Human Cytokeratin 18(CK-18) ELISA Kit

(96 T)

This immunoassay kit allows for the in vitro quantitative determination of human CK-18 concentrations in serum, plasma.

Expiration date six months from the date of manufacture

 

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Cytokeratins (CK) are intermediate filaments of epithelial cells, both in keratinizing tissue (ie., skin) a-n-d non keratinizing cells (ie. mesothelial cells). Although not a traditional marker for endothelial cells, cytokeratins have also been found in some microvascular endothelial cells. Atleast 20 different cytokeratins (CK) in the molecular range of 40-70 kDa a-n-d isoelectric points of 5-8.5 can be identified using two dimensional gel electrophoresis. Biochemically, most members of the CK family fall into one of two classes, type I (acidic polypeptides) a-n-d type II (basic polypeptides). At least one member of the acidic family a-n-d one member of the basic family is expressed in all epithelial cells. Monoclonal antibodies to cytokeratin proteins can be useful markers for tumor identification a-n-d classification. Cytokeratin 18 is an acidic keratin which is found primarily in non squamous epithelia a-n-d is present in a majority of adenocarcinomas a-n-d ductal carcinomas but not in squamous cell carcinomas. Cytokeratin 18 exists in combination with Cytokeratin 8, a basic keratin. Hepatocellular carcinomas have been reportedly defined by the use of antibodies that recognize only Cytokeratins 8 a-n-d 18.

The microtiter plate provided in this kit has been pre-coated with an antibody specific to CK-18. Sta-n-dards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP)-conjugated antibody preparation specific for CK-18 a-n-d incubated. Then substrate solutions are added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution a-n-d 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 sta-n-dard curve.

0.625 ng/ml-15 ng/ml. The sta-n-dard curve concentrations used for the ELISA’s were 15 ng/ml, 7.5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625ng/ml.

This assay recognizes human CK-18. No significant cross-reactivity or interference was observed.

The minimum detectable dose of human CK-18 is typically less than 0.5 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.

Assay plate 1 Sta-n-dard(S0-S5) 6 HRP-conjugate 1 x 6ml

1 x15 ml

Wash Buffer

(20×concentrate) Substrate A 1 x 7 ml Substrate B 1 x 7 ml Stop Solution 1 x 7 ml

 

1.    Unopened test kits should be stored at 2-8?C upon receipt a-n-d 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 ba-n-dwidth of 10 nm or less a-n-d an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.

 

1         Bring all reagents a-n-d plate to room temperature for at least 30 minutes before use. Unused wells need store at 2-8°C a-n-d avoid sunlight.

2         Wash Buffer If crystals have formed in the concentrate, warm to room temperature a-n-d mix gently until the crystals have compley dissolved. Dilute 15 ml of Wash Buffer Concentrate with deionized or distilled water to prepare 300 ml of Wash Buffer.

 

 

1          Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 540 nm or 570 nm.

2          Pipettes a-n-d pipette tips.

3          Deionized or distilled water.

4          Squirt bottle, manifold dispenser, or automated microplate washer.

 

Serum Use a serum separator tube (SST) a-n-d allow samples to clot for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove serum a-n-d assay immediay or aliquot a-n-d 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 immediay or aliquot a-n-d store samples at -20°C. Centrifuge the sample again after thaw before the assay. Avoid repeated freeze-thaw cycles.

 

1. Set a Blank well without any solution. Add 50μl of Sta-n-dard 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 a-n-d 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 a-n-d blot it against clean paper towels.

1         Add 50μl of HRP-conjugate to each well (not to Blank well). Mix well a-n-d then incubate for 20 minutes at 37°C.

2         Repeat the aspiration a-n-d wash five times as step 2.

3         Add 50μl of Substrate A a-n-d 50μl of Substrate B to each well, mix well. Incubate for 8-15 minutes at room temperature. Keeping the plate away from drafts a-n-d other temperature fluctuations in the dark.

4         Add 50μl of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.

5         Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm.

 

Average the duplicate readings for each sta-n-dard, control, a-n-d sample a-n-d subtract the average zero sta-n-dard optical density. Create a sta-n-dard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a sta-n-dard curve by plotting the mean absorbance for each sta-n-dard on the y-axis against the concentration on the x-axis a-n-d 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. a-n-d 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 sta-n-dard curve must be multiplied by the dilution factor.

 

1          The kit should not be used beyond the expiration date on the kit label.

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

3          It is important that the Calibrator Diluent selected for the sta-n-dard curve be consistent with the samples being assayed.

4          If samples generate values higher than the highest sta-n-dard, dilute the samples with the appropriate Calibrator Diluent a-n-d repeat the assay.

5          Any variation in Sta-n-dard Diluent, operator, pipetting technique, washing technique, incubation time or temperature, a-n-d kit age can cause variation in binding.

6          This assay is designed to eliminate interference by soluble receptors, binding proteins, a-n-d other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

7          Centrifuge vials before opening to collect contents.

8          When mixing or reconstituting protein solutions, always avoid foaming.

9          To avoid cross-contamination, change pipette tips between additions of each sta-n-dard level, between sample additions, a-n-d between reagent additions. Also, use separate reservoirs for each reagent.

10     When using an automated plate washer, adding a 30 second soak period following the addition of wash buffer, a-n-d/or rotating the plate 180 degrees between wash steps may improve assay precision.

11     To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.

12     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.

13     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.