Chemicon Human sICAM-1 ELISA试剂

Human sICAM-1 ELISA

Chemicon (Millipore)

Intercellular Adhesion Molecule-1 (ICAM-1) is a member of the immunoglobulin supergene family [Rothlein et al., 1988] and functions as a ligand for integrin alphaLbeta2 or Lymphocyte Function-Associated Antigen-1 (LFA-1), a member of the leukocyte integrin family [Marin & Springer 1987] which mediates lymphocyte adhesion.

ICAM-1 is a single-chain glycoprotein with a polypeptide core of 55kDa expressed on non-hematopoietic cells of many lineages including vascular endothelial cells, thymic epithelial cells, other epithelial cells and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T-lymphoblasts, germinal center B-cells and the dendritic cells from tonsils, lymph nodes and Peyer's patches. ICAM-1 is inducible on fibroblasts and endothelial cells by the inflammatory mediators IL-1, TNF and IFN-gamma. Its presence correlates with infiltration of lymphocytes into inflammatory lesions [Dustin et al., 1989; Prober et al., 1986; Rothlein et al., 1986]. ICAM-1 seems to be the initial marker of inflammatory reactions and is expressed prior to and to a greater extent than HLA-DR.

The role of ICAM-1 as a disease marker has been demonstrated for a number of different pathologies, including allergic rhinitis, allergic contact dermatitis [Vejlsgaard et al. 1989], gastrointestinal and bladder cancer, lymphoproliferative disorders [Lal et al., 1992; Rothlein et al., 1991], melanoma [Altomone et al., 1992; Becker et al., 1992; harning et al., 1991], HIV-1 infection [Most et al., 1993], malaria, tissue or organ transplant rejection [Adams et al., 1989; Adams et al., 1993], diabetes mellitus [Lampeter et al., 1992], glomerulonephritis, asthma [Wegner et al., 1990], rheumatoid arthritis [Popocnik et al., 1990] and psoriasis [Lisby et al., 1989].

A panel of 50 sera from healthy blood donors (male and female) was tested for sICAM-1. The detected sICAM levels ranged between 129.9 and 297.4 ng/ml with a mean of 230.3 ng/ml and a standard deviation of 47.4 ng/ml. Normal sICAM-1 levels may vary depending on the serum collective used ranging up to 400ng/ml or greater. Depending upon the study population ranges from 219ng/ml to 1050ng/ml have been reported in the literature.

Analytical Sensitivity and Detection Limits:

Sensitivity:3.3 ng/mL

Range of Detection:.25-100 ng/mL

Intra-Assay Variation:4.1%

Inter-Assay Variation:7.7%

Recovery:98.6% average

Assay Time: 75 minutes

Crossreactivity: no interference from soluble TNF-R (60 or 80 kDa), IL-8/NAP-1, TNF-alpha, TNF-beta, IFN-gamma, IFN-alpha2C, IFN-w, IL-6, IL-2R, E-selectin-1 or L-selectin-1.

The ICAM-1 ELISA is an enzyme-linked immunosorbent assay for quantitative detection of soluble Intercellular Adhesion Molecule-1 in cell culture supernatants, serum, plasma, or other biological fluids. The ICAM-1 ELISA is for research use only. Not for use in diagnostic procedures.

Test Principle:

An anti-ICAM-1 monoclonal antibody is adsorbed onto microwells. The pair of monoclonal antibodies use in this ELISA assay detect the soluable form of ICAM-1 present in serum, plasma or other biological fluids.

Soluble ICAM-1 present in a sample or standard then binds to antibodies adsorbed to the microwells. A second, HRP-conjugated monoclonal anti-ICAM-1 antibody is added and binds to ICAM-1 captured by the first antibody.

Unbound enzyme-conjugated anti-ICAM-1 is removed with a wash step and HRP substrate solution is added to the wells.

An amount of colored product is formed, proportional to the amount of soluble ICAM-1 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from five ICAM-1 standard dilutions and the ICAM-1 sample concentration is determined.

Application:

The ICAM-1 ELISA is an enzyme-linked immunosorbent assay for quantitative detection of soluble Intercellular Adhesion Molecule-1 in cell culture supernatants, serum, plasma, or other biological fluids. The ICAM-1 ELISA is for research use only. Not for use in diagnostic procedures.

ELISA

Human

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

ELISA Assays

• · 1 aluminum pouch with Microwell plate coated with Monoclonal Antibody (murine) to human ICAM-1
• · 1 vial (0.1 mL) HRP-Conjugated Anti-ICAM-1 Monoclonal (murine) Antibody
• · 2 vials (0.5 mL each) 100 ng/mL soluble ICAM-1 Standard
• 1 vial lyophilized control (high).
• 1 vial lyophilized control (low).
• · 1 bottle (50 mL) Wash Buffer Concentrate 20X (PBS with 1% Tween 20)
• · 1 vial (5 mL) Assay Buffer Concentrate 20X (PBS with 2% Tween 20 and 10% BSA)
• · 1 bottle (12 mL) Sample Diluent (buffered protein matrix)
• · 1 vial (15 mL) Substrate Solution
• · 1 vial (15 mL) Stop Solution (1M Phosphoric Acid)
• · 1 Microwell Strip Holder
• · 2 Adhesive Plate Covers

Store kit reagents, except the control, between 2° and 8°C. Store the lyophilized control at -20°C. Immediately after use remaining reagents should be returned to cold storage (2° to 8°C) with the exception of reconstituted control (-20°C). Expiration date of the kit is stated on outside label.

Precautions:

· Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.

· Do not mix or substitute reagents with those from other lots or other sources.

· Do not use kit reagents beyond expiration date on label.

· Do not expose kit reagents to strong light during storage or

incubation.

· Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.

· Some reagents contain thimerosal as preservative, which is highly toxic by inhalation, ingestion, or contact with skin. Thimerosal is a possible mutagen and should be handled accordingly.

· Avoid contact of substrate solutions with oxidizing agents and metal.

· In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes.

· Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagents.

· Exposure to acids will inactivate the conjugate.

· Glass-distilled water or deionized water must be used for reagent

preparation.

· Substrate solutions must be at room temperature prior to use.

· Since exact conditions may vary from assay to assay, a standard

curve must be established for every run.

· Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.

· Disposable pipette tips, flasks or glassware are preferred. Reusable glassware must be washed and thoroughly rinsed of all detergents before use.

· Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Wash Buffer, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods.

· The use of radioimmunotherapy has significantly increased the number of patients with human anti-mouse IgG antibody (HAMA). HAMA may interfere with assays utilizing murine monoclonal antibodies leading to both false positive and false negative results. HAMA interference can be reduced by adding murine immunoglobulins (serum, ascitic fluid, or monoclonal antibodies of irrelevant specificity) to the Sample Diluent.

Chromogenic

• ICAM1
• P3.58
• BB2
• CD54
• ICAM-1

CD54

- 5 mL and 10 mL graduated pipettes

- 5 μL to 1000 μL adjustable single channel micropipettes with

disposable tips

- 50 μL to 300 μL adjustable multichannel micropipette with disposable tips

- Multichannel micropipette reservoir

- Beakers, flasks, cylinders necessary for preparation of reagents

- Device for delivery of wash solution (multichannel wash bottle or

automatic wash system)

- Microwell strip reader capable of reading at 450 nm (620 nm as

optional reference wavelength)

- Glass-distilled or deionized water

- Statistical calculator with program to perform linear regression

analysis.

sICAM-1