1. Preparation work:

1. Kit preparation

Each component of the kit was left to equilibrate at room temperature for 30 minutes before operation was started. ?

1. Required consumables and equipment (Note: You need to bring your own)

(1) Microplate reader, constant temperature oscillator (or constant temperature incubator), plate washer (the plate washing method is hand washing or not), vortex oscillator, timer

(2) High precision pipettes and disposable tips (0.5-10 μ L, 10-100 μ L, 30-300 μ L, 100-1000 μ L)

(3) Deionized water

(4) Absorbent paper, EP tube, disposable gloves

1. Reagent preparation (Note: prepare according to the requirements according to the components provided in the kit)

(1) Wash solution (1 ×): Take the wash solution (20 ×) and add deionized water to dilute it 20 times for later use. For example, take 10mL of wash solution (20 ×) and add 190mL of deionized water to mix well.

Note

If crystals are formed in the wash solution (20 ×), shake gently in room temperature or 37 ℃ water bath, and then dilute after the crystals are completely dissolved)

(2) Preparation of enzyme-labeled antibody solution: Dilute enzyme-labeled antibody (10 ×) 10 times with diluent to prepare enzyme-labeled antibody solution (1 ×). For example: 100μL enzyme-labeled antibody is added to 900μL diluent.

(3) Preparation of color development solution: Mix color development solution A, color development solution B and other volumes, mix well and place it in the dark from light (Note: The time cannot be left for too long, generally prepare it 10min before use. If the color development solution has turned blue after mixing, please do not use it). ?

4. Preparation of standards

(Note: It is prepared according to the requirements according to the standard provided by the kit. In order to further ensure the accuracy of the results, you can also use collagenase from specific sources actually used in the production process to establish the standard curve by yourself)

Dilute the standard (1 μg/mL) to 20 ng/mL with a diluent and then prepare the standard by serial dilution (dilution factor: 2 times) as shown in the following figure:

Note

In order to ensure the validity of the experimental results, a freshly prepared standard solution is used for each experiment. ?

5. Sample preparation

Return the sample to room temperature and mix well before adding the sample (the sample should be a homogeneous solution, and if there is precipitation, centrifuge the supernatant); If users need to dilute samples or high-concentration standards in the box, they can use the diluent in the box for dilution.

2. Operation process

1. Detailed operation steps

All operations are performed at room temperature and repeat assays are recommended for all spiked wells. ?

(1) Return each component of the kit to room temperature for 30min, take out the slats required for the test from the aluminum foil bag that has been equilibrated to room temperature, mark the sequence of the slats with a marker, seal the remaining slats with a sealing film and put them back in the aluminum foil bag, seal them well, and store them at 2-8 ℃. ?

(Note: The slats are easy to fall off during the washing process, so be careful to mark them.)

(2) Sample incubation: Add the diluted standard substance and the sample to be tested to the enzyme label plate (recommended addition sequence: standard well, blank well, sample well, standard substance is added according to the concentration gradient), 100 μ L/well, seal the plate with a plate sealing membrane, and then place it at 37 ℃ for incubation for 1h. ?

(Note: The sample addition time should be controlled within 10min to avoid drift over time. If the plate is not sealed or the plate is incomplete during incubation, the reaction solution will evaporate, resulting in errors in the experiment.)

(3) Plate washing: After incubation, carefully peel off the sealing film, discard the liquid in the wells, wash the plates three times (250 μ L/well) with washing solution (1 ×), and pat dry the residual liquid in the sample wells. (If the plate washing method is hand washing, it can be allowed to stand for 1min after adding washing solution (1 ×); if the plate washing machine is used to wash the plates, it can be slightly shaken for 5s after adding washing solution.)

(4) Incubation of enzyme-labeled antibody solution: Add enzyme-labeled antibody solution to each well, 100 μ L/well, seal the plate with a plate sealing membrane, and then place it at 37 °C for 1h. ?

(Note: Before adding liquid after each plate washing, check whether the slats are fixed to prevent liquid splashing caused by fixing the slats after adding liquid.)

(5) Plate washing: same as step 3). ?

(6) Color development: Add the pre-prepared color development solution to the enzyme labeled plate, 100 μ L/well, seal the plate with a sealing membrane, and incubate at 37 °C in the dark for 20 minutes. ?

(7) Termination: Add the stop solution, 100 μ L/well, and the reading can be made after the color is uniform (Note: Generally, the reading can be completed within 20 minutes after adding the stop solution). ?

(8) Reading: Put the microplate into the microplate reader, set the wavelength to dual wavelength 450/630 nm, and read the absorbance value (Note: It is recommended to set a 5-10s shock in the microplate reader reading program).

3. Data processing:

1. Calculation of absorbance value

The light absorption calibration values for each standard or sample are:

Standard/sample well absorbance values (OD450nm-OD630nm)-Blank control well absorbance value (OD450nm-OD630nm）?

1. Draw the standard curve with the standard concentration calibration value as the abscissa (X) and the standard light absorption calibration value as the ordinate (Y). It is recommended to use a four-parameter Logistic mathematical model to fit the equation:

Y = ((A-D)/(1 + (X/C) ^ B)) + D

Light absorption calibration value [standard/sample well absorbance value (OD450nm-OD630nm)-Blank control well absorbance value (OD450nm-OD630nm)] Substitute into the formula to calculate the content of collagenase in the sample. ?

1. If the OD value of the sample to be tested exceeds the OD value of the highest point of the standard curve, the sample needs to be diluted and re-measured

The following standard curve is for reference only, and the standard curve drawn by the same experimental standard shall prevail.

This kit uses double antibody sandwich enzyme-linked immunosorbent assay to determine the trace residue of Collagenase Type I (hereinafter referred to as collagenase) derived from Clostridium histolyticum in samples. Coating 96-well plates with capture antibodies to make solid phase antibodies, then adding standard and test samples, and then adding horseradish peroxidase (HRP) labeled labeled antibodies to form a solid phase antibody-collagenase-labeled antibody sandwich conjugate. Its absorbance (OD value) was measured at wavelengths of 450 nm and 630 nm, where 630 nm is the corrected wavelength. Results Calculate the standard/sample well absorbance value (OD450nm-OD630nm)-blank control well absorbance value (OD450nm-OD630nm), and calculate the content of collagenase in the sample to be tested through the standard curve (it is recommended to use the four-parameter Logistic mathematical model fitting equation).

Collagenase can be used to separate various tissues into single cells in vitro, and is suitable for single cell dissociation of organs and tissues for various scientific research purposes. The regulations related to the production of biological products clarify the necessity of detection of cell culture-related materials, so the detection of collagenase residues has attracted more and more attention. ?

This product uses a double antibody sandwich enzyme-linked immunosorbent assay based on polyclonal antibody, developed for collagenase mixtures obtained from Clostridium histolyticum, which can quickly and effectively detect collagenase residues in various samples.

Product Features:

Accurate detection

Polyclonal antibodies prepared against collagenase mixtures can perform effective detection

Strong specificity

no cross-reaction with other non-target proteins commonly used in cell culture

Excellent performance

detection limit as low as 0.1 ng/mL, high accuracy and good repeatability

Wide applicability

You can use collagenase from specific sources actually used in the production process to establish a standard curve by yourself to further ensure the accuracy of the results.

Product performance indicators:

Linearity range: 0.625-20ng/mL

Limit of quantitation: 0.625 ng/mL

Limit of detection: ≤ 0. 5 ng/mL

Accuracy (spiked recovery): 80%-120%

Accuracy (measurement deviation): ≤ 15%

Repeatability (intra-batch difference): ≤ 10%

Collagenase is a collagenase mixture obtained from Clostridium histolyticum, which can be used to separate a variety of tissues into single cells in vitro, and is suitable for single cell dissociation of organs and tissues for various scientific purposes. Collagenase is widely used in the isolation and acquisition of mesenchymal stem cells, 3D and organoid culture, single cell sequencing, tumor research and other fields. The bacterial collagenase commercially available on the market is mainly derived from Clostridium histolyticum. It is a crude enzyme extract that not only contains collagenase (clostridiopeptidase A), but also can degrade natural collagen and reticular fibers. It also contains some other proteases, polysaccharide enzymes, lipases, etc. According to the difference of collagenase activity, it can be divided into collagenase type I, type II, type III, type IV and type V, which can act on different types of tissue dissociation. Bacterial collagenases differ from vertebrate collagenases in that they have a wider substrate singularity. Unlike animal collagenase, which only decomposes the natural three-dimensional helical structure of collagen, bacterial collagenase is unique in that it can degrade both water-insoluble natural collagen and water-soluble denatured proteins. Bacterial collagenase can degrade almost all collagen types, and there are many kinds of divisions in the three-dimensional helical region. Collagenase is an endopeptidase, which can specifically hydrolyze the three-dimensional helical structure of natural collagen under physiological temperature and PH conditions. Collagen is the main fibrous component of animal extracellular connective tissue. This function of collagenase is mainly due to its ability to specifically recognize the Pro-X-Gly-Pro sequence and cleave the peptide bond between the neutral amino acid (X) and glycine (Gly) of this sequence, which is present in collagen with high frequency. Collagenase is also the only protease that can degrade natural collagen fibers with triple helices widely existing in connective tissue. Collagenase is also efficient enough to hydrolyze other proteins, polysaccharides and lipids within the extracellular matrix of connective tissue and epithelial tissue, making collagenase products well suited for tissue dissociation. In the treated biological products, there may be trace collagenase residues, which will have a certain impact on the subsequent application of biological products. The residue of collagenase in biological products is one of the important indexes to measure the quality of biological products.

1. All components in the kit must be restored to room temperature (20-25 ℃) before use. ?
2. Each component should be thoroughly mixed before use to ensure the uniformity of the reagents. The standard product should be centrifuged briefly for 5 seconds. All the liquid on the tube wall and lid should be concentrated at the bottom of the tube. Immediately after use, all the reagents should be put back to 2-8 ℃. ?
3. The kit must be used within the validity period, and the corresponding standard curve must be re-prepared for each test. It is not recommended to mix different standard curves, and it is not recommended to mix different batches of related reagents. ?
4. When adding liquid to the microplate, be careful not to touch the bottom of the microplate to prevent damage to the coating layer. Change the sample loading tank and tip in time between different samples to avoid cross-contamination.
5. When patting the slats dry after washing, be careful to prevent the slats from falling off, and be careful not to reuse the sealing film. ?
6. High concentration may produce black flocs during color development. It is recommended to dilute the sample for retest. ?
7. When reading, pay attention to check whether the detection wavelength and fitting equation are correct. ?
8. Only by strictly abiding by the operation methods of the instructions and using all the reagents supporting this kit can the best detection effect be guaranteed. ?
9. The kit uses accurate quantification of high-purity and high-activity collagenase as the standard product, which is suitable for most type I collagenases derived from Clostridium histolyticum. It can directly detect the residual levels of collagenase from different sources and can be used for more accurate detection of Merck Sigma-Aldrich collagenase. To further guarantee the accuracy of the results, it is also possible to self-establish standard curves using specific sources of collagenase actually used in the manufacturing process. ?
10. Differences in test results can be caused by many factors, including the operation of the experimenter, the use of the pipette, the plate washing technique, the reaction time or temperature, the storage of the kit, etc. ?
11. The company is only responsible for the kit itself, and is not responsible for the sample consumption caused by the use of the kit. Users are requested to fully consider the possible usage of samples and reserve sufficient samples before use. ?
12. The termination solution in this kit is acid solution, so special attention should be paid to operation. ?
13. For safety reasons, operators should wear personal protective equipment, such as lab coats, gloves, masks and goggles.

The Collagenase Type I quantitative detection kit (enzyme-linked immunosorbent assay) produced by our company is a highly sensitive collagenase detection kit, which is developed for the collagenase mixture obtained from Clostridium histolyticum, and can sensitively, specifically and accurately detect the residue of type I collagenase in intermediate products, semi-finished products and finished products of various tissue dissociation samples. The kit uses accurate quantification of high-purity and high-activity collagenase as the standard product, which is suitable for most type I collagenases derived from Clostridium histolyticum, and can directly detect the residual levels of collagenase from different sources. The standard can be used for more accurate detection of Merck Sigma-Aldrich collagenase type I. To further guarantee the accuracy of the results, it is also possible to self-establish standard curves using specific sources of collagenase actually used in the manufacturing process.