Fibronectin (FN) ELISA Kit

Antigen: Fibronectin

Synonyms: fibronectin, fn1, FN1, FN, cig, msf, finc, lets, MGC97508, CIG, FNZ, MSF, ED-B, FINC, GFND, LETS, GFND2, DKFZp686H0342, DKFZp686I1370, DKFZp686F10164, DKFZp686O13149, Fn, Fn-1, MGC117493, E330027I09, fn-1, FIBNEC

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN365794

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human FN concentrations in serum, plasma and other biological fluids.

Alternative name: Fibronectin (FN)

Description: Fibronectin is a high-molecular weight (~440kDa) extracellular matrix glycoprotein that binds to membrane-spanning receptor proteins called integrins. In addition to integrins, fibronectin also binds extracellular matrix components such as collagen, fibrin and heparan sulfate proteoglycans (e.g. syndecans). Fibronectin exists as a dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds. The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms. Fibronectin plays a major role in cell adhesion, growth, migration and differentiation, and it is important for processes such as wound healing and embryonic development. Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis.

Specificity: This assay recognizes recombinant and natural human FN. No significant cross-reactivity or interference was observed.

Synonyms: fibronectin, fn1, FN1, FN, cig, msf, finc, lets, MGC97508, CIG, FNZ, MSF, ED-B, FINC, GFND, LETS, GFND2, DKFZp686H0342, DKFZp686I1370, DKFZp686F10164, DKFZp686O13149, Fn, Fn-1, MGC117493, E330027I09, fn-1, FIBNEC

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to FN antibody. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated monoclonal antibody preparation specific for FN and 3 incubated. Then substrate solution A and B are added to each well. Only those wells that contain FN, HRP-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of FN in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Preparation of Reagents: Sample Collection and Storage: Serum Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum and assay immediately or aliquot and store samples at -20° C. Avoid repeated freeze-thaw cycles. 6 Plasma Collect plasma using citrate, EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of collection. Assay immediately or aliquot and store samples at -20°C. Avoid repeated freeze-thaw cycles. Assay Procedure: Calculation of Results: Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the FN concentrations versus the log of the O.D. and the best 8 fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. Limitations of the Procedure: The kit should not be used beyond the expiration date on the kit label. Do not mix or substitute reagents with those from other lots or sources. If samples generate values higher than the highest standard, dilute the samples and repeat the assay. Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding. This assay is designed to eliminate interference by soluble receptors, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

Application Notes: 9 Centrifuge vials before opening to collect contents. When mixing or reconstituting protein solutions, always avoid foaming. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent. When using an automated plate washer, adding a 30 second soak period following the addition of ddH 2 O, and/or rotating the plate 180 degrees between wash steps may improve assay precision. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Substrate Solution should remain colorless until added to the plate. Keep Substrate Solution protected from light. Substrate Solution should change from colorless to gradations of blue. Stop Solution should be added to the plate in the same order as the Substrate Solution. The color developed in the wells will turn from blue to yellow upon addition of the Stop Solution. Wells that are green in color indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.

Components: Reagent Quantity Assay plate 1 Standard(S 1 -S 5 ) 5 x 1 ml HRP-conjugate 1 x 6 ml Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml STORAGE 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 5 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

Storage: 1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag. The test kit may be used throughout the expiration date of the kit. Refer to the package label for the expiration date. 5 2. Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 3. A microtiter plate reader with a bandwidth of 10 nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. Expiration date: six months from the date of manufacture.

Restrictions: For Research Use only