HtrA1 ELISA Assay Kit

Additional Information

Assay Background

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Human HtrA1 protease (previously termed PRSS11) was initially identified in human fibroblasts and belongs to the high temperature requirement factor A (HtrA) family of serine proteases that can be distinguished from other serine proteases by sequence homology, by the presence of a trypsin-type protease domain and one or two carboxyterminal PDZ domains and by their oligomeric architecture.

The loss of mammalian HtrA activity is correlated with severe diseases, including arthritis, cancer, familial ischemic cerebral small vessel disease and age related macular degeneration, as well as Parkinson’s disease and Alzheimer’s disease. Epigenetic silencing occurs in various cancers, and the loss of HtrA1 correlates with decreased sensitivity to anticancer drugs and increased cell migration. In addition, overexpression of HtrA1 inhibited proliferation in vitro and tumour growth in vivo. These data suggest that HtrA1 might function as a tumour suppressor.

In the extracellular matrix, HtrA1 cleaves numerous secreted proteins, such as fibronectin, decorin, fibromodulin, aggrecan, type II collagen, biglycan, clusterin, a disintegrin and metalloproteinase domain-containing 9 (ADAM9), vitronectin, α-2-macroglobulin and the amyloid precursor protein fragment Aβ. The degradation of extracellular matrix components and the strong upregulation of HtrA1 in samples from patients implicate HtrA1 in arthritic diseases, in which it might affect the degradation of cartilage as well as inflammation.

It is unknown, how the cellular distribution of HtrA1 is regulated. In addition, little is known about the interaction partners of mammalian HtrAs. It will be important to identify proteins that function as determinants of the cellular localization, substrate specificity and regulation of HtrA proteases.

Assay Procedure

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Prepare reagents and standards as described in the sections above. For assessing serum samples, prepare the standard using the Serum Diluent. Remind that it is necessary to equilibrate the reagents to room temperature before use.

1. Prepare the unknown samples by appropriate dilution with Diluent Buffer.
2. Prepare the Microtiter plate by inserting the required amount of wells into the frame. Note that you need 16 wells for the standard curve.
3. Pipette 100 μl of the reconstituted standards (25 ng/ml) in duplicate in the wells using a clean pipette tip for each standard. Diluent Buffer serves as zero blank.
4. Pipette 100 μl of the prepared unknown samples in duplicate into the wells.
5. Seal the plate with the provided foil and incubate on a shaker at room temperature for exactly 120 minutes.
6. Wash by filling each well with Wash Buffer (200 μl), then remove by discarding/drying by taping inverted plate against clean paper towels. Take care that all wells are completely filled and emptied at each wash. Wash the wells 4 times with Wash Buffer.
7. Add 100 μl of diluted Biotinylated Antibody into each well.
8. Seal the plate and incubate on a shaker at room temperature for exactly 90 minutes.
9. Wash by filling each well with Wash Buffer (200 μl), then remove by discarding/drying by taping inverted plate against clean paper towels. Take care that all wells are completely filled and emptied at each wash. Wash the wells 4 times with Wash Buffer.
10. Add 100 μl of diluted Conjugate Solution into each well.
11. Seal the plate with the provided foil and incubate on a shaker at room temperature for exactly 30 minutes.
12. Wash by filling each well with Wash Buffer (200 μl), then remove by discarding/drying by taping inverted plate against clean paper towels. Take care that all wells are completely filled and emptied at each wash. Wash the wells 6 times with Wash Buffer.
13. Add 100 μl of TMB Solution to each well.
14. Seal the plate with foil provided and incubate in the dark at room temperature without shaking for 5-15 minutes.
15. Stop the reaction by adding 100 μl of Stop Solution to each well.
16. Read the plate at 450 nm (620 nm reference filter) within 30 minutes. Reading of the plate without reference may yield higher absorbances and thus may be less accurate.

Typical Standard Curve

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