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Content/Vial	Preparation	Storage and Stability
Standards, Vials 9 to 14	Add 500 μl of water to each of the vials and mix thoroughly for 30 min to completely dissolve the lyophilizate.	At +2 to +8°C for 4 weeks.
Controls, Vials 15, 16	Add 500 μl of water to each of the vials and mix thoroughly for 30 min to completely dissolve the lyophilizate.	At +2 to +8°C for 4 weeks.
Wash Buffer Vial 5	Dilute the appropriate volume 1:10 with Incubation Buffer from Vial 1. For a complete 96-well plate, prepare 500 μl of Biotin Conjugate stock solution + 4.5 ml of Incubation Buffer. If using only a part of the 96 wells, then prepare a proportionally reduced volume of this working solution.	At +2 to +8°C for 4 weeks.
DIG Conjugate, Vial 4	Dilute the appropriate volume 1:10 with Incubation Buffer from Vial 1. For a complete 96-well plate, prepare 500 μl of DIG Conjugate stock solution + 4.5 ml of Incubation Buffer. If using only a part of the 96 wells, then prepare a proportionally reduced volume of this working solution.	At +2 to +8°C for 4 weeks.
Anti-DIG POD Reagent, Vial 6	1. Add 500 μl water to the vial and mix thoroughly for 30 min to completely dissolve the lyophilizate.The concentration of the reconstituted solution is 4 U/ml. 2. Dilute the 4 U/ml solution with Conjugate Buffer (Vial 2) to achieve a working concentration of 100 mU/ml, as follows: 250 μl reconstituted anti-DIG-POD reagent + 9.75 ml Conjugate Buffer	At +2 to +8°C for 4 weeks.
Biotin DIG Working Solution	Combine equal volumes of the prepared working solutions: Biotin Conjugate and DIG Conjugate (from Steps 4 and 5 above). Mix thoroughly, for example, using a roller mixer for 10 min	Use within 30 min after preparation.

 ", "Country": "XG", "Code": "Storage Conditions (Working Solution)", "Name": "Storage Conditions (Working Solution)" }, { "Language": "en", "Value": "Store the kit at +2 to +8°C.", "Country": "XG", "Code": "Storage Conditions (Product)", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "0.1 ng/ml", "Country": "XG", "Code": "Sensitivity", "Name": "Sensitivity" }, { "Language": "en", "Value": "This ELISA kit detects the constituents of Liberase Blends containing Collagenase I, Collagenase II, and Thermolysin, specifically the formulations found in the following Liberase Blends available from Roche Diagnostics: Liberase MNP-S, Liberase MNP-S GMP Grade, and Liberase MTF C/T GMP Grade.
\nThis ELISA kit has been developed specifically for above-mentioned Liberase Blends from Roche Diagnostics, other applications have not been verified.", "Country": "XG", "Code": "Specificity", "Name": "Specificity" }, { "Language": "en", "Value": "0.1 ng/ml up to approximately 5 ng/ml.", "Country": "XG", "Code": "Detection range", "Name": "Detection range" }, { "Language": "en", "Value": "Intra-assay precision: ≤10% (typically ≤ 5%)
Inter-assay precision: ≤15% (typically ≤10%)", "Country": "XG", "Code": "Precision", "Name": "Precision" }, { "Language": "en", "Value": "Unit of measure is \"piece\".", "Country": "XG", "Code": "CB - Order Information", "Name": "CB - Order Information" }, { "Language": "en", "Value": "

\n\t
• Polypropylene tubes for preparation of antibody reagents
\n\t
• 1 liter bottle for wash buffer preparation
\n\t
• Micropipettes
\n\t
• Centrifuge
\n\t
• Multi-well plate shaker
\n\t
• Roller mixer
\n\t
• ELISA reader
\n\t
• Multi-well plate washer
\n

", "Country": "XG", "Code": "Additional Equipment and Reagent Required", "Name": "Additional Equipment and Reagent Required" }, { "Language": "en", "Value": "A quantitative, functional assay is performed to assess background signal, and the lot-specific concentrations of Standards A to F and Controls X and Y.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "\n\n\nFirst test a sample of fresh, unused media for a potential background absorbance as some media may contain residual trypsin from production procedures.When testing fresh, unused media, ensure that all supplements required for the culture conditions have been added to the media.\n\n\nNo sample preparation is required.\n\n\nThe samples must be centrifuged and the supernatants can be used for Liberase Blend determination.
\nIf there are samples with an expected Liberase Blend concentration of approximately > 5 ng/ml, or the photometric absorbance of a sample measurement is higher than the measured absorbance of the highest Standard F, then dilute the sample (e.g., 1:20 and 1:100) with Incubation Buffer from Bottle 1 and test the diluted sample.
\nProcedure\n\n\n\n\n\n\n\n\n\n\n\n

\n\t
1. Transfer 30 μl per well of the following materials in duplicate to 2 wells each on the coated Multi-well Plate:- Standards A to F (Bottles 9 to 14)- Positive Controls X and Y (Bottles 15 and 16)- Negative control of your sample matrix (cell culture medium or aqueous buffer solution)- Samples (centrifuged cell culture supernatants or aqueous solutions from your biotechnological process step)
\n\t
2. Add 70 μl of the fresh Biotin DIG Working Solution to each well.
\n\t
3. Seal the Multi-well Plate with an MWP-sealing film.
\n\t
4. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 1 hour.
\n\t
5. Remove the film carefully and wash the Multi-well Plate 3 times with 300 μl per well of the diluted 1x Wash Buffer using a multi-well plate washer.
\n\t
6. Pipette 100 μl of the diluted working solution of the Anti-Dig POD Reagent (100 mU/ml) into each well.
\n\t
7. Seal the Multi-well Plate with an MWP-sealing film.
\n\t
8. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 30 minutes.
\n\t
9. Remove the film carefully and wash the Multi-well Plate 3 times with 300 μl per well of the diluted 1x Wash Buffer using a multi-well plate washer.
\n\t
10. Pipette 100 μl Detection Substrate (TMB) (Bottle 7) into each well.
\n\t
11. Seal the Multi-well Plate with an MWP-sealing film.
\n\t
12. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 15 minutes.
\n\t
13. Remove the film carefully and pipet 50 μl of Stop Solution (Bottle 8) into each well.
\n\t
14. Seal the Multi-well Plate with an MWP-sealing film.
\n\t
15. Incubate the sealed plate on a multi-well plate shaker with 300 rpm for 1 minute at +15 to +25°C.
\n\t
16. Place the plate in a multi-well plate reader and measure at 450 nm, reference wavelength 620 nm.
\n

\n\n\n\n

\n\t
1. Calculate the mean of the absorbance values from the replicate measurements. The mean values are used for the calculations in the following steps.
\n\t
2. If the background absorbance value of the negative control is higher than the absorbance value of Standard A, the absorbance values of the samples must be corrected.- Subtract the absorbance value of Standard A from the absorbance value of the negative control.- Subsequently, subtract the corrected negative control absorbance value from the absorbance values of the samples.- Use the obtained absorbance values to calculate the trypsin concentrations of the samples.
\n\t
3. Generate a calibration curve using the concentrations and absorbances of Standards A to F. Use the information from the lot-specific standard concentrations in the value table in section Lot-specific Data. Use a curve fitting software with a 4 parameter non-linear fit model to calculate the standard curve using the results of Standards A to F.
\n\t
4. Use the absorbance values of the controls, as well as the corrected absorbance values of the samples from Step 2 to determine the concentrations based on the standard curve created in Step 3.
\n

\n\n\n\n\nCheck the recovery of the controls. The determined control concentrations should be within ± 25% of the lot-specific target concentration (see section Lot-Specific Data for the concentrations of Controls X and Y). If the results of the control concentrations are not within ± 25% of the lot-specific target concentration, the sample results may be invalid.\n\n\nIf the mean absorbance of a sample is higher than the absorbance of the highest Standard F, then the sample concentration is higher than the concentration of Standard F. An exact concentration value for a sample cannot be calculated when the sample has an absorbance value that is higher than the absorbance of the Standard F. To determine the concentration of such a sample, repeat the test with a diluted sample. Dilute the sample with Incubation Buffer (Bottle 1) at, for example, 1:20 and 1:100. Take the dilution factor into consideration when calculating the concentration with the absorbance values of the diluted sample.", "Country": "XG", "Code": "Protocols", "Name": "Protocols" }, { "Language": "en", "Value": "

Vial / Bottle	Cap	Label	Function/Description	Content
1	white	Residual Protein Liberase Kit, Incubation Buffer	Ready-to-use solution For preparation of the Biotin DIG working Solution	1 bottle, 100 ml
2	blue	Residual Protein Liberase Kit, Conjugate Buffer	Ready-to-use solution For preparation of the Anti-DIG POD Reagent, Vial 6	1 bottle, 100 ml
3	yellow	Residual Protein Liberase Kit, BiotinConjugate	Biotin labeled for target capture Polyclonal sheep antibody against Liberase Blend 20-fold stock solution	1 vial, 0.75 ml
4	purple	Residual Protein Liberase Kit, DIG conjugate	Digoxigenin labeled for target marking Polyclonal sheep antibody against Liberase Blend 20-fold stock solution	1 vial, 0.75 ml
5	red	Residual Protein Liberase Kit, Wash Buffer	For washing steps 10-fold stock solution	1 bottle, 100 ml
6	red	Residual Protein Liberase Kit, Anti-DIG PODReagent	Peroxidase conjugated to anti-DIG antibody Lyophilizate	1 bottle
7	black	Residual Protein Liberase Kit, Detection Substrate (TMB)	For color development nd detection Ready-to-use solution	1 bottle, 15 ml
8	colorless	Residual Protein Liberase Kit, Stop Solution	For stopping the color development Ready-to-use solution	1 bottle, 15 ml
9	sand	Residual Protein Liberase Kit, Standard A	For calibration of the assay Lyophilizate Liberase free	1 bottle
10	beige	Residual Protein Liberase Kit, Standard B	For calibration of the assay Liberase Blend Lyophilizate	1 bottle
11	mustard	Residual Protein Liberase Kit, Standard C	For calibration of the assay Liberase Blend Lyophilizate	1 bottle
12	olive	Residual Protein Liberase Kit, Standard D	For calibration of the assay Liberase Blend Lyophilizate	1 bottle
13	caramel	Residual Protein Liberase Kit, Standard E	For calibration of the assay Liberase Blend Lyophilizate	1 bottle
14	rosewood	Residual Protein Liberase Kit, Standard F	For calibration of the assay Liberase Blend Lyophilizate	1 bottle
15	white	Residual Protein Liberase Kit, Control X	Positive control Liberase Blend Lyophilizate	1 bottle
16	green	Residual Protein Liberase Kit, Control Y	Positive control Liberase Blend Lyophilizate	1 bottle
17	-	Residual Protein Liberase Kit, Coated Micro Titer Plate	Target capture Streptavidin- coated multi-well plate Ready-to-use	12 strips in frame
18	-	Residual Protein Liberase Kit, Multi-well SealingFilm	Sealing MWP during incubations	10 pieces of self-adhesive film

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

\n\t
• Incubation time: 1 hour 55 minutes.
\n\t
• Total assay time: approximately 2 hours 30 minutes.
\n

", "Country": "XG", "Code": "Assay Time", "Name": "Assay Time" }, { "Language": "en", "Value": "\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\t\n\t\t\t\n\t\t\t\n\t\t\n\t\n

Observation	Possible cause	Recommendation
Absorbance of samples too low	Insufficient TMB reaction	Increase the incubation time with the Detection Substrate (TMB) for color development (Step 12). The incubation time can be increased until the wells with the higher concentrated standards show a clearly observable blue color, up to 45 minutes.
POD conjugate has a reduced enzymatic activity.	Use freshly prepared POD conjugate.
Low concentration after dilution of highly concentrated samples	Reduce dilution factor for highly concentrated samples.
Absorbance of samples too high	Concentration exceeds measuring range.	Dilute samples to ensure that they are within the specified detection range.
Background of sample matrix.	Check analyte-free sample matrix for background absorbance value. Correct sample values for background absorbance of matrix.
Contamination of samples with Liberase Blend from lab workspace.	Check absorbance values of Standard A and negative control. If the absorbance values of Standard A and the negative control are significantly increased, repeat the experiment in a different workspace using a new kit. Be careful when reconstituting lyophilizates. Thoroughly clean contaminated workspace.
Absorbance of negative control too high	Contamination of samples with Liberase Blend from lab workspace.	Check absorbance values of Standard A and negative control. If the absorbance values of Standard A and the negative control are significantly increased, repeat the experiment in a different workspace using a new kit. Be careful when reconstituting lyophilizates. Thoroughly clean contaminated workspace.
Absorbance of the background too high	Substrate shows color development without enzymatic activity.	Use a newly opened vial of Detection Substrate (TMB).
Contamination of samples with Liberase Blend from lab workspace.	Check absorbance values of Standard A and negative control. If the absorbance values of Standard A and the negative control are significantly increased, repeat the experiment in a different workspace using a new kit. Be careful when reconstituting lyophilizates. Thoroughly clean contaminated workspace.
Variations too high	Insufficient mixing of sample and incubation mix.	Ensure mixing on the multi-well plate mixer is done with 300 rpm.
Residual buffer after washing	Check performance of multi-well plate washer. Wells should not contain residual buffer after washing. Try tapping the Multi-well Plate on an absorbent paper towel to remove traces of Wash Buffer.
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.
Precision of micropipettes	Check the precision of the micropipettes.
Multi-well plate washer not washing correctly.	Check the multi-well plate washer for tip blockage or salt crystallization, which may affect the evenness and effectiveness of the washing steps.

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

• Aqueous buffer solutions from biotechnology processes.
• Cell culture supernatant

", "Country": "XG", "Code": "Sample Materials", "Name": "Sample Materials" }, { "Language": "en", "Value": "Control X and Y are positive controls and are provided with the kit. For preparation of the positive controls, see section Preparation of Working Solutions.Analyte-free matrix of your sample material, such as buffer or cell culture medium.", "Country": "XG", "Code": "Control Reactions", "Name": "Control Reactions" }, { "Language": "en", "Value": "In addition to the ready-to-use solutions supplied with this kit, you will need to prepare the following working solutions:

Solution	Content	Reconstitution/Preparation of Working Solution	Storage and Stability	For use in...
9 to 14	Standards A to F (Bottles 9 to 14)	Add 500 μl of double-distilled water to each of the vials and mix thoroughly for 30 minutes to completely dissolve the lyophilizate.	Store 4 weeks at +2 to +8°C	Calibration of the assay
15 and 16	Controls X and Y (Bottles 15 and 16)	Add 500 μl of double-distilled water to each of the vials and mix thoroughly for 30 minutes to completely dissolve the lyophilizate. The controls supplied with the kit are ready to use once reconstituted. Do not further dilute the Positive Controls X and Y.	Store 4 weeks at +2 to +8°C	Positive controls
5	Wash Buffer (Bottle 5)	Dilute the entire contents of the bottle (100 ml) with 900 ml water and mix thoroughly.	Store 4 weeks at +2 to +8°C	Washing steps
3	Biotin Conjugate (Vial 3)	Dilute the appropriate volume 1:10 with Incubation Buffer from Bottle 1. For a 96-well plate, prepare 500 μl of Biotin Conjugate stock solution + 4.5 ml of Incubation Buffer. If using only a part of the 96 wells, then prepare a proportionally reduced volume of this working solution.	Store 4 weeks at +2 to +8°C	Preparation of Biotin DIG Working SolutionTarget capture
4	DIG Conjugate (Vial 4)	Dilute the appropriate volume 1:10 with Incubation Buffer from Bottle 1. For a 96-well plate, prepare 500 μl of DIG Conjugate stock solution + 4.5 ml of Incubation Buffer. If using only a part of the 96 wells, then prepare a proportionally reduced volume of this working solution.	Store 4 weeks at +2 to +8°C	Preparation of Biotin DIG Working SolutionTarget marking
6	Anti-DIG POD Reagent (Bottle 6)	Add 500 μl double-distilled water to the vial and mix thoroughly for 30 minutes to completely dissolve the lyophilizate. The concentration of the reconstituted solution is 4 U/ml. Dilute the 4 U/ml solution with Conjugate Buffer (Bottle 2) to achieve a working concentration of 100 mU/ml, as follows: 250 μl Anti-DIG POD Solution (4 U/ml) + 9.75 ml Conjugate Buffer.	Store 4 weeks at +2 to +8°C	Detection
-	Biotin DIG Working Solution	Combine equal volumes of the prepared working solutions: Biotin Conjugate (Solution 3) and DIG Conjugate (Solution 4).Mix thoroughly, for example, using a roller mixer for 10 minutes. Always prepare the Biotin DIG Working Solution just before use, and use the solution within 30 minutes after preparation.	Use within 30 minutes after preparation.	ELISA Step 6

", "Country": "XG", "Code": "Working Solution", "Name": "Working Solution" }, { "Language": "en", "Value": "Immunologic assay for sensitive detection of the compounds of the Liberase blend", "Country": "XG", "Code": "Positioning", "Name": "Positioning" }, { "Language": "en", "Value": "

• Fast: Total assay time of 2-2.5 h
• High sensitivity: Detection limit ≤ 0.1 ng/mL, measuring range 0.1-10 ng/mL
• High specificity: Specific detection of the components of the Liberase blend (Collagenases I + II, Thermolysin) and fragments thereof by use of polyclonal antibodies

", "Country": "XG", "Code": "Benefits", "Name": "Benefits" }, { "Language": "en", "Value": "The kit is designed for sensitive detection of residual Liberase in in-process control and release testing.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "The Roche Residual Protein Liberase Kit, based on an immuno assay with tailor-made polyclonal antibodies against the components of the Liberase blend makes testing for residual Liberase fast and reliable. The assay is perfectly suited for process development, in-process control and final quality control.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Sensitivity: ≤0.1 ng/mL
Measuring range: 0.1 - 5 ng/mL
Precision: ≤15% (typically <5%)
Accuracy (vs. weight): ≤25%
Specificity: Roche Liberase Blends; Collagenases I and II from Clostridium histolyticum, Thermolysin from Bacillus thermopoteolyticus, and fragments
Assay time (h:min): 2 - 2:30 total assay time
Test format: 96 reactions", "Country": "XG", "Code": "Specification", "Name": "Specification" } ] } } ] }