Product Description

                                    CA72-4 ELISA Kit

Catalog No.: L5E1007

Intended use

This immunoassay kit allows for the in vitro quantitative determination of  tumor associated glycoprotein TAG-72 (CA72-4) in serum, plasma and other biological fluids.

Introduction

CA72-4 antigen as TAG-72 is a high-molecular-weight,  mucin-like complex which is predominantly expressed in malignant tumors of the gastrointestinal tract. This antigen has been found in a variety of human adenocarcinomas (colorectal, gastric, ovarian, breast, and lung), but rarely expressed in benign and normal adult tissue. The level of TAG 72 may be elevated in the serum or plasma from patients with GI malignancies as well as with other malignancies, such as lung cancer, and with some non-malignant disorders.

Test principle

The CA72-4 antigen is an antigenic determinant of TAG-72 which is recognized by B72.3 and CC-49 monoclonal antibodies. The LINC-BIOI CA 72-4 ELISA is based upon two monoclonal antibodies, cc49 and B72.3, which react with distinct antigenic determinants expressed on the circulatingTAG 72 antigen in a forward sandwich format. The microtiter plate provided in this kit has been pre-coated with cc49. Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase (HRP) -conjugated  antibody B72.3 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 TAG-72, biotin-conjugated antibody and enzyme-conjugated Avidin 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 TAG-72 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Materials and components

Reagent                                          Quantity

Assay plate                                           1

CALs                                               5 x 500ul

With concentrations of 0 U/ml，4 U/ml，20 U/ml，100U/ml，300 U/ml

HRP conjugate                                                                         1 x 10ml

Wash Buffer   (20 x concentrate)                        1 x 30ml

Substrate                                                                                        1 x 10ml

Stop Solution                                                                                 1 x 5ml

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 approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C or -80° C.

Plasma – Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for

15 minutes at 1000 x g at 2 – 8° C within 30 minutes of collection. Store samples at -20° C or -80° C. Avoid repeated freeze-thaw cycles.

Other biological fluids – Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20° C or -80° C. Avoid repeated freeze-thaw cycles.

Note: Serum, plasma, and cell culture supernatant samples to be used within 7 days may be stored at 2-8 ° C, otherwise samples must stored at -20° C (≤ 3 months) or -80° C (≤ 6 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

It is recommended that all samples be assayed in duplicate.

DO NOT USE HEAT-TREATED SPECIMENS.

Limitations of the procedure

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

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. If samples generate values higher than the highest standard, further dilute the samples with the Assay 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.
4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.

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.

1. Add 40 uL of Standard, Blank, or Sample per well. Cover with the adhesive strip. Incubate for 45 mins at 37° C.
2. 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.

3. Add 100 uL of HPP conjugate solution to each well. Incubate for 45mins at 37°C.
4. Aspirate each well and wash, repeating the process five times for a total of three washes. Refer to step2.
5. Add 100 uL of Substrate Solution to each well. Incubate for 10 minutes at room temperature. Protect from light.
6. Add 50 uL 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 at of each well within 15 minutes, using a microplate reader set to 450 nm.

Specificity
This assay recognizes recombinant and natural human TAG-72. No significant cross-reactivity or interference was observed.

Sensitivity

The minimum detectable dose of human TAG-72 is typically less than 1 U/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

4-300 U/ml..

 Important Note:

1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available.
3. Duplication of all standards and specimens, although not required, is recommended.
4. When mixing or reconstituting protein solutions, always avoid foaming.
5. 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.
6. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.
7. Do not substitute reagents from one kit lot to another. Standard, conjugate  a microtiter plates are matched for optimal performance.  Use only the reagents supplied by manufacturer.

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 TAG-72 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.

Storage of test kits and instrumentation

1. Unopened test kits should be stored at 2-8°C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). 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. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
4. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
5. Use fresh disposable pipette tips for each transfer to avoid contamination.
6. Substrate Solution is easily contaminated. If bluish prior to use, do not use.

Precaution

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

FOR RESEARCH USE ONLY