The ProAxsis K-Postn immunoassay is used for the in vitro quantitative determination of K-Postn in serum samples. K-Postn is a Cathepsin K-mediated degradation fragment of the matricellular protein periostin. Periostin regulates bone formation, microstructure, metabolism, and strength. Circulating levels of periostin has been suggested to be an indicator of incident fractures, particularly in postmenopausal women. However, periostin is secreted in elevated levels by a range of tissues during various morbidities including lung disease, inflammation, and several types of cancer. This limits the ability of periostin to accurately indicate bone health and fragility.
In contrast, K-Postn is a fragment of periostin resulting from cathepsin K digestion that is specific to bone health and metabolism. Periostin is a substrate of cathepsin K that undergoes degradation to produce K-Postn fragments in a mechanism of bone resorption that is specific to bones. Serum levels of K-Postn have potential use as a a predictor of incident fractures in postmenopausal subjects, independent from bone mineral density (BMD), fracture risk assessment tool (FRAX), and bone turnover markers (BTMs). Thus, serum K-Postn levels may aid in the research and identification of high risk of fracture and metabolic bone disorders.
Key Features and Benefits
- Osteoporotic fracture risk biomarker
- Bone health and fragility research
- Quantitative readout with detection range of 20 – 1280 ng/ml
- Competitive assay with inverse association between K-Postn concentration and absorbance signal.
- 4 hour run time
The ProAxsis K-Postn immunoassay has been designed to detect and quantify K-Postn within serum samples. The plate is washed and then coated with biotinylated peptide which is incubated before washing to remove any unbound material. Standards, samples, and primary antibody are then added and incubated. The primary antibody competitively binds to either the immobilized biotinylated peptide or soluble K-Postn fragments/non-biotinylated peptide (K-Postn Standard). Unbound primary antibody together with K-Postn fragments/non-biotinylated peptide are removed by a further wash step. A secondary horseradish peroxidase (HRP) conjugated antibody is then incubated on the plate and will bind to the captured primary antibody. A further wash step is performed prior to the addition of a colorimetric substrate, which results in the formation of a blue colored product in the presence of HRP. This enzymatic reaction is subsequently stopped by the addition of acidic stop solution to each test well (a yellow solution is formed). The color intensity (absorbance) is read at 450 nm using a plate reader. The absorbance reading is inversely proportional to K-Postn concentration i.e., a high absorbance measurement denotes a low K-Postn concentration.
|Freezer Box Components|
|K-Postn Biotinylated Peptide||1 vial containing 30 μL (concentration: 1.6 μg/ml) of Biotinylated peptide in N,N-dimethylformamide (DMF)|
|K-Postn Standard (Non-Biotinylated Peptide)||1 vial containing 30 μL of Non-Biotinylated peptide (concentration: 32 μg/mL) in N,N-dimethylformamide (DMF)|
|K-Postn Primary Antibody||1 vial containing 20 μL of K-Postn primary antibody (concentration: 41 μg/mL) in glycerol solution|
|K-Postn Secondary Antibody (HRP)||1 vial containing 30 μL of Goat Anti-Rabbit Antibody (HRP) (100 μg/mL) in glycerol solution|
|Main Box Components|
|Nunc Immobilizer Plate||96-well streptavidin coated microtitre plate (pre-blocked)
|K-Postn Wash Buffer Concentrate||80 mL of a 10-fold concentration of buffered surfactant with preservatives|
|K-Postn Dilution Buffer Concentrate||6 mL of a 10-fold concentration of buffer surfactant with preservatives|
|TMB Substrate||12 ml of tetramethylbenzidine solution (ready to use)|
|Stop Solution||6 ml of 2 N sulfuric acid (ready to use)|
|Plate Sealers||4 adhesive strips|
The range of the standard curve of this immunoassay is 20 – 1280 ng/mL.
Limit of Detection:
The lowest concentration of K-Postn giving an absorbance reading greater than two standard deviations (SD) above the mean zero (blank matrix) reading (n=32) was determined to be 18.11 ng/ml.
Intra-Assay CVs (within assay precision) = 12.27%
Inter-Assay CV (between assay precision) = 15.13%
Specificity / Interference:
To assess specificity of the K-Postn immunoassay, serum samples were spiked with potentially interfering substances listed in the table below. No significant difference was observed between baseline serum levels of K-Postn, and levels upon spiking with each substance.
|Substance||Concentration tested||Average Recovery (%)|
|Cathepsin K||220 pg/ml||97.26|
|Bilirubin, unconjugated||4.0 μg/ml||114.18|
|Bilirubin, conjugated||4.0 μg/ml||101.67|
|Human Serum Albumin||400 μg/ml||93.07|
|DMSO||1.0 % v/v||100.92|
|Water||1.0 % v/v||92.59|
Bonnet N, Biver E, Chevalley T, Rizzoli R, Garnero P, Ferrari SL. Serum Levels of a Cathepsin-K Generated Periostin Fragment Predict Incident Low-Trauma Fractures in Postmenopausal Women Independently of BMD and FRAX. J Bone Miner Res. 2017;32(11):2232-2238. doi:10.1002/jbmr.3203