Classical Complement Pathway Assay Kit [Rat] (CAT#: CB-P066-K) Datasheet

Product Type
Kit
Description
The kit can measure the classical pathway activity mediated by IgM. Complement defects or other defects in the complement system can be easily screened by analyzing each pathway in parallel or separately. ELISA contains a positive control based on the Wistar strain, which can be used as a process control to ensure that the classical complement cascade is fully performed. This positive control cannot be used to determine the activation level between batches. We recommend that you include a reference sample of 100% complement activity and a negative control (such as unpreserved activated serum) in your study to determine the activation level of the sample. Note that the level of complement activity depends on the rat strain and may be affected by the way the sample is collected and processed. The innate immune system is important in the important defense against foreign pathogens. The main component of this response is the complement system. The complement system consists of a complex family of proteins and receptors, which are found in the circulatory system, tissues and other body fluids. Today, some connections with adaptive immunity are also described. The system consists of three defined pathways, which are activated by pathway-specific molecules. Complement activation proceeds in a sequential manner through the proteolytic cleavage of a series of proteins, resulting in the production of activation products that mediate various biological activities through interactions with specific cell receptors and other serum proteins. These three pathways are the classical pathway, the lectin pathway and the alternative pathway, which converge into the final universal pathway at the central component. That is, the activation of C3 leads to the formation of C3a and C3b. This cleavage activates the terminal complement pathway, which ultimately leads to the formation of the terminal C5b-9 complement complex (TCC). The classical pathway is initiated by the binding of C1q to the antibody complex, while the alternative pathway and the lectin pathway are through the interaction of complement components with specific carbohydrate groups and lipopolysaccharides (LPS) on the surface of foreign pathogens, respectively. The antibody is activated in an independent manner. Alternative pathways also serve as amplification loops for other pathways. Under certain conditions, the complement system may be detrimental to the host, causing for example. Autoimmune diseases and infections. The lack of C3 is, for example, related to SLE. Changes in alternative pathways, such as properdin or fibronectin deficiency, can increase susceptibility to infection. Mannose-binding lectin (MBL) is the main component of the lectin pathway and is related to bacterial, fungal and viral infections. A common method to measure the activity of the classical or alternative pathway is hemolysis of red blood cells. In addition, several assays have been described to measure the activity of the MBL pathway. Not all determinations are easy to perform. By using a combination of specialized coatings and buffers, the rat approach ELISA is easy to use and specific. Complement defects or other defects in the complement system can be easily screened by analyzing each pathway in parallel or separately.
Applications
The rat classical complement pathway ELISA is a qualitative/semi-quantitative ELISA used to determine the activation of the classical pathway of the complement system in serum and plasma samples in vitro.
Principle
The Rat Classical Complement ELISA kit is a ready-made solid-phase enzyme-linked immunosorbent assay based on the sandwich principle. The working time is 3½ hours. The effective form of the plate with twelve disposable 8-well plates allows free choice of batch size for determination. Samples and positive controls are incubated in microtiter wells coated with antibodies. The biotinylated tracer antibody will bind to the bound C5b-9 in the sample and control. The streptavidin-peroxidase conjugate will bind the biotinylated tracer antibody. Streptavidin peroxidase will react with the substrate tetramethylbenzidine (TMB). The enzyme reaction is stopped by adding oxalic acid. Measure the absorbance at 450nm with a spectrophotometer.
Size
1 x 96 det.
Detection Method
Enzyme immunoassay (ELISA) technique
Sample
Serum, plasma, tissue homogenates, cell lysates, cell culture supernates and other biological fluids
Specimen Collection
Blood samples were collected using sterile venipuncture techniques, and serum was obtained using standard procedures. At least 5 ml of whole blood is recommended. Let the blood clot in the serum test tube for 60-65 minutes at room temperature (20-25°C). Centrifuge the blood sample and transfer the cell-free serum to a clean test tube. The serum must be handled correctly to prevent complement activation in vitro. Serum should be frozen in a sealed tube at -70°C or lower for long-term storage or transport on dry ice. The sample should not be frozen and thawed more than once. Do not use jaundice, lipemia and hemolytic serum. You cannot use heat-inactivated serum. Plasma cannot be used.
Sample Volume
100 µl/well
Species Reactivity
Rat
Protocol Length
2 hours
Storage
Product should be stored at 4 °C. Under recommended storage conditions, product is stable for at least six months.
Research Use
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.

All products and services are for Research Use Only. Do Not use in humans.

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