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Content/VialPreparationStorage 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.













 ", "Name": "Storage Conditions (Working Solution)" }, { "Language": "en", "Value": "Store the kit at +2 to +8°C.", "Name": "Storage Conditions (Product)" }, { "Language": "en", "Value": "0.1 ng/ml", "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.", "Name": "Specificity" }, { "Language": "en", "Value": "0.1 ng/ml up to approximately 5 ng/ml.", "Name": "Detection range" }, { "Language": "en", "Value": "Intra-assay precision: ≤10% (typically ≤ 5%)
Inter-assay precision: ≤15% (typically ≤10%)", "Name": "Precision" }, { "Language": "en", "Value": "Unit of measure is \"piece\".", "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
", "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.", "Name": "Quality Control" }, { "Language": "en", "Value": "
Preparation of Sample Material
\n\n
Culture supernatant
\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
Cell-free aqueous solution
\nNo sample preparation is required.\n\n
Cell culture material
\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
ELISA Procedure
\n\n
The ELISA was developed and evaluated using 30 μl sample and 70 μl immunoreagent per well of the Multi-well Plate. Use the recommended volumes.
\n\n
Perform measurements of at least duplicates of each sample, standards, and controls.
\n\n
Include the standards provided with the kit on the same plate in each run to generate quantitative results. For quality control of the correct function of all components of the assay, measure the positive controls provided with the kit (Controls X and Y; Bottles 15 and 16) together with the samples on the same plate in each run and include a negative control.
\n\n
Working temperature for all of the procedures described below is +15 to +25°.
\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)
  2. \n\t
  3. Add 70 μl of the fresh Biotin DIG Working Solution to each well.
  4. \n\t
  5. Seal the Multi-well Plate with an MWP-sealing film.
  6. \n\t
  7. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 1 hour.
  8. \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.
  10. \n\t
  11. Pipette 100 μl of the diluted working solution of the Anti-Dig POD Reagent (100 mU/ml) into each well.
  12. \n\t
  13. Seal the Multi-well Plate with an MWP-sealing film.
  14. \n\t
  15. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 30 minutes.
  16. \n\t
  17. 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.
  18. \n\t
  19. Pipette 100 μl Detection Substrate (TMB) (Bottle 7) into each well.
  20. \n\t
  21. Seal the Multi-well Plate with an MWP-sealing film.
  22. \n\t
  23. Incubate the sealed plate on a multi-well plate shaker at 300 rpm and +15 to +25°C for 15 minutes.
  24. \n\t
  25. Remove the film carefully and pipet 50 μl of Stop Solution (Bottle 8) into each well.
  26. \n\t
  27. Seal the Multi-well Plate with an MWP-sealing film.
  28. \n\t
  29. Incubate the sealed plate on a multi-well plate shaker with 300 rpm for 1 minute at +15 to +25°C.
  30. \n\t
  31. Place the plate in a multi-well plate reader and measure at 450 nm, reference wavelength 620 nm.
  32. \n
\n\n
Calculation of Concentrations
\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.
  2. \n\t
  3. 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.
  4. \n\t
  5. 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.
  6. \n\t
  7. 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.
  8. \n
\n\n
Checks and Controls
\n\n
Quality control of the test run
\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
Concentration out of range
\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.", "Name": "Protocols" }, { "Language": "en", "Value": "
Vial / BottleCapLabelFunction/DescriptionContent
1whiteResidual Protein Liberase Kit, Incubation Buffer
  • Ready-to-use solution
  • For preparation of the Biotin DIG working Solution
1 bottle,
100 ml
2blueResidual Protein Liberase Kit, Conjugate Buffer
  • Ready-to-use solution
  • For preparation of the Anti-DIG POD Reagent, Vial 6
1 bottle,
100 ml
3yellowResidual Protein Liberase Kit, BiotinConjugate
  • Biotin labeled for target capture
  • Polyclonal sheep antibody against Liberase Blend
  • 20-fold stock solution
1 vial,
0.75 ml
4purpleResidual 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
5redResidual Protein Liberase Kit, Wash Buffer
  • For washing steps
  • 10-fold stock solution
1 bottle,
100 ml
6redResidual Protein Liberase Kit, Anti-DIG PODReagent
  • Peroxidase conjugated to anti-DIG antibody
  • Lyophilizate
1 bottle
7blackResidual Protein Liberase Kit, Detection Substrate (TMB)
  • For color development nd detection
  • Ready-to-use solution
1 bottle,
15 ml
8colorlessResidual Protein Liberase Kit, Stop Solution
  • For stopping the color development
  • Ready-to-use solution
1 bottle,
15 ml
9sandResidual Protein Liberase Kit, Standard A
  • For calibration of the assay
  • Lyophilizate
  • Liberase free
1 bottle
10beigeResidual Protein Liberase Kit, Standard B
  • For calibration of the assay
  • Liberase Blend
  • Lyophilizate
1 bottle
11mustardResidual Protein Liberase Kit, Standard C
  • For calibration of the assay
  • Liberase Blend
  • Lyophilizate
1 bottle
12oliveResidual Protein Liberase Kit, Standard D
  • For calibration of the assay
  • Liberase Blend
  • Lyophilizate
1 bottle
13caramelResidual Protein Liberase Kit, Standard E
  • For calibration of the assay
  • Liberase Blend
  • Lyophilizate
1 bottle
14rosewoodResidual Protein Liberase Kit, Standard F
  • For calibration of the assay
  • Liberase Blend
  • Lyophilizate
1 bottle
15whiteResidual Protein Liberase Kit, Control X
  • Positive control
  • Liberase Blend
  • Lyophilizate
1 bottle
16greenResidual 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
", "Name": "Content" }, { "Language": "en", "Value": "
    \n\t
  • Incubation time: 1 hour 55 minutes.
  • \n\t
  • Total assay time: approximately 2 hours 30 minutes.
  • \n
", "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
ObservationPossible causeRecommendation
Absorbance of samples too low                        Insufficient TMB reactionIncrease 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 highConcentration 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 highContamination 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 highSubstrate 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 highInsufficient mixing of sample and incubation mix.Ensure mixing on the multi-well plate mixer is done with 300 rpm.
Residual buffer after washingCheck 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 micropipettesCheck 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.
", "Name": "Troubleshooting" }, { "Language": "en", "Value": "
  • Aqueous buffer solutions from biotechnology processes.
  • Cell culture supernatant
", "Name": "Sample Materials" }, { "Language": "en", "Value": "
Always ensure that positive and negative controls are tested on each plate together with samples.
Positive controls
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.
Negative control
Analyte-free matrix of your sample material, such as buffer or cell culture medium.", "Name": "Control Reactions" }, { "Language": "en", "Value": "
Use double-distilled or deionized water of equivalent quality, for reconstitution of the lyophilizates. Ensure that the lyophilizates are carefully reconstituted to avoid Liberase Blend contamination of the environment. Trypsin contamination of the workspace may potentially contaminate samples to be tested.
A roller mixer may be used to effectively dissolve lyophilizates
In addition to the ready-to-use solutions supplied with this kit, you will need to prepare the following working solutions:
SolutionContentReconstitution/Preparation of Working SolutionStorage and StabilityFor use in...
9 to 14Standards 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°CCalibration of the assay
15 and 16Controls 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°CPositive controls
5Wash 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°CWashing steps
3Biotin 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°CPreparation of Biotin DIG Working SolutionTarget capture
4DIG 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°CPreparation of Biotin DIG Working SolutionTarget marking
6Anti-DIG POD Reagent (Bottle 6)
  1. 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.
  2. 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°CDetection
-Biotin DIG Working SolutionCombine 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
", "Name": "Working Solution" }, { "Language": "en", "Value": "Immunologic assay for sensitive detection of the compounds of the Liberase blend", "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
", "Name": "Benefits" }, { "Language": "en", "Value": "The kit is designed for sensitive detection of residual Liberase in in-process control and release testing.", "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.", "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", "Name": "Specification" } ] } } ] }

Residual Protein Liberase Kit

ELISA kit for determination of Liberase

Residual Protein Liberase Kit