ImmunoDiagnostics Human Proteinase 3 (PR3) ELISA kit

Reagents Supplied

Each kit is sufficient for one 96-well plate and contains the following components:

1. Microtiter Strips (96 wells) – Coated with a polyclonal antibody against human PR3, sealed.
2. 10×Wash buffer, 40ml.
3. 5×Assay buffer, 30ml.
4. 100×Detection antibody solution – A polyclonal antibody against human PR3 conjugated to horseradish peroxidase, 0.12ml.
5. Human PR3 standard, 10ng of recombinant human PR3 in a buffered protein base, lyophilized.
6. Substrate solution, 12ml, ready for use.
7. Stop solution, 12ml, ready for use.

Other Materials Required, But Not Provided

1. Pipettes and pipette tips.
2. 96-well plate or manual strip washer.
3. Buffer and reagent reservoirs.
4. Paper towels or absorbent paper.
5. Plate reader capable of reading absorbency at 450nm.
6. Distilled water or deionized water.

Storage

The kit should be stored at 2-8°C upon receipt, and all reagents should be equilibrated to room temperature before use. Remove any unused antibodycoated strips from the micro-plate, return them to the foil pouch and reseal. Once opened, the strips may be stored at 2-8°C for up to one month.

Preparation of Reagents

Bring all reagents and materials to room temperature before assay.

1. 1×Assay buffer
   Prepare 1×Assay buffer by mixing the 5×Assay buffer (30ml) with 120ml of distilled water or deionized water. If precipitates are observed in the 5×Assay buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1×Assay buffer may be stored at 2-8°C for up to one month.
2. 1×Wash buffer
   Prepare 1×Wash buffer by mixing the 10×Wash buffer (40ml) with 360ml of distilled water or deionized water. If precipitates are observed in the 10×Wash buffer bottle, warm the bottle in a 37°C water bath until the precipitates disappear. The 1×Wash buffer may be stored at 2-8°C for up to one month.
3. 1×Detection antibody solution
   Spin down the 100×Detection antibody solution briefly and dilute the desired amount of the antibody 1:100 with 1×Assay buffer, 100µl of the 1×Detection antibody solution is required per well. Prepare only as much 1×Detection antibody solution as needed. Return the 100×Detection antibody solution to 2-8°C immediately after the necessary volume is pipetted.

Preparation of Standards and Samples

Human PR3 standards: Reconstitute the lyophilized standard with 1ml of 1×Assay buffer to generate a standard stock solution of 10ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare serially diluted standards using 1×Assay buffer as follows:

1x Assay buffer serves as the zero standard (0ng/ml).

Note: The reconstituted standard stock should be aliquoted and stored at -20ºC for up to one month. Avoid repeating freezing/thawing cycles. Please do not store the diluted standard solutions.

Sample Preparation

Serum or plasma sample is generally required a 100-fold dilution in 1×Assay buffer. A suggested dilution step is to add 10µl of sample to 990µl of 1×Assay buffer. If a sample has a PR3 level greater than the highest standard, the sample should be diluted further and the assay should be repeated.

Assay Principle

This assay is a quantitative sandwich ELISA. The microtiter strips are precoated with a polyclonal antibody specific for human PR3. Standards and samples are pipetted into the wells and any human PR3 present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase (HRP) labelled polyclonal antibody specific for human PR3 is added to the wells. After wash step to remove any unbound reagents, an HRP substrate solution is added and color develops in proportion to the amount of human PR3 bound initially. The assay is stopped and the optical density of the wells is determined using a microplate reader. Since the increase in absorbance is directly proportional to the amount of captured human PR3, the unknown sample concentration can be interpolated from a reference curve included in each assay.

Assay Procedure

It is recommended that all standards and samples be assayed in duplicate.

1. Add 100µl of standard or sample per well, incubate at room temperature for 1 hour.
2. Discard the content and tap the plate on a clean paper towel to remove residual solution in each well. Add 300µl of 1×Wash buffer to each well and incubate for 1 minute. Discard the 1×Wash buffer and tap the plate on a clean paper towel to remove residual wash buffer. Repeat the wash step for a total 3 washes.
3. Add 100µl of 1×Detection antibody solution to each well, incubate at room temperature for 1 hour.
4. Wash each well 4 times as described in step 2.
5. Add 100µl of Substrate solution to each well, incubate at room temperature for 15 minutes. Protect from light.
6. Add 100µl of Stop solution to each well, gently tap the plate frame for a few seconds to ensure thorough mixing.
7. Measure absorbance of each well at 450nm immediately.

Calculation

1. Subtract the absorbance of the blank from that of standards and samples.
2. Generate a standard curve by plotting the absorbance obtained (y-axis) against human PR3 concentrations (x-axis). The best fit line can be generated with any curve-fitting software by regression analysis. Any curve of 4-parameter or log-log curve fitting can be used for calculation.
3. Determine human PR3 concentration of samples from standard curve and multiply the value by the dilution factor.

Assay Procedure Summary

Add 100µl of Standard or sample to each well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well three times.
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Add 100µl of 1× Detection antibody solution to each well.
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Incubate at room temperature for 1 hour.
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Aspirate and wash each well four times.
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Add 100µl of Substrate solution to each well.
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Incubate at room temperature for 15 minutes.
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Add 100µl of Stop solution to each well.
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Measure absorbance of each well at 450nm.
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Calculation

The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.

Human PR3 Standard Curve (4-Parameter)

Proteinase 3 (PR3), also known as myeloblastin, Wegener autoantigen, PRTN3 and NP-4, is one of the hematopoietic serine proteases localized in the primary granules of polymorphonuclear neutrophils (PMNs). The primary function of PR3 is recognized as to participate in direct intracellular killing of phagocytosed pathogens in phagolysosomes and degradation of extracellular matrix components at inflammatory sites. PR3 has also been proven to be able to process some pro-inflammatory cytokines, such as IL-1β, IL-18 and TNF-α, activate mitogen activated protein kinase (MAPK) signaling through proteinase activated receptor-1 (PAR1), and induce endothelial cell apoptosis through NF-κB signaling pathways. PR3 is identified as the target autoantigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in Wegener granulomatosis. Increased PR3 levels have been reported in samples with acute myocardial infarction, and in subjects with type 1 diabetes.

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