{ "ProductData": { "ID": "3.5.3.3.14.3", "ProductType": "Others", "BrandName": "Collagenase Activity Test", "ProductNameAddition": "Kit for determination of the enzymatic activity of Collagenases I and II", "RegulatoryDisclaimer1": "For use in quality control/manufacturing process only.", "DisclaimerGroup1": "", "RegulatoryDisclaimer2": null, "DisclaimerGroup2": null, "RegulatoryDisclaimer3": null, "SampleType": [ ], "LicenseDisclaimers": [ ], "RelatedLinks": "", "Clone": "", "ControlTissue": [ "" ], "ISOtypes": "", "Species": [ "" ], "StainLocalization": [ "" ], "ProductNameGlobal": "Collagenase Activity Test" }, "ProductImageDetails": { "ImagePath": "https://pim-media.roche.com/Images/Article_08074461001_im_en.png", "ImageType": "Image main" }, "Product2Taxonomy": { "Product2TaxonomyReferences": [ { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0133", "StructureNodeStatus": "valid", "NodeName": "Cell and Gene Therapy" }, { "StructureSystemIdentifier": "OWP_Organization", "StructureSystemName": "Organization", "NodeID": "01-01-01-07-00", "StructureNodeStatus": "valid", "NodeName": "CustomBiotech and LifeScience" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0014", "StructureNodeStatus": "valid", "NodeName": "Enzymes" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0131", "StructureNodeStatus": "valid", "NodeName": "Quality Control Testing" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0060", "StructureNodeStatus": "valid", "NodeName": "Impurity Testing" }, { "StructureSystemIdentifier": "Applications", "StructureSystemName": "Applications", "NodeID": "10-00-00", "StructureNodeStatus": "valid", "NodeName": "Impurity Testing" }, { "StructureSystemIdentifier": "Product_Solutions", "StructureSystemName": "Product Solutions", "NodeID": "520", "StructureNodeStatus": "valid", "NodeName": "CustomBiotech" }, { "StructureSystemIdentifier": "OWP_Product_Types", "StructureSystemName": "Product Types", "NodeID": "20-000-00", "StructureNodeStatus": "valid", "NodeName": "Assays Reagents and Strips" }, { "StructureSystemIdentifier": "Lab_Type", "StructureSystemName": "Lab Types", "NodeID": "180-00", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "OWP_Techniques", "StructureSystemName": "Techniques", "NodeID": "999-00", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "OWP_Family", "StructureSystemName": "Product Families", "NodeID": "999", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "Health_Topics", "StructureSystemName": "Health Topics", "NodeID": "99-00-00", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "Disease_Areas", "StructureSystemName": "Disease Areas", "NodeID": "99-00-00", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "Pathogens", "StructureSystemName": "Pathogens", "NodeID": "99-00-00", "StructureNodeStatus": "invalid", "NodeName": "" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0008", "StructureNodeStatus": "valid", "NodeName": "Biologics" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0112", "StructureNodeStatus": "valid", "NodeName": "Protease Portfolio" }, { "StructureSystemIdentifier": "Product_Grouping", "StructureSystemName": "Product Grouping", "NodeID": "02-0090", "StructureNodeStatus": "valid", "NodeName": "Vaccine Testing" } ] }, "Product2Materials": { "P2MaterialReferences": [ { "MaterialNum": "08074461001", "MaterialDescription": "Collagenase Activity Test", "RegisteredProductName": "Collagenase Activity Test", "GTIN": "07613336147016", "ProductCategoryText": "Reagents, kits", "OldMaterialNumber": "", "PackSizePIM360": "1 kit", "PackSizeDescPIM360": "96 reactions", "MaterialAnnotation": "CustomBiotech product. Please contact your local representative.", "ReadyForUse": "", "OrderInformation": "Unit of measure is \"piece\"." } ] }, "Product2Products": { "Product2ProductReference": [ ] }, "ProductSpec": [ { "ProductSpecVariant": { "Chapters": [ { "Name": "Storage Conditions (Product)", "Value": "Store the kit at −15 to −25°C.", "Language": "en", "Country": "XG", "Code": "Storage Conditions (Product)" }, { "Name": "Unit Conversion", "Value": "

Enzyme	Factor
Collagenase I	9.1
Collagenase II	2.7
Liberase Blends	3.0

", "Language": "en", "Country": "XG", "Code": "Unit Conversion" }, { "Name": "Quality Control", "Value": "A quantitative, functional assay is performed to determine the lot-specific activities of Calibrators A to E and recover the activity of an internal standard.", "Language": "en", "Country": "XG", "Code": "Quality Control" }, { "Name": "Additional Equipment and Reagent Required", "Value": "

• Polypropylene tubes for preparation of sample dilutions
• 0.5 to 10 μL and 10 to 100 μL micropipettes
• 96-well transparent flat-bottom microplate treated for cell culture from the following list or equal product:
  • Thermo Scientific Nunclon Delta, 167314
  • Corning Falcon TC-treated, 353075
  • Corning Costar TC-treated, 3997
  • Charles River Endosafe, M9005
  • Greiner Cellstar, 655180
• Multiwell plate reader capable of determining initial maximal rates (Vmax) of absorbance at 340 nm with at least two data points per well per minute

• Purified water for the blank
• Diluent for preparation of sample dilutions

• Liberase Blend*, see Section Ordering Information

", "Language": "en", "Country": "XG", "Code": "Additional Equipment and Reagent Required" }, { "Name": "Assay Time", "Value": "Total assay time: approximately 30 minutes.", "Language": "en", "Country": "XG", "Code": "Assay Time" }, { "Name": "Troubleshooting", "Value": "

Observation	Possible cause	Recommendation
Activity of samples too low.	Low concentration after dilution of highly concentrated samples.	Reduce dilution factor for highly concentrated samples.
Activity of samples too high.	Activity exceeds measuring range.	Dilute samples to ensure that they are within the specified measuring range.
Background of sample matrix.	Check analyte-free sample matrix for background activity value (negative control). Correct sample values for background activity of matrix if the background Vmax value of the negative control is higher than the Vmax value of Calibrator A.
Contamination of samples with Liberase Blend from lab workspace.	Repeat the experiment in a different workspace using a new kit. Thoroughly clean contaminated workspace.
Variations too high.	Insufficient mixing of calibrator or sample and FALGPA Solution.	Thoroughly mix by pipetting up and down several times and/or including an agitation/shaking step in the multiwell plate reader software.
Cross-contamination between wells during the agitation/shaking step.	Ensure that the agitation/shaking step is performed at the lowest possible setting on the plate reader.
Unsuitable microplate.	Use transparent flat-bottom 96-well plates.
Sample matrix difficult to pipette due to viscosity or composition.	Carefully pipette samples without droplets on outside of tip. Avoid high aspiration or ejection velocity when pipetting the samples.
Pipetting precision is not optimal.	Check the precision of the pipetting.
Determination of Vmax values disturbed by air bubbles or other artifacts.	Check the measured absorbance rates in the instrument software. Make sure Vmax values are determined in a sufficiently linear phase near the start of the enzymatic reaction. Vary the measurement time range and/or increase the number of data points to improve linearity. The time between sample pipetting and measuring should be minimized to achieve the highest accuracy and precision. If the high variations persist, repeat the test run and avoid/remove air bubbles in the wells by carefully agitating the microplate.

", "Language": "en", "Country": "XG", "Code": "Troubleshooting" }, { "Name": "Principle", "Value": "The product is a test for the quantitative determination of the enzymatic activity of Collagenase I, Collagenase II, and blends thereof (Roche Diagnostics Liberase Blends), and is based on the principle of a continuous hydrolytic reaction of a colorimetric peptide substrate.
FALGPA = N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala
FAL = N-(3-[2-Furyl]acryloyl)-Leu
GPA = Gly-Pro-Ala

The hydrolysis of FALGPA is observed as a decrease in optical absorbance at 340 nm, recorded as continuous absorbance rate, and expressed as enzymatic units, where one unit of Collagenase activity is defined as hydrolysis of 1.0 μmole of FALGPA per minute at +25°C and pH 8.8 in the presence of calcium ions.

The format is a photometric assay using a 96-well plate and is applicable with most commercially available flat-bottom plates and plate readers.", "Language": "en", "Country": "XG", "Code": "Principle" }, { "Name": "Protocols", "Value": "

All solutions supplied with the kit are ready to use once thawed and centrifuged. Do not further dilute the Calibrators A to E (Vials 1 to 5) or FALGPA Solution (Vial 6) provided with the kit.

Sample preparation

If cell-free aqueous solutions are used as samples, no sample preparation is required. For testing of cell culture samples, the samples must be centrifuged; the supernatants can be used in the test.
In case of samples with an expected Collagenase activity of approximately >1 FALGPA U/mL, or if the rate of photometric absorbance of a sample measurement is higher than the measured rate of absorbance of the highest Calibrator E, then dilute the sample, for example, 1:20 and 1:100 with 5 mM HEPES, 1 mM CaCl₂ pH 7.5, and test the diluted sample.

Test procedure

The test was developed and evaluated using 5 μL sample or calibrator and 95 μL FALGPA Solution per well of the microplate. Volumes should not be changed.

• Use at least triplicates of each sample, calibrator, and controls. Include the calibrators provided with the kit on the same plate in each run to generate quantitative results. Positive and negative controls should also be measured on the sample plate.
• Only thaw the number of 1 mL vials of FALGPA Solution (Vial 6) required for the procedure. For ease of use, the FALGPA Solution can be pooled into a multichannel reagent reservoir and pipetted via 8- or 12-channel pipettes.

1. Thaw kit contents at +15 to +25°C. As soon as kit contents are thawed, store Calibrators A to E (Vials 1 to 5) on ice and FALGPA Solution (Vial 6) at +15 to +25°C. During the subsequent steps, the solutions must be stored as described here.
2. Centrifuge all vials for 10 seconds using a tabletop microcentrifuge. Transfer 95 μL of FALGPA Solution (Vial 6) into each well of a microplate that will be used for the reactions, based on the total number of replicates of blank, calibrators, controls, and samples.
3. Pipette 5 μL per well of each of the following reagents in triplicate to 3 wells each on the microplate in this order:
   - Blank (purified water)
   - Negative control(s) of the sample matrix (cell culture medium or aqueous buffer solution)
   - Positive control(s) of a Liberase Blend
   - Calibrators A to E (Vials 1 to 5)
   - Samples (centrifuged cell culture supernatants or aqueous solutions from your biotechnological process).
4. Remove air bubbles in the wells by gently tapping the microplate on an even surface. If possible, include a 10 second agitation (shaking) step on the multiwell plate reader on the lowest possible agitation setting to remove any remaining air bubbles before measuring.
5. Immediately measure the rate of absorbance, for example, mAbs/min at 340 nm for 10 minutes.

", "Language": "en", "Country": "XG", "Code": "Protocols" }, { "Name": "Content", "Value": "

Vial / Bottle	Cap	Label	Function / Description	Content
1	brown	Collagenase Activity Test, Collagenase I Calibrator A	Collagenase I in buffered solution. Ready-to-use solution for calibration of the assay.	1 vial, 250 μL
2	brown	Collagenase Activity Test, Collagenase I Calibrator B	Collagenase I in buffered solution. Ready-to-use solution for calibration of the assay.	1 vial, 250 μL
3	brown	Collagenase Activity Test, Collagenase I Calibrator C	Collagenase I in buffered solution. Ready-to-use solution for calibration of the assay.	1 vial, 250 μL
4	brown	Collagenase Activity Test, Collagenase I Calibrator D	Collagenase I in buffered solution. Ready-to-use solution for calibration of the assay.	1 vial, 250 μL
5	brown	Collagenase Activity Test, Collagenase I Calibrator E	Collagenase I in buffered solution. Ready-to-use solution for calibration of the assay.	1 vial, 250 μL
6	brown	Collagenase Activity Test, FALGPA Solution	1 mM FALGPA in buffered solution. Ready-to-use solution for colorimetric detection of Collagenase activity.	10 vials, 1 mL each

", "Language": "en", "Country": "XG", "Code": "Content" }, { "Name": "Product Description", "Value": "The Collagenase Activity Test kit is based on the chromogenic substrate FALGPA, which is a labeled tetrapeptide resembling the primary structure of collagen, and which is specifically hydrolyzed by all known collagenases but not by other proteases. This test offers a better precision and more convenience compared to other existing tests, and it is perfectly suited for process development and in-process control.", "Language": "en", "Country": "XG", "Code": "Product Description" }, { "Name": "Sample Materials", "Value": "This kit is intended for use with the following types of sample material:

• Aqueous buffer solutions from biotechnological processes.
• Cell culture supernatant.
  If cell culture supernatant is used as sample material, first test a sample of fresh media for potential background reactivity. When testing fresh, unused media, ensure that all supplements required for the culture conditions have been added to the media.

", "Language": "en", "Country": "XG", "Code": "Sample Materials" }, { "Name": "Control Reactions", "Value": "

Positive control

As a positive control, use a freshly prepared solution of any Liberase Blends*. The working concentration should be approximately 0.25 Wünsch Units/mL.

Negative control

As a negative control, use a Collagenase-free matrix of your sample material, such as cell culture medium or aqueous buffer solution.", "Language": "en", "Country": "XG", "Code": "Control Reactions" }, { "Name": "Benefits", "Value": "

• Reliability
  Accurate and reproducible results due to included calibration standards and high lot-to-lot consistency of the kit.
• Convenience
  Solutions of FALGPA substrate and Collagenase standards are ready-to-use; no manual preparation of solutions or extraction of dyes needed.
• High sensitivity
  Limit of detection: 0.32 FALGPA Units/mL.
• Fast turnaround time
  Time-to-result: 30 minutes.

", "Language": "en", "Country": "XG", "Code": "Benefits" }, { "Name": "Positioning", "Value": "Test kit for determination of collagenase activity for in-process control.", "Language": "en", "Country": "XG", "Code": "Positioning" }, { "Name": "Applications", "Value": "The kit is designed for determination of collagenase activity in in-process control.", "Language": "en", "Country": "XG", "Code": "Applications" }, { "Name": "Specification", "Value": "Measuring range: 0.32 - 1.15 FALGPA Units/mL
Precision: ≤15% (typically <10%)
Specificity: Collagenases I and II from Clostridium histolyticum
Assay time: 30 min approximately
Test format: 96-well plate, absorption at 340 nm
Stability: At -15 to -25°C within specification range for 12 months.", "Language": "en", "Country": "XG", "Code": "Specification" } ] } } ] }