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Product description of human collagenase ELISA kit
Detection range: 20IU/L - 900IU/L
Purpose of use:
This reagent kit is used to determine the activity of collagenase in human serum, plasma, and related liquid samples.
Experimental principle
This reagent kit employs a double-antibody sandwich method to measure the level of human collagenase (Collagenase) in specimens. Purified human collagenase (Collagenase) antibody is used to coat microplates, forming solid-phase antibodies. Collagenase (Collagenase) is sequentially added to the microplates coated with monoclonal antibodies, and then binds to HRP-labeled collagenase (Collagenase) antibody, forming an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted to blue color under the catalysis of HRP enzyme and to the final yellow color under the action of acid. The intensity of color is positively correlated with the collagenase (Collagenase) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using a microplate reader, and the activity concentration of human collagenase (Collagenase) in the sample is calculated through a standard curve.
Specimen requirements
1. Extract the specimens as soon as possible after collection, following relevant literature. After extraction, conduct experiments as soon as possible. If experiments cannot be conducted immediately, store the specimens at -20℃, but avoid repeated freeze-thaw cycles
2. Samples containing NaN3 cannot be tested because NaN3 inhibits the activity of horseradish peroxidase (HRP).
Operational steps
1. Dilution of standard: This reagent kit provides one vial of undiluted standard. Users can dilute it in a small test tube according to the following table.
800IU/L Standard 5: Add 150μl of the original standard to 150μl of standard diluent
400IU/L Standard 4 Add 150μl of Standard 5 to 150μl of Standard Diluent
200IU/L Standard 3: Add 150μl of Standard 4 to 150μl of Standard Diluent
100IU/L Standard 2: Add 150μl of Standard 3 to 150μl of Standard Diluent
50IU/L Standard 1: Add 150μl of Standard 2 to 150μl of Standard Diluent
2. Sample addition: Set up blank wells (no sample and enzyme-linked immunosorbent assay (ELISA) reagent in the blank control wells, with the same operation for the remaining steps), standard wells, and test sample wells. Accurately add 50 μl of standard to the ELISA plate wells. Add 40 μl of sample diluent to the test sample wells first, and then add 10 μl of the test sample (the final dilution of the sample is 5 times). Add the sample to the bottom of the ELISA plate wells, avoiding contact with the well walls as much as possible, and gently shake to mix.
3. Incubation: After sealing the plate with a sealing film, incubate at 37℃ for 30 minutes.
4. Preparation of solution: Dilute the 30-fold concentrated detergent with distilled water to 30 times its original volume for use
5. Washing: Carefully remove the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry.
6. Add enzyme: Add 50μl of enzyme assay reagent to each well, except for the blank well.
7. Incubation: The operation is the same as in 3.
8. Washing: The operation is the same as that in step 5.
9. Color development: Add 50μl of developer A to each well, followed by 50μl of developer B. Gently shake to mix, and then incubate at 37℃ in the dark for 10 minutes for color development
10. Termination: Add 50μl of stop solution to each well to terminate the reaction (at this point, the blue color will turn yellow).
11. Measurement: Adjust to zero with a blank well, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The measurement should be conducted within 15 minutes after adding the stop solution.
You can obtain the full version of the instruction manual by consulting with our salesperson. Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory and a professional technical research and development team. We have long been committed to the research, development, and sales of various biological reagents, cells, sera, and ELISA kits. We provide free testing services and offer excellent technical services. If you have any experimental questions, please feel free to consult us. We will provide you with professional one-on-one technical guidance.
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