assay was tested by testing 3 EDTA plasma samples in quadruple and was between 2.8 and 9.7%.

Product type
Assays
Quantity
1 x 96 det., 2 x 96 det.
Standard range
7.8 to 500 ng/ml
Detection level
7.8 ng/ml
Working volume
100 µl/well
Species
Human
Cross reactivity
Horse – No, Mouse – No, Pig – No, Rat – No
Alias
CFB
Storage and stability
Product should be stored at 4 °C. Under recommended storage conditions, product is stable for at least six months.
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 with the use of or derivation of this product.
Assay Manual
HK367
367 kb
Safety Data Sheet

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