MUC1 ELISA Kit

Catalog No. ABIN649074

The Human MUC1 ELISA Kit (ABIN649074) is a Colorimetric ELISA Kit designed to quantify Human MUC1.

Quick Overview for MUC1 ELISA Kit (ABIN649074)

Target: MUC1 (Mucin 1 (MUC1))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Product Details MUC1 ELISA Kit

Purpose: Immunoenzymometric sequential assay (TYPE 4): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monoclonal anti-CA15-3 antibody. Upon mixing monoclonal biotinylated antibody, and a serum containing the native antigen, a reaction results between the native antigen and the antibody, forming an antibody-antigen complex. After a suitable incubation period, the antibody-antigen bound fraction is separated from unbound antigen by decantation or aspiration. Another antibody (directed at a different epitope) labeled with an enzyme is added. Another interaction occurs to form an enzyme labeled antibody-antigen-biotinylated-antibody complex on the surface of the wells. Excess enzyme is washed off via a wash step. A suitable substrate is added to produce color measurable with the use of a microplate spectrophotometer. The enzyme activity on the well is directly proportional to the native free antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained.

Analytical Method: Quantitative

Components: A. CA 15-3 Calibrators (1. 0 ml/vial). Six vials of human serum based reference calibrators at concentrations of 0 (A), 10 (B), 40 (C), 100 (D), 200 (E) and 400 (F) U/ml. Store at 2-8°C. A preservative has been added. Note 1: The calibrators are provided prediluted. Note 2: The calibrators, human serum based, were made using a purified preparation of CA 15-3. The preparation was calibrated against Centocor CA 15-3 IRMA test. B. CA15-3 Biotin Reagent 12 ml/vial (One vial contains biotinylated anti-human CA15-3 mIgG in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C. C. CA15-3 Enzyme Reagent 12 ml/vial. One vial contains horseradish peroxidase incorporated anti-human CA15-3 mIgG in a protein-stabilized matrix. A preservative has been added. Store at 2-8°C. D. Streptavidin Coated Plate (96 wells). One 96-well microplate coated with 1 µg/ml streptavidin and packaged in an aluminum bag with a drying agent. Store at 2-8°C. E. Wash Solution Concentrate (20ml). One vial contains surfactant in buffered saline. A preservative has been added. Store at 2-30°C. F. CA 15-3 Dilution Matrix (50 ml). One vial of serum diluent contains buffer salts, protein, surfactants. Store at 2-8°C. G. Substrate Solution (12ml/vial). One bottle contains tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) in buffer. Store at 2-8°C. H. Stop Solution (8ml/vial). One vial contains a strong acid Store at 2-30°C. I. Product Instructions: Note 1: Do not use reagents beyond the kit expiration date. Note 2: Opened reagents are stable for 60 days when stored at 2-8°C. Note 3: Above reagents are for a single 96-well microplate.

Material not included: 1. Pipette capable of delivering 25ml and 50ml with a precision of better than 1. 5%. 2. Dispenser(s) for repetitive deliveries of 0. 100ml and 0. 350ml volumes with a precision of better than 1. 5%. 3. Pipette (1000µl) used for serum diluent in patient dilutions. 4. Microplate washer or a squeeze bottle (optional). 5. Microplate Reader with 450nm and 620nm wavelength absorbance capability. 6. Absorbent Paper for blotting the microplate wells. 7. Plastic wrap or microplate cover for incubation steps. 8. Vacuum aspirator (optional) for wash steps. 9. Timer. 10. Quality control materials.

Application Details

Application Notes: Precautions: All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, Biosafety in Microbiological and Biomedical Laboratories, 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.

Plate: Pre-coated

Reagent Preparation:

1. Wash Buffer: Dilute contents of wash solution to 1000ml with distilled or deionized water in a suitable storage container. Diluted buffer can be stored at room temperature (20-27°C) for up to 60 days. 2. Patient Sample Dilution (1:21): Dispense 25 (l of each control and/or patient specimen into 0. 500 ml of CA 15-3 dilution matrix appropriately labeled, clean container(s) and mix thoroughly before use. Store refrigerated at 2-8°C. for up to 48 hours.

Sample Collection: The specimens shall be blood, serum or heparanised plasma in type and taken with the usual precautions in the collection of venipuncture samples. For accurate comparison to establish normal values, a fasting morning serum sample should be obtained. The blood should be collected in a redtop (with or without gel additives) venipuncture tube or for plasma use evacuated tube(s) containing heparin. Allow the blood to clot for serum samples. Centrifuge the specimen to separate the serum or plasma from the cells. Samples may be refrigerated at 2-8°C for a maximum period of five days. If the specimen(s) cannot be assayed within this time, the sample(s) may be stored at temperatures of -20 °C for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0. 050ml of the diluted specimen is required.

Calculation of Results:

A dose response curve is used to ascertain the concentration of CA15-3 in unknown specimens. 1. Record the absorbance obtained from the printout of the microplate reader. 2. Plot the absorbance for each duplicate serum reference versus the corresponding CA 15-3 concentration in U/ml on linear graph paper (do not average the duplicates of the serum references before plotting). 3. Connect the points with a best-fit curve. 4. To determine the concentration of CA 15-3 for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in U/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated).

Restrictions: For Research Use only

Handling

Handling Advice: Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20-27°C). 1. Format the microplates' wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8°C. 2. Pipette 0. 025 ml (25 µl) of the appropriate diluted serum reference, control or specimen into the assigned well. 3. Add 0. 100 ml (100µl) of the biotinylated labeled antibody to each well. It is very important to dispense all reagents close to the bottom of the coated well. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 60 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 8. Add 0. 100 ml (100µl) of the Ca15-3 Enzyme Reagent to each well. DO NOT SHAKE THE PLATE AFTER ENZYME ADDITION. 9. Cover and incubate 60 minutes at room temperature. 10. Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper. 11. Add 350µl of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two additional times for a total of three washes. An automatic or manual plate washer can be used. Follow the manufacturer's instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and Repeat two additional times. 12. Add 0. 100 ml (100µl) of substrate reagent to all wells. Always add reagents in the same order to minimize reaction time. DO NOT SHAKE THE PLATE AFTER SUBSTRATE ADDITION. 13. Incubate at room temperature for 20 minutes. 14. Add 0. 050ml (50µl) of stop solution to each well and gently mix for 15-20 seconds. 15. Read the absorbance in each well at 450nm (using a reference wavelength of 620-630nm to minimize well imperfections) in a microplate reader. The results should be read within 30 minutes of adding the stop solution.

Storage: 4 °C/-20 °C

Target Details for MUC1

Target: MUC1 (Mucin 1 (MUC1))

Alternative Name: CA 15-3

Background: Summary and Explanation of the test: Although multiple serum based tumor markers have been described for breast cancer, such as CA 15-3, BR 27-29, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA), tissue polypeptide specific antigen, and HER-2 (the extracellular domain), the most widely used are CA 15-3 and CEA. CA 15-3 is considered to be one of the first circulating prognostic factors for breast cancer (1). Preoperative concentrations thus might be combined with prognostic factors for predicting outcome in patients with newly diagnosed breast cancer(2). At present the most important clinical application of CA 15-3 is in monitoring therapy in patients with advanced breast cancer that is not accessible by existing clinical or radiologic procedures(3). The CA 15-3 assay measures the protein product of MUC1 gene. MUC1 protein is a large transmembrane glycosylated molecule containing three main domains, a large extracellular region, a membrane spanning sequence, and a cytoplasmic domain (4). Although the physiologic function of MUC1 is unclear, the glycoprotein has been implicated in cell adhesion, immunity and metastasis. Compared with healthy breast tissue, MUC1 is present in higher concentrations but less glycosylated in breast carcinoma (5-8). In this method, a prediluted CA15-3 calibrator diluted patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal antibody (specific for CA15-3) is added and the reactants mixed. Reaction between the CA15-3 antibodies and native CA15-3 forms complex that binds with the streptavidin coated to the well. The excess serum proteins are washed away via a wash step. Another enzyme labeled antibody specific for a different epitopic recognition of CA15-3 is added to the wells. The enzyme labeled antibody binds to the CA15-3 already immobilized on the well through its binding with the biotinylated monoclonal antibody. Excess enzyme is washed off via a wash step. A color is generated by the addition of a substrate. The intensity of the color generation is directly proportional to the concentration of the CA15-3 in the sample. Intended Use: The Quantitative Determination of Cancer Antigen (CA 15-3) Concentration in Human Serum by a Microplate Immunoenzymometric assay. Q. C. Parameters: In order for the assay results to be considered valid the following criteria should be met: 1. The absorbance (OD) of calibrator F should be greater than 1.3. 2. Four out of six quality control pools should be within the established ranges.

Pathways: Negative Regulation of intrinsic apoptotic Signaling