This serum amyloid A (SAA) ELISA kit applies a technique called quantitative sandwich immunoassay. This SAA ELISA kit is a 2.5-hour solid phase immunoassay readily applicable to measure SAA in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 80 ng/mL. Serum amyloid A (SAA) proteins are a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma. Different isoforms of SAA are expressed constitutively (constitutive SAAs) at different levels or in response to inflammatory stimuli (acute phase SAAs). These proteins are predominantly produced by the liver. The conservation of these proteins throughout invertebrates and vertebrates suggests SAAs play a highly essential role in all animals. This SAA ELISA kit recognises both natural and recombinant human SAA. This SAA ELISA kit exhibits no detectable cross-reactivity with human; IL-8, IL-1ß, MCAF, FGF, TGF-ß, EGF, GM-CSF, M-CSF, MCP-3, TNF-α, RANTES, and EPO.
The microtiter plate provided in this SAA ELISA kit has been pre-coated with a monoclonal antibody specific for SAA. Standards or samples are then added to the appropriate microtiter plate wells and incubated. SAA if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells of this SAA ELISA kit are thoroughly washed to remove any unbound SAA and other components of sample. In order to quantitate the amount of SAA present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for SAA is added to each well to "sandwich" the SAA immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate solution are allowed to react over a short incubation period. Only those wells that contain SAA and enzyme-substrate reaction will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the colour change measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.
In order to measure the concentration of SAA in the samples, this SAA ELISA kit includes two calibration diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing). According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D) versus SAA (ng/mL). The concentration of SAA in the samples is then determined by comparing the O.D. of the samples to the standard curve.
The minimum detectable dose of SAA using this SAA ELISA kit was determined by adding two standard deviations to the mean optical density value of 20 zero standard replicates and calculating the corresponding concentration from the standard curve. The minimum detectable dose of human SAA using a standard curve generated with Calibrator Diluent I is 1.1 ng/mL and using Calibrator Diluent II is 0.6 ng/mL. This SAA ELISA kit showed no cross reactivity with other cytokines tested. This SAA ELISA kit is expected to be effectively used for further investigations into the relationship between SAA and the various conditions mentioned.
This human SAA ELISA kit is to be used for the in vitro quantitative determination of human serum amyloid A (SAA) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This Human SAA ELISA kit assay is calibrated against NIBSC SAA 1st international standard (code 92/680). This human SAA ELISA kit is intended FOR LABORTORY RESEARCH USE ONLY and is not for use in diagnostic or therapeutic procedures. Fourteen apparently healthy, normal individuals were evaluated in this Human SAA ELISA kit assay. The SAA concentration of serum/plasma samples is ranged from 1000-5000 ng/mL and urine samples is less than 2.5 ng/mL.
Acute phase serum amyloid A proteins (A-SAAs) are secreted during the acute phase of inflammation.
These proteins have several roles, including the transport of cholesterol to the liver for secretion into the bile, the recruitment of immune cells to inflammatory sites and the induction of enzymes that degrade extracellular matrix. A-SAAs are implicated in several chronic inflammatory diseases, such as amyloidosis, atherosclerosis, and rheumatoid arthritis. Three acute phase SAA isoforms have been reported in mice, called SAA1, SAA2 and SAA3. During inflammation, SAA1 and SAA2 are principally expressed and induced in the liver, while SAA3 is induced in many distinct tissues. SAA1 and SAA2 genes are regulated in liver cells by the proinflammatory cytokines IL-1, IL-6, and TNF-α. Both SAA1 and SAA2 are induced up to a 1000-fold in mice under acute inflammatory conditions following exposure to bacterial lipopolysaccharide (LPS).Three A-SAA genes have also been identified in humans, although the third gene, SAA3, is believed to represent a pseudogene that does not generate messenger RNA or protein.
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