reagents kits:LINC-BIO CA15-3 EIA

LINC-BIO CA153 EIA Catalog No. L5E1010 Intended use This immunoassay kit allows for the in vitro quantitative determination of cancer associated antigen MUC-1 (CA153) in serum, plasma and other biological fluids. Introduction The MUC-1 antigen is a membrane-anchored mucin-type glycoprotein present in malignant and normal epithelial cells of certain organs, e.g. breast, lung, ovary, pancreas and colon. The apoprotein of the MUC-1 mucin contains a transmembrane domain, a cytoplasmic domain, and an extracellular carbohydrate rich domain. The extracellular domain is characterized by polymorphism with respect to the number of a 20 amino acid tandem repeat (VNTR polymorphism). The MUC-1 breast cancer mucin (CA15-3 antigen) is secreted from tumor cells and is a well-established serological marker for monitoring the clinical course of breast cancer patients. The serum CA153 concentration is less than 28 units per ml in normal personels. Materials and components Reagent Quantity Assay plate 1 Calibrators 6 x 500ul With concentrations of 0 U/ml,10 U/ml,30 U/ml,100U/ml,300 U/ml. Controls With concentrations of 20-40 U/ml and 80-120 U/ml relatively. 2 x 500ul CA153-HRP conjugate 1 x 10ml Sample Diluent 1 x 50ml Wash Buffer (20 x concentrate) 1 x 30ml Substrate Solution 1 x 10ml Stop Solution 1 x 5ml Assay procedure Allow all reagents to reach room temperature. All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Arrange and label required number of strips. Prepare all reagents, working standards and samples as directed in the previous sections. Sample preparation Dilute the samples 1/40 by mixing 10ul of sample with 390ul of Sample Diluent. Note Calibrators and Controls are ready for use, should not be diluted.Add 40 uL of Calibrators, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 1 hours at 37° C. Aspirate the liquid of each well and wash. repeating the process five times.Wash by filling each well with Wash Buffer (350 uL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. 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. Add 100 uL of HRP conjugate solution to each well. Incubate for 45 mins at 37°C. Aspirate each well and wash, repeating the process five times for a total of three washes. Refer to step2. Add 100 uL of Substrate Solution to each well. Incubate for 5 minutes at 37°C. Protect from light.Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.Determine the optical density of each well within 15 mins, using a microplate reader set to 450 nm.Calculation of results Average the duplicate readings for each calibrator, control, and sample and subtract the average zero standard optical density. Create a calibration curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit or liner curve. As an alternative, construct a calibration curve by plotting the mean absorbance for calibrator 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 CA153 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. Sensitivity The minimum detectable dose of human CA153 is typically less than 5 U/ml. Detection Range 11-300 U/ml. Limitations of the procedure FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.