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CA72-4 ELISA Kit Instructions
CA72-4 ELISA Kit Instructions
(German DRG imported original: EIA5071)
German DRG China exclusive agent "Shenzhen Kerunda Biological Engineering Co., Ltd." dedicated to serve you
1 , introduction
The DRG CA72-4 ELISA reagent provides materials for the quantitative detection of the tumor associated antigen CA72-4 (TAG-72) in human serum or plasma. This experiment is for in vitro diagnostic use only.
CA72-4 (cancer antigen 72-4) was originally described as an antigenic factor recognized by B72.3. B72.3 is a murine monoclonal antibody produced by immunological hybridization of the corresponding membrane extract after primary tumor metastasis. CA72-4 was confirmed to be a 1MDa mucin-like glycoprotein, and the composition was called TAG72 (tumor-associated antigen 72). The molecular weight of the TAG72 protein is 48 KD. The elevated levels of CA72-4 in serum and plasma are found in certain malignant diseases, including tumors of the pancreas, stomach, gallbladder, colon, breast, ovary, cervix, and endometrium. It has a high diagnostic sensitivity to gastrointestinal and ovarian tumors. Although elevated levels of CA72-4 have been found in some benign diseases such as rheumatism and ovarian cysts, clinical studies have shown that specific diagnoses are up to 95% for gastrointestinal and ovarian tumors. The level of CA72-4 is closely related to the size and grade of the tumor. CA72-4 is an indicator of the choice of therapeutic monitoring and subsequent care for patients with gastrointestinal tumors. Another suitable indicator is CA199 and CEA. In addition, CA72-4 is a reliable indicator of the choice of therapeutic monitoring and subsequent care for patients with ovarian tumors, especially for CA125-negative patients.
2 , the principle of experiment
DRG's CA72-4 enzyme-free assay is a solid-phase enzyme-free adsorption assay (ELISA) based on the sandwich method principle. The micro-detection well on the reaction plate is coated with a monoclonal mouse antibody that binds to a unique antigenic site on the CA72-4 molecule. A patient sample containing endogenous CA72-4 was incubated with the enzyme conjugate in a coated well, which is an anti-CA72-4 antiserum linked to horseradish peroxidase. After incubation, the unbound enzyme conjugate was washed away with washings. The total amount of horseradish peroxidase bound was positively correlated with the concentration of CA72-4 in the sample. After the addition of the substrate solution, the apparent color intensity is proportional to the concentration of CA72-4 in the patient sample.
3 , kit components
1) Microplate: 12x8 well, detachable, coated with anti-CA72-4 monoclonal antibody in microwell.
2) Standard (standard product 0-4): 5 0.5 ml, ready-to-use, concentration 0, 3, 20, 50, 100 U/ml, containing anhydrous silver preservative.
3) Quality control high & low: 2 lyophilized powder, 0.5ml, see “Reagent Preparationâ€. For the concentration and range of quality control products, please refer to the reagent bottle label or quality control sheet. Contains anhydrous silver preservatives.
4) Sample diluent: one, 3ml, ready to use, containing anhydrous silver preservative.
5) Enzyme conjugate: 1 14 ml of anti-CA72-4 antibody labeled with horseradish peroxidase containing anhydrous silver preservative.
6) TMB substrate solution: a 14ml, ready to use.
7) Stop solution: 0.5M sulfuric acid, a 14ml, ready to use, avoid contact with the stop solution, so as not to stimulate or burn the skin.
8) Washing solution (40X): a 30ml.
Note: Sample dilutions for sample dilution should be as needed.
4 , the experiment requires equipment (but the kit does not provide)
1) Microplate reader (450 ± 10 nm).
2) Pipette
3) Absorbent paper
4) distilled water
5) Timer
6) Coordinate paper or data processing software
5 , reagent storage and stability
Unopened reagents are stored at 2-8 ° C and maintain their stability during the shelf life. Do not use expired reagents. All open reagents should be stored at 2-8 ° C. Microplates should be stored at 2-8 ° C. Once the microplate bag is opened, it should be carefully resealed.
6. Preparation of reagents:
All reagents and the required strips were equilibrated to room temperature prior to use.
Control: Add 0.5ml of distilled water to the lyophilized powder to reconstitute it, and leave it for at least 10 minutes. Shake well before use. The reconstituted control should be stored at -20 °C.
Washing liquid: 1170 ml of distilled water was added to 30 ml of the concentrated washing liquid, and the volume of the final washing liquid was 1200 ml. The diluted wash solution remained stable for two weeks at room temperature.
7 , sample collection and storage
This experiment can use serum or plasma (using EDTA or heparin anticoagulation) samples, do not use samples of hemolysis, lipemia, and jaundice. Note: Samples containing sodium azide cannot be used in this experiment.
7.1 sample collection
Serum : venous blood collection, coagulation is allowed, and serum is separated by centrifugation at room temperature. Do not centrifuge before the whole blood has completely coagulated. Patients who have received anticoagulant therapy may require longer clotting time.
Plasma: Whole blood was collected into a centrifuge tube containing an anticoagulant and collected by centrifugation immediately after collection.
7.2 Storage of samples
After the sample is covered, it can be stored for 5 days at 2-8 °C before the experiment. For longer storage, it should be stored at -20 °C. Avoid repeated freeze-thaw samples before starting the experiment.
7.3 dilution of the sample
In the initial experiment, if the concentration of the sample was higher than the highest standard, the sample was applied 10 times or 100 as described in the experimental procedure.
Double dilution. The dilution factor is multiplied when calculating the concentration.
example:
a) Dilute 1:10: 10 ul of serum + 90 ul of sample dilution (shake well).
b) Discharge at 1:100: 10 ul of diluted serum according to a) +90 ul of sample-releasing solution (shake well)
8 , experimental steps
8.1 General
1) All reagents and samples should be equilibrated to room temperature before use. All reagents should be well mixed but not foamed.
2) Once the experiment begins, all steps must be carried out without interruption.
3) Use disposable nozzles for each reagent, standard, and sample to avoid cross-contamination.
4) The absorbance is determined by the incubation time and temperature. Before starting the experiment, it is recommended to prepare all the reagents and fix the microplates to be tested on the plate. These steps ensure that the time required for each dosing step is the same. No interruptions.
5) According to the general rule, the enzyme reaction time is linearly proportional to the incubation time and temperature.
8.2 Operation steps
Each test should contain a standard curve
1) Fix the number of slats required for the experiment on the plate holder.
2) Use a disposable nozzle to add 20 ul of standards, controls, and samples to the appropriate test wells.
3) Add 100 ul of enzyme conjugate per well.
4) Mix thoroughly for ten seconds. In this step, it is important to shake well.
5) Incubate for 120 minutes at room temperature without sealing.
6) Quickly discard the contents of the test wells, wash the plate five times with 400 ul of wash solution, and force the plate on the blotting paper to remove the residual liquid. Note: The correctness of the washing step will directly affect the sensitivity and accuracy of the experiment.
7) Add 100 ul of substrate solution to each well.
8) Incubate for 30 minutes at room temperature.
9) Stop the reaction by adding 100 ul of stop solution to each well.
10) Read the absorbance at 450 nm at room temperature for ten minutes after the addition of the stop solution.
9 , the result of calculation
1) Calculate the average absorbance of standards, controls, and patient samples.
2) A standard curve is drawn by taking the absorbance as the Y-axis and the concentration as the X-axis according to the average absorbance obtained from the standard and its corresponding concentration value.
3) Using the average absorbance of the sample, the corresponding concentration value is obtained on the standard curve.
4) Automatic method: The result can be obtained using a cubic function, a four-parameter mode or a double-log computer program.
5) The concentration of the sample can be obtained directly from the standard curve. Further concentration is required if the concentration of the sample is higher than the concentration of the highest standard. When calculating the concentration of the sample, multiply by the dilution factor.
The following is a typical example of the standard curve for the CA72-4 ELISA.
Standard
Absorbance (450nm)
Zero standard (0 U/ml)
0.08
Standard 1 (3U/ml)
0.19
Standard 2 (20 U/ml)
0.59
Standard 3 (50 U/ml)
1.16
Standard 4 (100 U/ml)
2.02
This translation is for reference only, please refer to the original for details!
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