Technical Data
A2275-60U
Amyloid A, Serum, Human (Serum Amyloid A, SAA) BioAssay™ ELISA Kit
192Tests
Kits and Assays Storage: 4°C/-70°CShipping: Blue Ice
Background:
Serum amyloid A (SAA) proteins comprise a family of small (12-14kD, 104-112 amino acid residues), differentially expressed proteins that are highly conserved among vertebrates. SAA proteins are involved in the acute phase responses; these are the immediate early host responses to inflammation. During the acute phase, circulating SAA levels are increased by 100-1000 fold, reaching concentrations of up to 1mg/ml. SAAs have also been implicated in several disease states including rheumatoid arthritis, atherosclerosis, AA amyloidosis, and coronary artery disease. Liver is the major site of SAA synthesis, although extrahepatic expression has also been reported.

The human SAA gene family maps to chromosome 11. In humans, four SAA genes and three protein products have been identified; human SAA1 and SAA2 are designated the acute phase SAA (A-SAA) isoforms; while, SAA4 is constitutively expressed, and SAA3 is a pseudogene. Mouse SAA gene family maps to chromosome 7. In mouse, five SAA genes and four protein products have been identified; mouse SAA1, SAA2, SAA3 are the acute phase isoforms, SAA5 is constitutively expressed, and SAA4 is a pseudogene. A-SAAs belong to a category of acute phase proteins that also includes C-reactive protein (CRP), haptoglobin, ceruloplasmin, complement components C3 and C4, a1-acid glycoprotein, a1-proteinase inhibitor, and fibrinogen. In humans, both CRP and SAA are synthesized during the acute phase; whereas, in mouse, SAA is the predominant acute phase protein.

According to current models, severe physiological challenges such as tissue injury, microbial infection, surgical trauma, bone fracture, burns, and exhaustion, result in immediate inflammatory responses in the host. The role of the acute phase response is to counteract these challenges by preventing further pathogen entry, minimizing tissue damage, promoting repair, and restoring homeostasis. At the onset of the acute phase, macrophages and monocytes which are recruited to the site of inflammation, release the primary inflammatory mediators, IL-1 and TNF-alpha. These cytokines, in turn, signal the stromal cells to express secondary inflammatory mediators, IL-6, IL-8, and monocyte chemoattractant protein. Leukocytes attracted to the area produce additional cytokines. The cytokines produced at the site of injury, in particular IL-1 and IL-6, enter the plasma and provide stimuli for the liver to produce A-SAAs. Other cytokines that have been implicated in the regulation of SAA synthesis include LIF, CNTF, oncostatin M, cardiotropin, and possibly NGF. Analysis of SAA promoter regions reveals control by several transcription factors, including NF-kB, C/EBP, YY1, SAF/Sp1, and AP-2. AP-2 acts to repress transcription in non-liver tissues. NF-kB and C/EBP act synergistically to enhance transcription, while YY1 antagonizes the function of NF-kB and, therefore, serves to silence transcription.

Upon synthesis, SAA is released into the bloodstream where it immediately binds the HDL particles. Binding to HDL is known to protect the SAAs from degradation by proteolytic enzymes. There are a number of important homeostatic functions associated with the circulating SAA-HDL complexes; these functions have been categorized as immune modulation, lipid transport, and anti-inflammatory. The circulating SAA is known to act as a chemoattractant and recruit additional monocytes, leukocytes, mast cells, and T lymphocytes to the site of inflammation. It also aides the tissue regeneration process by activating matrix metalloproteinases such as collagenase and strolemysin. Binding of SAA to HDL alters the reverse cholesterol transport function of HDL, allowing for the delivery of cholesterol for the site of repair. The SAA-HDL complex also serves to remove excess cholesterol released by the damaged tissue. The anti-inflammatory activities of circulating SAA include inhibition of lymphocyte cell function, inhibition of TNF- alpha and IL-1-induced fevers, and inhibition of platelet aggregation. SAA is also known to bind neutrophils, abrogate the oxidative burst response, and prevent oxidative tissue damage.

Chronically elevated SAA levels are implicated in a group of protein misfolding disorders known as the amyloid A amyloidoses. These include reactive amyloidosis, caused by chronic inflammation or recurrent acute inflammation, familial Mediterranean fever, systemic AA amyloidosis, and visceral AA amyloidosis. Amyloid A amyloidoses are characterized by the deposition of insoluble plaques composed principally of proteolytically cleaved A-SAA. These plaques are primarily deposited in organ sites of inflammation and ultimately result in the degeneration of the affected organ. Despite the potential for causing fatal amyloidoses, SAAs are believed to play a protective role for the host. This hypothesis is supported by the observations that there is immediate, robust induction of SAAs in response to inflammatory signals and that SAAs are highly conserved through evolution.

Intended Use:
The United States Biological Human SAA ELISA Kit is to be used for the in vitro quantitative determination of human SAA in serum, plasma, buffered solution and tissue culture medium. The assay will recognize both natural and recombinant human SAA.

Principle of the Method:
The United States Biological Human SAA Kit is a solid phase sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA). A highly purified monoclonal antibody against Human SAA has been coated onto the wells of the microtiter strips provided. During the first incubation, standards of known human SAA content, controls, and unknown samples are pipetted into the coated wells, followed by the addition of biotinylated secondary monoclonal antibody. After washing, Streptavidin-Peroxidase (enzyme) is added. This binds to the biotinylated antibody to complete the four-member sandwich. After a second incubation and washing to remove all the unbound enzyme, a substrate solution is added which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of human SAA present in the original specimen.

Specifications:
Sensitivity: <4ng/ml
Range: 9.4-600ng/ml
Incubation Time: 3 hours.
Simple: Pre-coated strip-well plates.
Liquid stable conjugate, chromogen and stop reagents.
Sample size: 50ul (1:1000 dilution).
Economical: Two 96-well plates, plus reagents for 192 determinations.
Reagents are stable for multiple runs.
Versatile: Serum, plasma, cell culture medium or buffered solutions.

Kit Components:
*A2275-60U1 SAA Standard, 4x600ng. Recombinant human SAA. Reconstitute with 3.83ml A2275-60U2 Standard Diluent Buffer. Once reconstituted, aliquot and store at -80°C or below. Avoid repeated freeze-thaws.
A2275-60U2 Standard Diluent Buffer. 2x100ml. Contains 0.5% ProClin® 300.
*A2275-60U3 Low Control, 1x1vial. Rrecombinant human SAA in tissue culture matrix, lyophilized. Reconstitute with A2275-60U2 Standard Diluent Buffer. Once reconstituted, aliquot and store at -70°C or below. Avoid repeated freeze-thaws.
*A2275-60U4 High Control, 1x1vial. Recombinant human SAA in tissue culture matrix, lyophilized. Reconstitute with A2275-60U2 Standard Diluent Buffer. Once
Once reconstituted, aliquot and store at -70°C or below. Avoid repeated freeze-thaws.
A2275-60U5 Microtiter Plate, 2x96wells
A2275-60U6 SAA (Biotin), 2x6ml. Biotin-labeled anti-human SAA. Contains 15mM sodium azide.
A2275-60U7 Streptavidin (HRP) (100x), 2x125ul Contains 3.3mM thymol.
A2275-60U8 HRP Diluent, 1x25ml. Contains 3.3mM thymol.
A2275-60U9 Wash Buffer (25x), 1x100ml
A2275-60U10 TMB, 1x25ml
A2275-60U11 Stop Solution, 1x25ml

Storage and Stability:
Store *A2275-60U1, A2275-60U3 and A2275-60U4, powder at 4°C. Store reconstituted controls at -70°C. Do not store reconstituted standards. Store other components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial prior to removing the cap.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
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Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.