cytokeratin 18 (CK-18) ELISA Kit

Antigen: Cytokeratin 18 (CK-18)

Reactivity: Human

Application: ELISA

Certificates: ISO 9001:2008

Catalog no.: ABIN366976

Additional Information

Characteristics: This immunoassay kit allows for the in vitro quantitative determination of human CK-18 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Description: Cytokeratins are intermediate filament keratins found in the intracytoplasmic cytoskeleton of epithelial tissue. There are two types of cytokeratins: the low weight, acidic type I cytokeratins and the high weight, basic or neutral type II cytokeratins. Cytokeratins are usually found in pairs comprising a type I cytokeratin and a type II cytokeratin. The high molecular weight cytokeratins, which are the basic or neutral cytokeratins, comprise subtypes CK1, CK2, CK3, CK4, CK5, CK6, CK7, CK8 and CK9. The low molecular weight cytokeratins, which are the acidic cytokeratins, comprise subtypes CK10, CK12, CK 13, CK14, CK16, CK17, CK18, CK19 and CK20. Expression of these cytokeratins is frequently organ or tissue specific. The subsets of cytokeratins which an epithelial cell expresses depends mainly on the type of epithelium, the moment in the course of terminal differentiation and the stage of development. Thus this specific cytokeratin fingerprint allows the classification of all epithelia upon their cytokeratin expression profile. Furthermore this applies also to the malignant counterparts of the epithelia (carcinomas), as the cytokeratin profile tends to remain constant when an epithelium undergoes malignant transformation. The main clinical implication is that the study of the cytokeratin profile by immunohistochemistry techniques is a tool of immense value widely used for tumor diagnosis and characterization in surgical pathology.

Specificity: This assay recognizes recombinant and natural human CK-18. No significant cross-reactivity or interference was observed.

Sensitivity: The minimum detectable dose of human CK-18 is typically less than 0.12 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. Detection Range: The minimum detectable dose of human CK-18 is typically less than 0.12 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero.

Application Details

Principle: The microtiter plate provided in this kit has been pre-coated with an antibody specific to CK-18. Standards or samples are then added to the appropriate microtiter plate wells. And a HRP-conjugated antibody is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain CK-18, 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 CK-18 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protocol: Reagent Preparation:
1. Bring all reagents and plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8°C and avoid sunlight. 2. Wash Buffer If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 15 ml of Wash Buffer Concentrate with deionized or distilled water to prepare 300 ml of Wash Buffer. Precaution: The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. 6.
Assay Procedure:
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate. 1. Set a Blank well without any solution. Add 5 0μl of Standard or Sample per well. Cover with the adhesive strip. Incubate for 30 minutes at 37° C. 2. Complete remove the liquid. Then fill each well with Wash Buffer (about 200μl), stay for 10 seconds and Spinning. Repeat the process for a total of five washes. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. 3. Add 50μl of HRP-conjugate to each well (not to Blank well). Mix well and then incubate for 20 minutes at 37°C. 4. Repeat the aspiration and wash five times as step 2. 5. Add 50μl of Substrate A and 50μl of Substrate B to each well, mix well. Incubate for 8-15 minutes at room temperature. Keeping the plate away from drafts and other temperature fluctuations in the dark. 8 6. Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.
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 CK-18 concentrations versus the log of the O.D. and the best 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. 9.
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.It is important that the Calibrator Diluent selected for the standard curve be consistent with the samples being assayed.If samples generate values higher than the highest standard, dilute the samples with the appropriate Calibrator Diluent and repeat the assay.Any variation in Standard Diluent, 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. 10.
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. Centrifuge the sample again after thaw before the assay. Avoid repeated freeze-thaw cycles. 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. Centrifuge the sample again after thaw before the assay. Avoid repeated freeze-thaw cycles. Note: Grossly hemolyzed samples are not suitable for use in this assay.

Application Notes: 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 wash buffer, 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(S1-S5) 5 HRP-Conjugate (1x6ml), Wash Buffer (20×concentrate) (1x15ml), Substrate A (1x7ml), Substrate B (1x7ml), Stop Solution (1x7ml)

Material not included: Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm. Pipettes and pipette tips. Deionized or distilled water. Squirt bottle, manifold dispenser, or automated microplate washer.

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. 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.

Restrictions: For Research Use only

Publications

Publications: Grattagliano, Palmieri, Portincasa et al.: "Long-term ursodeoxycholate improves circulating redox changes in primary biliary cirrhotic patients." in: Clinical biochemistry, 2011 (PubMed).