The mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a cell-surface Ig superfamily member composed of two extracellular Ig domains, followed by a mucin-like domain, a transmembrane domain and a short cytoplasmatic domain. It interacts via its N-terminal Ig domain with the lymphocyte homing receptor integrin alpha4beta7. MAdCAM-1 promotes the adhesion of T and B cells, monocytes/macrophages, and potentially eosinophils, basophils, and differentiated mast cells to the vascular endothelium and is critical for lymphocyte homing to the gut. RNA transcripts are predominantly expressed in the small intestine, mesenteric lymph nodes, colon and spleen. MAdCAM-1 transcripts are weakly expressed in human pancreas and brain. The MAdCAM-1 protein (60 kDa) is widely expressed on endothelia in both lymphoid and non-lymphoid tissues. Furthermore, it is expressed in thymic medulla and on high endothelial venules (HEV). This expression increases upon response to several cytokines including TNF-alpha, IL1b and IFN-gamma. MAdCAM-1 expression is upregulated on HEV-like vessels in a variety of chronic inflammatory diseases, and may mediate increased leukocyte trafficking into inflamed tissue. In utero and during early childhood, MAdCAM-1 plays a dominant role in lymphocyte-endothelial cell adhesion at both mucosal and nonmucosal sites. In contrast, in the adult the expression of MAdCAM-1 is restricted to mucosal tissues and has been shown to be dramatically up-regulated during intestinal inflammation. In the gut, MADCAM-1 is basically expressed on follicular dendrites in Peyer’s patches. Its expression is dramatically increased in inflammatory bowel disease (IBD). It is expressed in animal models of IBD and in human tissue samples from patients with Crohn’s disease and ulcerative colitis. MAdCAM-1 is strongly expressed in the synovium of osteoarthritis patients, predominantly on the endothelial lining of blood vessels, but also within the vessel lumen. The MAdCAM-1/integrin alpha4beta7 homing system possibly participates in gastric inflammation in response to Helicobacter pylori infection and contributes to mucosa-associated lymphoid tissue (MALT) formation typically leading to the development of nodular gastritis. Higher expression of MAdCAM-1 is reflected in elevated levels of the circulating soluble form of MAdCAM-1 (sMAdCAM-1). Since MAdCAM-1 is elevated in inflammatory, infectious and malignant diseases, sMAdCAM-1 serves as a perfect non-invasive biomarker for disease acitivity. In sera of healthy donor, sMAdCAM-1 was detected at 236.5 ± 55.8 ng/ml. In urine of healthy donors, sMAdCAM-1 was detected at 20-123 ng/ml. Measurement of sMAdCAM-1 levels is potentially useful to monitor disease activity and the results of therapy.
The human sMAdCAM-1 ELISA kit is to be used for the in vitro quantitative determination of human sMAdCAM-1 in serum, plasma, urine, breast milk and cell culture supernatant samples.
The human sMAdCAM-1 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 2 plates with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples and standards are captured by a solid bound specific antibody.
Biotinylated tracer antibody will bind to captured sMAdCAM-1.
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 sMAdCAM-1 standards (log).
The human sMAdCAM-1 concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The linearity of the assay was determined by serially diluting a sample containing 50 ng/ml human sMAdCAM-1. The diluted samples were measured in the assay. The line obtained a slope of 0.96 and a correlation coefficient of 0.994.
Normal human blood samples (plasma), containing baseline levels of 10 ng/ml sMAdCAM-1, were spiked with sMAdCAM-1 in concentrations of 10 and 25 ng/ml. Samples with and without sMAdCAM-1 were incubated for 1 hour at room temperature. Samples were measured using the ELISA. Values for sMAdCAM-1 ranged between 67 % and 122 % (mean 93%).