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QIA99 Soluble Tissue Plasminogen Activator ELISA Kit

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
QIA99
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Overview

Replacement Information

Key Spec Table

Species ReactivityDetection Methods
HColorimetric

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      Description
      OverviewUseful for the quantitative detection of soluble tissue-type plasminogen activator (t-PA). Altered levels of t-PA have been implicated in the progression of Grave’s disease, liver disorders, diabetic retinopathy, cardiovascular disease, thrombosis, myocardial infarction, and stroke.
      Catalogue NumberQIA99
      Brand Family Calbiochem®
      Application Data
      Materials Required but Not Delivered• 5 and 10 ml graduated pipettes
      • 10 to 1,000 µl adjustable single channel micropipettes with disposable tips
      • 50 to 300 µl adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Multichannel wash bottle or automatic wash system for delivery of wash solution
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis
      References
      ReferencesLi, Y., et al. 1998. Eur. J. Clin. Invest. 28, 1050.
      Skrha, J., et al. 1994. Clin. Chim. Acta. 229, 5.
      Okabe, K., et al. 1992. Gastroenterol. Jpn. 27, 61.
      Dano, K., et al. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects (Kluft, C.,
      ed.) pp. 20-46, CRC Press, Boca Raton, Florida.
      Kluft, C. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clincal Aspects(Kluft, C., ed.) pp. 47-49, CRC Press, Boca Raton, Florida.
      Kruithof, E.K.O. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clincal Aspects (Kluft, C.,
      ed.) pp. 190-210, CRC Press, Boca Raton, Florida.
      Mehta, J., et al. 1987. J. Am. Coll. Cardiol. 9, 263.
      Wilman, B., et al. 1986. J. Lab. Clin. Med. 105, 265.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsPre-Coated Microtiter Plate Strips, Anti-t-PAI/HRP-Conjugate, t-PA Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Sample Diluent, Substrate Solutions I and II, Stop Solution, Plate Covers, and a user protocol
      Quality LevelMQ100
      Applications
      Biological Information
      Assay range16 - 1000 pg/ml
      Assay time2.5 h
      Sample TypeCell culture supernatant, serum, plasma, or other biological fluids
      Species Reactivity
      • Human
      Physicochemical Information
      Sensitivity8 pg/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 36/37/38

      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36-45

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Yes
      Mercury prohibited To comply with ban of sale of mercury-added products required by The Interstate Mercury Education and Reduction Clearinghouse (IMERC), this product is prohibited to be sold in the following US states: Rhode Island and Connecticut.
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsPre-Coated Microtiter Plate Strips, Anti-t-PAI/HRP-Conjugate, t-PA Standard, Wash Buffer Concentrate, Assay Buffer Concentrate, Sample Diluent, Substrate Solutions I and II, Stop Solution, Plate Covers, and a user protocol
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      QIA99-1KIT 04055977209266

      Documentation

      Soluble Tissue Plasminogen Activator ELISA Kit SDS

      Title

      Safety Data Sheet (SDS) 

      Soluble Tissue Plasminogen Activator ELISA Kit Certificates of Analysis

      TitleLot Number
      QIA99

      References

      Reference overview
      Li, Y., et al. 1998. Eur. J. Clin. Invest. 28, 1050.
      Skrha, J., et al. 1994. Clin. Chim. Acta. 229, 5.
      Okabe, K., et al. 1992. Gastroenterol. Jpn. 27, 61.
      Dano, K., et al. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clinical Aspects (Kluft, C.,
      ed.) pp. 20-46, CRC Press, Boca Raton, Florida.
      Kluft, C. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clincal Aspects(Kluft, C., ed.) pp. 47-49, CRC Press, Boca Raton, Florida.
      Kruithof, E.K.O. 1988. In Tissue-Type Plasminogen Activator (t-PA): Physiological and Clincal Aspects (Kluft, C.,
      ed.) pp. 190-210, CRC Press, Boca Raton, Florida.
      Mehta, J., et al. 1987. J. Am. Coll. Cardiol. 9, 263.
      Wilman, B., et al. 1986. J. Lab. Clin. Med. 105, 265.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision04-April-2016 MJ
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman
      StorageUpon arrival store the entire contents of the kit at 4°C.
      BackgroundTissue-type plasminogen activator (t-PA) is a serine protease found in blood plasma, serum, other body fluids, and tissues. It converts the inactive proenzyme plasminogen to the active protease plasmin. Plasmin degrades the fibrin that makes up blood clots in a process known as fibrinolysis, which leads to the dissolution of the clot. Plasminogen activation is also implicated in the metastatic spread of malignant cells and in tissue remodeling. Fibrin has been shown to accelerate the conversion of plasminogen to plasmin, which is mediated by t-PA. Through this pathway fibrin promotes its own degradation. Inhibitors to t-PA have been found in blood preparations, cell culture media, and tissues. Plasminogen activator inhibitors -1 and -2 react with t-PA to form inactive complexes and the availability of free active t-PA is regulated through this interaction. Altered levels of soluble t-PA have been implicated in the progression of Graves disease, liver disease, diabetic retinopathy, cardiovascular disease, thrombosis, myocardial infarction, and stroke.
      Principles of the assayThe Calbiochem® brand t-PA ELISA kit is suitable for quantitative detection of soluble human Tissue-type plasminogen activator levels in cell culture supernatants, human serum, plasma, urine, or other body fluids.
      Materials provided• Anti-t-PA Coated Plate (Kit Component No. JA6700): Coated with polyclonal antibody (sheep) to human t-PA and includes strip holder
      • HRP-Conjugate anti-t-PA Monoclonal Antibody (Mouse) (Kit Component No. JA6701): 1 vial
      • t-PA Standard (Kit Component No. JA6702): 2 vials, lyophilized, 2 ng/ml upon reconstitution with preservative
      • 20X Wash Buffer Concentrate (Kit Component No. JA6703): 1 bottle, 50 ml, PBS with 1% Tween®-20 Detergent and preservative
      • 20X Assay Buffer Concentrate (Kit Component No. JA6704): 1 bottle, 5 ml, PBS with 1% Tween®-20 Detergent, 10% BSA, and preservative
      • Sample Diluent (Kit Component No. JA6712): 1 bottle, 12 ml
      • Substrate Solution (Kit Component No. JA6713): 1 bottle, 15 ml
      • Stop Solution (Kit Component No. JA6707): 1 bottle, 15 ml, 1 M Phosphoric Acid
      • Adhesive Plate Cover (Kit Component No. JA6711): 2 adhesive strips
      Materials Required but not provided• 5 and 10 ml graduated pipettes
      • 10 to 1,000 µl adjustable single channel micropipettes with disposable tips
      • 50 to 300 µl adjustable multichannel micropipette with disposable tips
      • Multichannel micropipette reservoir
      • Beakers, flasks, and cylinders necessary for preparation of reagents
      • Multichannel wash bottle or automatic wash system for delivery of wash solution
      • Microwell strip reader capable of reading at 450 nm with 620 nm as optional reference wavelength
      • Glass-distilled or deionized water
      • Calculator with program to perform linear regression analysis
      Precautions and recommendations• As conditions may vary from assay to assay a standard curve must be established for every run.
      • Bacterial, fungal, or cross contamination of samples or reagents may cause erroneous results.
      • While disposable pipette tips, flasks, or glassware are preferred, reusable glassware must be thoroughly washed and rinsed prior to use.
      • Insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty and fill wells as indicated for each wash cycle and do not allow wells to dry out at any time.
      • The limit of detection of t-PA defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be 8 pg/ml (mean of 6 independent assays).
      • When a panel of 7 sera from healthy blood donors was tested for t-PA the detected t-PA levels ranged between 500 and 5500 pg/ml with a mean level of 2060 pg/ml.
      PreparationRemove serum or plasma from the clot or red cells, as soon as possible after clotting and separation. Note: samples containing a visible precipitate must be clarified prior to use. Do not use extensively hemolyzed or lipemic specimens. Store all samples at -20°C. Allow to come to room temperature before use. Avoid freeze/thaw cycles.
      Reagent preparation1. Add distilled or deionized water to 50 ml Wash Buffer Concentrate for a final volume of 1,000 ml. Mix gently to avoid foaming and transfer to a clean wash bottle. Bacterial or fungal contamination of samples or reagents may cause false results. The pH of the final solution will be 7.4. Wash Buffer is stable for 30 days at 2° to 25°C.
      2. Mix the contents of the Assay Buffer Concentrate and add to 95 ml distilled or deionized water. Mix gently to avoid foaming. Assay Buffer is stable for 30 days at 4°C.
      3. The HRP-conjugated anti-t-PA should be diluted 1:100 as needed with Assay buffer and used within 30 min of dilution.
      4. Reconstitute t-PA standard with distilled water as stated on the label of the standard as needed and mix gently.
      Detailed protocol1. Mix all reagents gently to avoid foaming before use.
      2. Determine the number of Anti-t-PA Coated Plate required to test the desired number of samples and the appropriate number of blanks and standards. Each sample, standard, blank, and optional control sample should be assayed in duplicate. Remove extra Anti-t-PA Coated Plate (16 wells per strip) coated with polyclonal antibody (sheep) to human soluble t-PA from holder and store in sealed foil bag with the desiccant at 4°C.
      3. Wash the Anti-t-PA Coated Plate twice with approximately 300 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Please note that insufficient washing at any step in the assay will result in false results. Completely empty wells before each wash. Take care not to scratch the surface of the microwells. After the last wash, empty wells and tap Anti-t-PA Coated Plate on absorbent pad or paper towel to remove excess buffer. Use the Anti-t-PA Coated Plate immediately after washing or place upside down on a wet absorbent paper for not longer than 15 min. Do not allow wells to dry.
      4. Add 100 µl of Sample Diluent in duplicate to all standard wells. Prepare standard dilutions by pipetting 100 µl of the t-PA standard, in duplicate, into wells A1 and A2. Mix the contents of wells A1 and A2 by repeated aspiration and transfer 100 µl to wells B1 and B2, respectively. Take care not to scratch the inner surface of the wells. Continue this procedure five times, creating two rows of t-PA standard dilutions ranging from 16 to 1,000 pg/ ml. Discard 100 µl of the contents from the last wells used.
      5. Add 100 µl of Sample Diluent in duplicate to the blank wells.
      6. Add 90 µl of Sample Diluent, in duplicate, to the sample wells.
      7. Add 10 µl of each sample, in duplicate, to the designated wells.
      8. Prepare HRP-conjugate.
      9. Add 50 µl of 1:100 diluted HRP-conjugate to all wells including blanks.
      10. Cover with a plate cover and incubate at room temperature (18° to 25°C) for 2 h on a rotator platform.
      11. Remove plate cover and empty wells. Wash Anti-t-PA Coated Plate 3 times. Proceed to the next step.
      12. Pipette 100 µl of TMB Substrate Solution to all wells including blanks.
      13. Incubate the Microwell Strips at room temperature (18° to 25°C) for 15 min on a rotator platform. Avoid direct exposure to intense light. The point at which the substrate reaction should be stopped may be determined by the ELISA reader being used. Many ELISA readers record absorbance only up to 2 Abs Therefore the color development and the substrate reaction must be stopped before positive wells are no longer properly recordable.
      14. Stop the enzyme reaction by quickly pipetting 100 µl of Stop solution into each well including the blanks. It is important that the Stop solution be spread quickly and uniformly throughout the wells to completely inactivate the enzyme. Results must be read immediately after the Stop solution is added or within one h if the Anti-t-PA Coated Plate are stored at 4°C in the dark.
      15. Read absorbance of each microwell on a spectrophotometer at 450 nm as the primary wave length (620 nm as the reference wave length, 610 nm to 650 nm acceptable). Blank the plate reader using the blank wells. Determine the absorbance of the samples and the t-PA standards. Note: incubation without shaking may result in lower than expected Abs values.
      Calculations1. Calculate the average absorbance values for each set of duplicate standards and samples. 2. Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the t-PA concentration on the abscissa. 3. Determine the concentration of circulating t-PA for each sample by finding the mean absorbance value on the ordinate and extending a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding t-PA concentration. Calculation of samples with an ABS exceeding the range of the standard curve may result in incorrect/low t-PA levels. These samples should be re-analyzed at higher dilution (1:20 to 1:40) in Sample Diluent to accurately quantitate the t-PA level. Values obtained should be within the expected range of known t-PA controls. 4. A sample standard curve is shown below.

      Figure 1: Standard Curve
      Sensitivity8 pg/ml
      Assay Range16 - 1000 pg/ml
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Tween® is a registered trademark of ICI Americas, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.