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|Sample type||Average %||Range|
|Serum||103||99% - 110%|
|Plasma||113||96% - 126%|
|Cell culture media||98||91% - 102%|
|Extraction Buffer||87||82% - 97%|
Abcam’s ICAM1 (CD54) in vitro SimpleStepELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of ICAM-1 protein in Human cell culture supernatant, plasma, serum and cell lysate samples.
The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
ICAM1, is a cell surface glycoprotein typically expressed in endothelial cells and cells of the immune system. The extracellular portion of ICAM-1 forms five immunoglobulin like domains attached to a single hydrophobic transmembrane region and a short cytoplasmic tail. ICAM-1, binds to the Leukocyte Integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) as well as to non integrin ligands such as CD43, fibrinogen, hyaluronan, Rhinoviruses and Plasmodium falciparum-infected erythrocytes. Binding to LFA-1 facilitates trans-endothelial leukocyte migration to areas of inflammation via promotion of endothelial apical cups assembly.
ICAM-1 expression increases in the vascular endothelium, macrophages and lymphocytes in response to cytokine stimulation (IL-1 and TNF). Furthermore, ligation of ICAM-1 up-regulates its own expression in a positive-feedback loop to maintain a pro-inflammatory environment conducive to leukocytes endothelial transmigration.
Soluble ICAM-1 is reported to be present in normal serum, cerebrospinal fluid, urine and bronchoalveolar lavage fluid. Elevated levels of soluble ICAM-1 have been associated with multiple organ failure, atherosclerosis, autoimmune disorders, diabetes, obesity, hypertension, liver disease as well as cancer, and several studies have correlated serum levels of sICAM-1 with the severity of these diseases.
|Components||1 x 96 tests|
|10X ICAM-1 Capture Antibody||1 x 600µl|
|10X ICAM-1 Detector Antibody||1 x 600µl|
|10X Wash Buffer PT (ab206977)||1 x 20ml|
|50X Cell Extraction Enhancer Solution (ab193971)||1 x 1ml|
|5X Cell Extraction Buffer PTR (ab193970)||1 x 10ml|
|ab221824 - Antibody Diluent 4BI||1 x 6ml|
|ICAM-1 Human Lyophilized Recombinant Protein||2 vials|
|Plate Seals||1 unit|
|Sample Diluent NS (ab193972)||1 x 50ml|
|SimpleStep Pre-Coated 96-Well Microplate (ab206978)||1 unit|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab174445 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||Use at an assay dependent concentration.|
Background-subtracted data values (mean +/- SD) are graphed.
The curve was prepared by loading 5 µg/mL of Raji cell extracts, followed by a 1:2 titration series. Background-subtracted data values (mean +/- SD) are graphed.
Human PBMCs were cultured in RPMI supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were cultured for 2 days at 37⁰C in the presence or absence of PHA. The concentrations of ICAM-1 were interpolated from the calibration curve and corrected for sample dilution. The mean ICAM-1 concentration was determined to be 320 pg/mL in unstimulated PBMC supernatants and 2,654 pg/mL in stimulated PBMC supernatants.
The levels of ICAM-1 in serum samples were tested from ten individual healthy donors. Levels were interpolated from the standard curve and corrected for sample dilution. The levels of ICAM-1 are shown for the percentage of individuals within each 100 ng/mL bin center of the distribution. The mean level of ICAM-1 was 469 ng/mL with a range of 347 to 629 ng/mL and a standard deviation of 77 ng/mL.
The levels of ICAM-1 protein were assessed in three Human cell line lysates loaded at 2.5 µg/mL of protein. The raw OD 450 nm signal for each sample was interpolated from an ICAM1 standard curve.
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