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Description
Overview
Can be used to measure PMN elastase/α1-PI complexes released from granulocytes during an inflammatory response.
Catalogue Number
QIA96
Brand Family
Calbiochem®
Application Data
Materials Required but Not Delivered
• 5 and 10 ml graduated pipettes • 5 to 1000 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
References
Fretzayas, A., et al. 2000. Pediatrics 105, E28. Belorgey, D. and Bieth, J.G. 1998. Biochemistry 37, 16416. Panyutich, A.V., et al. 1995. Am. J. Respir. Cell Mol. Biol. 12, 351. Friedman, R.B. and Young, D.S. (Eds.) 1997. Effects of Disease on Clinical Laboratory Tests. AACC Press, Washington, 3rd Edition, pages 3-161.
Product Information
Detection method
Colorimetric
Form
96 Tests
Format
96-well plate
Kit contains
Anti-PMN Elastase coated 96-Well Plate, Anti-α1-PI/HRP Conjugate, PMN Elastase/α1-PI Complex Standard, Wash Buffer Concentrate, Sample Diluent, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
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
Multiple Storage Conditions
Toxicity
Multiple Toxicity Values, refer to MSDS
Storage
Multiple storage requirements
Storage Conditions
Please note that this kit is shipped in two parts. Upon arrival store the Control Low and Control High at -70°C and the remaining components of the kit at 4°C.
Avoid freeze/thaw
Avoid freeze/thaw
Do not freeze
Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit contains
Anti-PMN Elastase coated 96-Well Plate, Anti-α1-PI/HRP Conjugate, PMN Elastase/α1-PI Complex Standard, Wash Buffer Concentrate, Sample Diluent, Substrate Solution, Stop Solution, Plate Covers, and a user protocol.
Please note that this kit is shipped in two parts. Upon arrival store the Control Low and Control High at -70°C and the remaining components of the kit at 4°C.
Background
Polymorphonuclear (PMN) granulocytes play an important role in the inflammatory response of cells to attacks of invading pathogens such as microorganisms and viruses, as well as tissue damaged by accidents or surgery. Various bloodstream mediators like cytokines, leukotrienes, complement factors, bacterial endotoxins, and clotting and fibrinolysis factors stimulate these cells to phagocytize and destroy the foreign substances. The PMN granulocytes then use proteinases to digest these agents and any tissue debris. The proteinase PMN elastase is localized in the azurophilic granules of polymorphonuclear granulocytes. During the phagocytosis of foreign substances these enzymes are excreted into the extracellular surrounding, and the activity of the PMN elastase is regulated by inhibitors such as the a1-proteinase inhibitor (α1-PI). An overwhelming release of PMN elastase, however, can exceed the inhibitory potential of the α1-proteinase inhibitor, allowing enzymatically active PMN elastase and simultaneously produced oxidants to cause local tissue injury. Due to the actions of the bloodstream and the lymphatic system, however, α1-PI is eventually able to form a complex with the excreted elastase. Therefore, the concentration of the PMN elastase/α1-PI complex can be used to measure the activity of granulocytes during the inflammatory response. This kit is designed to detect PMN elastase/α1-PI complexes in cell culture supernatants, human serum, plasma, exudate, bronchoalveolar lavage fluid, and cerebrospinal fluid.
• 5 and 10 ml graduated pipettes • 5 to 1000 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. • Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells and fill wells as indicated for each wash cycle and do not allow wells to dry out at any time.
Preparation
The PMN elastase/α1-PI complex ELISA kit can be used to detect PMN elastase/α1-PI complexes in cell culture supernatants, human serum, plasma, exudate, bronchoalveolar, fluid, cerebrospinal fluid, and seminal plasma. Remove the 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 and warm to room temperature before use. Avoid freeze/thaw cycles. Dilute samples 1:100 with Sample Diluent just prior to assay.
Reagent preparation
• Wash Buffer: Add distilled or deionized water to 50 ml Wash Buffer Concentrate for a final volume of 500 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. Wash Buffer is stable for 30 days at 2° to 25°C.
• PMN Standard: Reconstitute lyophilized PMN elastase/α1-PI complex standard with Sample diluent 30 min before use. Refer to vial label for reconstitution volume. The concentration of reconstituted standard is 10 ng/ml. Mix gently. Store aliquots at -20°C.
• Controls: Reconstitute lyophilized Controls (high and low) with 1 ml Sample Diluent 30 min before use. Refer to vial labels for the concentration range of each control. Store in aliquots at -20°C. Controls are now ready to use and do not require further dilution.
• Standard Preparation: Label 6 tubes, one for each standard point: S2, S3, S4, S5, S6, S7. Then prepare 1:2 serial dilutions for the standard curve as follows: Pipette 225 µl of Sample Diluent into tubes S2 - S7. Pipette 225 µl of reconstituted standard (serves as the highest standard S1, concentration of standard 1= 10 ng/ml) into the first tube, labelled S2, and mix (concentration of standard 2 = 5 ng/ml). Pipette 225 µl of this dilution into the second tube, labelled S3, and mix thoroughly before the next transfer. Repeat serial dilutions 4 more times thus creating the points of the standard curve. Sample Diluent serves as blank.
Detailed protocol
Mix all reagents gently to avoid foaming before use. 1. Determine the number of microwell strips 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 Plate coated with polyclonal antibody to human PMN elastase from holder and store in sealed foil bag at 4°C with desiccant. 2. Add 100 µl of Sample Diluent, in duplicate, to the standard wells leaving the first wells empty. Prepare standard dilutions by pipetting 200 µl of PMN elastase/α1-PI Complex Standard, in duplicate, into wells A1 and A2. Transfer 100 µl from wells A1 and A2 to wells B1 and B2, respectively. Mix the contents of wells B1 and B2 and transfer 100 µl to wells C1 and C2, respectively. Do not scratch the inner surface of the microwells. Repeat step five times, creating two rows of PMN elastase/α1-PI complex standard dilutions. Discard 100 µl of the contents from the last microwells (G1, G2) used. The concentration range of the standards is 10-0.16 ng/ml. 3. Add 100 µl of Sample Diluent, in duplicate, to the blank wells. 4. Add 100 µl of each 1:100 diluted sample, in duplicate, to designated wells. Add 100 µl of optional Control, in duplicate, to the designated wells. 5. Cover with a Plate Cover and incubate at room temperature for 1 h on a rotator. 6. Remove Plate Cover. Wash the microwell strips four times with ~400 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Do not scratch the surface of the microwells. After the last wash, tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing or place upside down on wet absorbent paper for no longer than 15 min. Do not allow wells to dry. 7. Add 150 µl of HRP-conjugate to all wells. 8. Cover with a Plate Cover and incubate for 1 h at room temperature on a rotator. 9. Remove Plate Cover and empty wells. Wash microwell strips 4 times as outlined above. Proceed immediately to next step. 10. Add 200 µl of TMB substrate solution to all wells including blanks. 11. Incubate the microwell strips at room temperature for approximately 20 min on a rotator. 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 2.0, therefore the color development and the substrate reaction must be stopped before positive wells are no longer properly recordable. 12. Stop the enzyme reaction by quickly pipetting 50 µl of Stop Solution into each well, including blanks. The Stop solution must be spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results should be read immediately after addition of the Stop Solution or within 1 h if the microwell strips are stored at 4°C in the dark. 13. Read absorbance of each microwell using a microplate reader set at 450 nm (620 nm serves as the reference wave length; 610 nm to 650 nm is acceptable). Blank the plate reader using the blank wells. Determine the absorbance of the samples and the PMN elastase/a1-PI Complex Standards. Note: incubation without shaking may result in lower than expected absorbance values.
Calculations
1. 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 PMN elastase/a1-PI complex concentration on the abscissa.
3. Determine the concentration of circulating PMN elastase/α1-PI complex 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 PMN elastase/α1-PI complex concentration. Calculation of samples with an Abs exceeding the range of the standard curve may result in incorrect/low PMN elastase/α1-PI complex levels. Such samples may require 1:200 to 1:400 dilution to accurately quantitate the PMN elastase/α1-PI complex level. Values obtained should be within the expected range of known PMN elastase/α1-PI complex controls.
4. A sample standard curve is shown below.
Assay characteristics and examples
When a panel of 57 sera from healthy blood donors (male and female) was tested for PMN elastase the detected PMN elastase mean level was 350 ng/ml. Normal PMN elastase levels may vary depending on the serum used.
Standard curve
Figure 1: Standard Curve
Sensitivity
3.0 ng/ml
Sensitivity Notes
The limit of detection for PMN elastase, defined as the analyte concentration resulting in an absorption significantly higher than the absorption of the dilution medium (mean plus three standard deviations) was determined to be 3 ng/ml (mean of 10 independent assays).
Assay Range
15.6 - 1000 ng/ml
Registered Trademarks
Calbiochem® is a registered trademark of Merck, KGaA, Darmstadt. Inc. Tween® is a registered trademark of ICI Americas, Inc. Interactive Pathways™ is a trademark of Merck, KGaA, Darmstadt.
