Download biomedica product listFluoBolt™ Periostin FIA | FIA-1703
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Sample type
Serum
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Sample volume
20 µl / well
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Assay time
Overnight / 37°C
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Sensitivity
2 pmol/l (= 186.6 pg/ml)
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Standard range
0 – 180 pmol/l (= 0 – 16.4 ng/ml)
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Conversion factor
1 ng/ml = 11 pmol/l (MW: 93.3 kDa)
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Cross-reactivity
99-98% aa sequence identity with higher apes, 95% with bovine/ equine, 91% with mouse periostin.
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Regulatory status
CE marked – for IVD use in the EU
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Detection target
This assay detects human PERIOSTIN. No interference of BMP-1 or TGF-ß1 with the assay’s signal up to a 10-fold molar excess was monitored
Product Overview
The FluoBolt™-PERIOSTIN immunoassay is a o.n, Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well plate format for the quantitative determination of PERIOSTIN in serum. The assay employs human based serum standards to ensure the measurement of biologically reliable data.
Principle of the Assay
The FluoBolt™-PERIOSTIN immunoassay is a o.n, Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well plate format for the quantitative determination of PERIOSTIN in serum samples.
Figure explaining the principle of metal enhanced fluorescence:
In a first step, standard/sample/control and detection antibody (fluorescent labelled anti-Periostin) are pipetted into the wells of the microtiter plate, which are pre-coated with anti- Periostin antibody. Periostin present in the standard/sample/control binds to the pre-coated antibody in the well and forms a sandwich with the detection antibody.
The signal of the bound fluorescent detection antibody is enhanced several hundred fold by the metal nano-structures at the plate bottom and thus highly sensitive detectable with a standard microtiter plate fluorescence reader. Measurements can either been done without washing (bottom measurement) or after a final washing step (top measurement). The concentration of Periostin in the sample is determined directly from the dose response curve.
Typical Standard Curve
The figure below shows a typical standard curve for the FluoBolt™- Periostin ELISA. The immunoassay is calibrated against recombinant full length Periostin:
[caption id="attachment_6596"]
Standard Curve for the FluoBolt Periostin kit[/caption]
Kit components
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Contents |
Description |
Quantity |
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FM |
Anti-human PERIOSTIN antibody, pre-coated MEF-microtiter plate, packed in vacuum sealed aluminum bag |
1 x 96 well |
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WP |
20 X wash buffer concentrate |
1 x 25 ml |
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AF, FA3, FA5, FAA |
Anti-human PERIOSTIN antibody, black flask, labeled with FITC, Cy3, Cy5 or AlexaFluor680 |
1 x 4 ml |
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FS |
Serum-based standards 1-6, (0, 11.25, 22.5, 45, 90, 180 pmol/l), white caps, lyophilized |
6 vials, 0.25 ml |
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FCA/B |
Serum-based controls with expected concentrations after reconstitution (lyophilised) |
2 vials, 0.25 ml |
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FD |
Sample diluent, natural cap, ready to use |
1 x 10 ml |
Storage instructions: All reagents of the human soluble neuropilin-1 ELISA kit are stable at 4°C until the expiry date stated on the label of each reagent.
Sample Collection & Storage
Adding recombinant PERIOSTIN to serum and plasma resulted in 25% recovery in EDTA/Heparin-plasma and 80% in Citrate-plasma compared to serum (100%). This may be a result of the heparin and/or fibronectin binding properties of this molecule. Therefore we suggest to use only serum samples for optimal results. Collect venous blood samples by using standardized blood collection tubes for serum. We recommend performing serum separation by centrifugation as soon as possible, e.g. 10 min at 2000 x g, preferably at 4°C (2-8°C). The acquired serum samples should be measured as soon as possible. For longer storage aliquot samples and store at -25°C or lower. Do not freeze-thaw samples more than 4 times
Urine
Urine has not been evaluated as sample type for this assay.
Cell Culture Supernatant
Cell Culture Supernatant has not been evaluated as sample type for this assay.
Reagent Preparation
Wash Buffer
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1. |
Bring the WASHBUF concentrate to room temperature. Crystals in the buffer concentrate will dissolve at room temperature. |
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2. |
Dilute the WASHBUF concentrate 1:20, e.g., 50ml WASHBUF + 950ml distilled or deionized water. Only use diluted WASHBUF when performing the assay. |
The diluted WASHBUF is stable up to one month at 4°C (2-8°C).
Standards & Controls for Serum, Plasma, and Urine Measurements
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1. |
Add 250 µl of distilled or deionized water to the lyophilized FS (Standards) and FC (Controls). Reconstituted FS and FC are stable at -25°C or lower until expiry date stated on the label. Reconstituted FS and FC can undergo 4 freeze-thaw cycles. |
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2. |
Leave at room temperature (18-26°C) for 20 min. Swirl gently. |
Reconstituted FS and FC are stable at -25°C or lower until expiry date stated on the label. Reconstituted FS and FC can undergo 4 freeze-thaw cycles.
Sample Preparation
All reagents and samples must be at room temperature (18-26°C) before use in the assay.
Assay Protocol
Read the entire protocol before beginning the assay.
In standard format, the kit is delivered with an AlexaFluor680 labeled detection antibody (FAA) because serum background fluorescence is minimal within this wavelength range. Therefore if your reader is equipped with monochromatic optics, please set Excitation/Emission to 679/702 nm or if you are using an optical filter based reader, select a suitable filter pair (e.g. 670/720 nm). On request the kit can also be delivered with FITC, Cy3 or Cy5 (Ex/Em = 495/518 nm, 550/570 nm or 650/670 nm) labeled detection antibody
Mark positions for STD/SAMPLE/CTRL (Standard/Sample/Control) on the protocol sheet.
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1. |
Take the plasmonic enhanced microtiter plate out of the aluminum bag. Avoid touching the bottom of the plate with bare hands, because reading without washing is performed through the well bottom |
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2. |
Add 40 µl of the selected fluorescence labeled detection antibody (FAF or FA3 or FA5 or FAA) to all wells required. Swirl gently. |
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3. |
Add 20 µl of standard, control or sample to the wells according to the marked positions on the protocol sheet, swirl gently, |
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4. |
Cover tightly with the delivered adhesive film and incubate over night at 37°C in the dark. |
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5a. |
If your reader allows bottom reading, read the plate without any further processing at the Ex/Em wavelength fitting to the delivered detection antibody (495/518 nm for FAF, 550/570 nm for FA3, 650/670 nm for FA5, 679/702 nm for FAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0 pM and the 180 pM PERIOSTIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (FD |
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5b. |
If your reader has no bottom read option or if you want to store the plate for documentation purposes, discard or aspirate the content of the wells and wash 3x with diluted wash buffer. Use a minimum of 200 µl wash buffer per well. After the final wash, remove remaining fluid by strongly tapping the plate against a paper towel. Read the plate in top configuration without any further processing at the Ex/Em wavelength fitting to the chosen detection antibody (495/518 nm for FAF, 550/570 nm for FA3, 650/670 nm for FA5, 679/702 nm for FAA). |
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7. |
Hint: Quality of bottom reading (5a) may vary between microplate readers. For first time users we suggest to perform the washing step and follow protocol 5b. |
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8. |
The gain of oyur reader should be set to achieve at least 10000 fluorescence units (F.U.) between the signals of the 0 pM and the 180 pM PERIOSTIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with appropriate dilution using sample diluent (FD).. |
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9. |
Store the plate with the 2 desiccant bags supplied at 4°C (2-8°C) in the aluminum bag. Unused wells are stable until expiry date stated on the label. Fluorescence signals of standards, controls and samples remain detectable for at least two month at the plate surface, depending on signal intensity achieved |
Calculation of Results
Subtract the fluorescence intensity of the 0 pM standard from all other standards, samples and controls. Construct a calibration curve from the fluorescence units (F.U.) of the standards using commercially available software or graph paper. Read sample and control concentrations from this standard curve. Make sure to use appropriate curve fitting algorithm (e.g. linear or 4PL).
The quality control (QC) protocol supplied with the kit shows the results of the final release QC for each kit lot at production date.
Fluorescence intensity obtained by customers may differ due to various influences and/or due to the normal decrease of signal intensity during shelf life. However, this does not affect validity of results as long as the supplied kit controls read according to specifications (target ranges see labels)
Information on the Analyte
PERIOSTIN (UniProtKB - Q15063), also known as osteoblast specific factor 2 (OSF-2), is a cell adhesion protein belonging to the fasciclin domain-containing protein family. It consists of 836 amino acids (aa) starting with a 21 aa long signalling sequence, followed by a Emilin-like domain rich in cysteine, four repeated fasiclin 1 and a C-terminal variable domain, which is different among the 7 splice variants (isoforms) in humans.
PERIOSTIN is expressed during ontogenesis as well as in adult connective tissues submitted to mechanical stress such as bone, tendons, heart valves, skin and the periodontal ligaments. Further, it is expressed in aorta, stomach, lower gastrointestinal tract, placenta, uterus, thyroid and breast tissue. In bone, PERIOSTIN directly interacts with collagen type I, fibronectin, Notch 1, tenascin-C and BMP-1, resulting in enhanced proteolytic activation of lysyl oxidase for collagen cross-linking stabilising bone matrix. Next to developing, maintaining and repairing tissue, PERIOSTIN plays a vital role in tumorigenesis by interacting with various cell-surface receptors and signaling pathway, which e.g. results in inactivation of integrin- mediated signaling, leading to promoting cell adhesion and motility which is of relevance for tumor progression and metastasis. Elevated serum PERIOSTIN levels have been associated with pancreatic, ovarian, lung, breast, colon, gastric, thyroid and oesophageal tumours
All FluoBoltTM IMMUNOASSAYs are validated according to sensitivity, specificity, precision, accuracy, dilution linearity, sample stability, expected values in blood donor collections and sample matrix .
Calibration
The FluoBoltTM PERIOSTIN immunoassay is calibrated against recombinant human PERIOSTIN protein (AA 22-863 of Q15063 (Uniprot ID)).
Detection limit & Sensitivity
To determine the sensitivity of the FluoBoltTM PERIOSTIN immunoassay, experiments measuring the Lower Limit of Detection (LOD) and the lower limit of quantification (LLOQ) were conducted.
The LOD, also called the detection limit, is the lowest point at which a signal can be distinguished above the background signal, i.e. the signal that is measured in the absence of PERIOSTIN, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of PERIOSTIN, three independent measurements) plus three times the standard deviation of the measurements.
The LLOQ, or sensitivity of an assay, is the lowest concentration at which an analyte can be accurately quantified. The criteria for accurate quantification at the LLOQ are an analyte recovery between 75 and 125% and a coefficient of variation (CV) of less than 25%. To determine the LLOQ, standard 2, i.e. the lowest standards containing PERIOSTIN, is diluted, measured three times and its concentration back calculated. The lowest dilution, which meets both criteria, is reported as the LLOQ.
The following values were determined for the FluoBoltTM PERIOSTIN immunoassay:
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LOD |
2 pmol/l |
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LLOQ |
11 pmol/l |
Precision
The precision of an FluoBoltTM PERIOSTIN immunoassay is defined as its ability to measure the same concentration consistently within the same experiments carried out by one operator (within-run precision or repeatability) and across several experiments using the same samples but conducted by several operators at different locations using different FluoBoltTM PERIOSTIN immunoassay lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run precision was tested by measuring the same samples 3 times within one FluoBoltTM PERIOSTIN immunoassay lot. The experiment was conducted by one operator. Samples reading above the highest standard were diluted with the sample diluent provided in the kit.
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Sample 1 |
Sample 2 |
Sample 3 |
Sample 4 |
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Mean (pmol/l) |
29,44 |
25,3 |
40,5 |
126,6 |
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SD (pmol/l) |
1,03 |
1,90 |
2,10 |
6,96 |
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CV (%) |
3,5% |
7,5% |
5,2% |
5,5% |
In-Between-Run Precision
In-between-run precision was assessed by measuring the same samples 3 times within multiple FluoBoltTM PERIOSTIN immunoassay lots. The measurements were carried out by one operator.
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Sample 1 |
Sample 2 |
Sample 3 |
Sample 4 |
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Mean(pmol/l) |
29,9 |
25,0 |
37,4 |
101,0 |
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SD (pmol/l) |
2,84 |
2,18 |
2,28 |
13,2 |
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CV (%) |
10% |
9% |
6% |
13% |
Accuracy
The accuracy of an FluoBoltTM PERIOSTIN immunoassay is defined as the precision with which it can recover samples of known concentrations.
The recovery of the FluoBoltTM PERIOSTIN immunoassay was measured by adding recombinant PERIOSTIN to clinical samples containing a known concentration endogenous PERIOSTIN. The %recovery of the spiked concentration was calculated as the percentage of measured compared over the expected value.
This table shows the summary of the recovery experiments in the FluoBoltTM PERIOSTIN immunoassay for serum samples. When using plasma samples, analytic recovery of the protein strongly varies depending on the amount and type of coagulant used. Therefore, we recommend to only use serum samples for PERIOSTIN analysis.
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Sample ID |
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#1 |
#2 |
#3 |
#4 |
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Measured(pM) |
83,1 |
77,0 |
118,0 |
95,6 |
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Expected (pM) |
84,4 |
87,1 |
132,1 |
124,4 |
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% Recovery |
98% |
88% |
89% |
77% |
Dilution Linearity
Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing PERIOSTIN behave in a dose dependent manner and are not affected by matrix effects. Dilution linearity assesses the accuracy of measurements in diluted clinical samples spiked with known concentrations of recombinant analyte. By contrast, parallelism refers to dilution linearity in clinical samples and provides evidence that endogenous analyte behaves same way as the recombinant one. For dilution linearity and parallelism are assessed for each sample type and are considered good if the results are within 20% of the expected concentration.
Dilution linearity was assessed by serially diluting clinical samples spiked with recombinant PERIOSTIN with standard 1 (serum stripped of PERIOSTIN).
The figure and table below show the mean recovery and range of serially diluted recombinant PERIOSTIN in human samples. Linearty did not differ between human serum and plasma :
INSERT FIGURE DILUTION LINEARITY (x-axis: Dilutions, y-axis: % Mean Recovery, lines: solid, heading: Dilution linearity of recombinant X)
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Measured (pM) |
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Dilution |
Sample #1 |
Sample #2 |
Sample #3 |
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1+0 |
59,7 |
38,7 |
29,1 |
|
1+1 |
29,0 |
16,3 |
14,5 |
|
1+2 |
17,9 |
13,2 |
8,5 |
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Expected (pM) |
|||
|
1+1 |
29,8 |
19,2 |
14,6 |
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1+2 |
19,9 |
12,8 |
9,7 |
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Linearity (%) |
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1+1 |
103% |
118% |
100% |
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1+2 |
111% |
97% |
114% |
Specificity
Analyte Specificity:
This assay detects human PERIOSTIN. Addition of recombinant TGF-β1 or BMP-1, which are considered to be binding to PERIOSTIN, to the standards supplied with this kit did not reduce signal intensity.
Species Specificity:
Human PERIOSTIN shares around 98-99% aa sequence identity with higher apes (e.g. gorilla or chimpanzee), 95% with bovine/ equine and 91% with mouse PERIOSTIN. Reactivity of this assay with other species than human has not been tested. So using this assay for PERIOSTIN measurements in serum of species with high sequence homology may be possible, but must be evaluated by the user.
Sample Stability
The stability of endogenous PERIOSTIN was tested by incubation of clinical samples at room temperature for a specific period of time and by subjection samples to repeated freeze / thaw cycles.
Stability at Room Temperature:
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|
Fluorescence Units |
|||
|
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t=0 |
t=1h |
t=5h |
o.n. |
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Sample #1 |
20372 |
20372 |
20310 |
20318 |
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Sample #2 |
20430 |
20430 |
19700 |
19424 |
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Sample #3 |
43224 |
40184 |
39620 |
40184 |
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Sample #4 |
29390 |
28030 |
26873 |
26873 |
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|
% Signal retained |
|||
|
Sample #1 |
100% |
100% |
100% |
100% |
|
Sample #2 |
100% |
100% |
96% |
95% |
|
Sample #3 |
100% |
93% |
92% |
93% |
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Sample #4 |
100% |
95% |
91% |
91% |
|
Mean |
100% |
97% |
95% |
95% |
Samples show only slight losses of signal even when kept at Room Temperature over night (≈ 16 hrs)
Freeze/Thaw Stability
For freeze-thaw experiments, samples were collected according to the supplier’s instruction using blood collection devices and stored at -80°C. Reference samples were freeze-thawed once. The mean recovery of sample concentration after five freeze-thaw cycles is 84%.
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|
Fluorescence Units |
||||
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|
Cycle 1 |
Cycle 2 |
Cycle 3 |
Cycle 4 |
Cycle 5 |
|
Sample #1 |
12563 |
11755 |
11974 |
12206 |
12289 |
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Sample #2 |
3057 |
3081 |
3048 |
2615 |
2142 |
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% Signal retained |
||||
|
Sample #1 |
100% |
94% |
95% |
97% |
98% |
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Sample #2 |
100% |
101% |
100% |
86% |
70% |
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Mean |
100% |
97% |
98% |
91% |
84% |
Samples can be freez/thawed for at least three times.
Sample Values
PERIOSTIN Values in Apparently Healthy Individuals
To provide expected values for circulating PERIOSTIN, a panel of samples from a collection of blood donors (aged 18-69 years) was tested.
A summary of the results is shown below:
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PERIOSTIN (pmol/L) |
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Percentiles |
female (n=25) |
male (n=23) |
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90 |
10,1 |
10,9 |
|
70 |
6,4 |
5,1 |
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Median |
4,6 |
4,4 |
|
30 |
2,2 |
3,2 |
|
10 |
0,1 |
2,7 |
No dependency on age, or sex was found.
Matrix Comparison
Adding recombinant PERIOSTIN to serum and plasma resulted in 25% recovery in EDTA/Heparin-plasma and 80% in Citrate-plasma compared to serum (100%). This may be a result of the heparin and/or fibronectin binding properties of this molecule. Therefore we suggest to use only serum samples for optimal results.
- Age-related changes in serum periostin level in allergic and non-allergic children.Fujitani H, Kasuga S et al., Allergol Int. 2019: S1323-8930(19)30003-6.
PMID:30711305- Overexpression of periostin is positively associated with gastric cancer metastasis through promoting tumor metastasis and invasion.Zhong H et al. J Cell Biochem. 2019. [Epub ahead of print]
PMID:30637809
- Serum periostin levels and severity of fibrous dysplasia of bone.Guerin Lemaire H et al., Bone. 2019 ;121:68-71.
PMID:30616028- Elevated Periostin Concentrations in the Bronchoalveolar Lavage Fluid of Patients with Eosinophilic Pneumonia.Nakagome K et al., Int Arch Allergy Immunol. 4:1-8.
PMID:30612125- Effect of inhaled corticosteroids on serum periostin levels in adult patients with mild-moderate asthma.Solanki B et al. Allergy Asthma Proc. 2019;40(1):32-34.
PMID:30582493- Elevated serum periostin level in patients with chronic cough and airway hyperresponsiveness.Kono Y et al. , Respir Investig. 2019 ;57(2):122-125.
PMID:30553784- Longitudinal evaluation of serum periostin levels in patients after large-artery atherosclerotic stroke: A prospective observational study.He X et al., Sci Rep. 2018 ;8(1):11729.
PMID:30082879- Serum periostin levels following small bone fractures, long bone fractures and joint replacements: an observational study.Varughese R et al., Allergy Asthma Clin Immunol. 2018 ;14:30.
PMID:30065761- Identification of Serum Periostin as a Potential Diagnostic and Prognostic Marker for Colorectal Cancer.Dong D et al., Clin Lab. 2018 ;64(6):973-981.
PMID:29945311- The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis.Gineyts E et al., Calcif Tissue Int. 2018 ;103(5):567-580.
PMID:29916127