FluoBolt™ Periostin

Additional material supplied with the kit

• 2 self-adhesive plastic films
• QC data sheet
• Protocol sheet
• Instruction manual for use
• 2 desiccant bags for plate storage

Material and equipment required but not supplied

• Precision pipettes calibrated to deliver 10μl, 20μl, 50μl, 200μl, 500μl and disposable tips
• Distilled or deionized water
• Plate washer, multichannel pipette or manifold dispenser for washing
• Refrigerator with 4°C (2-8°C)
• Fluorescence microplate reader
• Graph paper or software for calculation of results

Reagents and sample preparation

All reagents of the kit are stable at 4°C (2-8°C) until expiry date stated on the label of each reagent.

Sample preparation

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 it is suggested 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. 10min 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.

Lipemic or hemolysed samples may give erroneous results. Samples should be mixed well before assaying.

Reagent preparation

Add 250μl of distilled or deionized water to the lyophilized FS (Standards) and FC (Controls). Leave at room temperature (18-26°C) for 20min. 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.

Bring WP (Wash buffer) concentrate (20x) to room temperature. Make sure that the solution is clear and without any salt precipitates before further dilution. Dilute the WP to working strength by adding the appropriate amount of distilled or deionized water, e.g. 25ml of WP + 475ml water, prior to use in the assay. Undiluted WP is stable at 4°C (2-8°C) until expiry date on the label. Diluted WP is stable at 4°C (2-8°C) up to one month. Only use diluted WP in the assay.

All reagents and samples must be at room temperature (18-26°C) before use in the assay.

Mark position for standards, controls and samples on the protocol sheet. It is recommended to run samples and standards in duplicates.

Remove 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.

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 679/702nm or if you are using an optical filter based reader, select a suitable filter pair (e.g. 670/72nm). On request the kit can also be delivered with FITC, Cy3 or Cy5 (Ex/Em = 495/518nm, 550/570nm or 650/670nm) labeled detection antibody.

1)      Add 40μl of the selected fluorescence labeled detection antibody (FAF or FA3 or FA5 or FAA) to all wells required. Swirl gently.

2)      Add 20μl of standard, control or sample to the wells according to the marked positions on the protocol sheet, swirl gently, cover tightly with the delivered adhesive film and incubate overnight at 37°C in the dark.

3a)    If your reader allows bottom reading, read the plate without any further processing at the Ex/Em wavelength fitting to the delivered detection antibody (495/518nm for FAF, 550/570nm for FA3, 650/670nm for FA5, 679/702nm for FAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0pM and the 180pM PERIOSTIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (FD).

3b)    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 200μl 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 (495/518nm for FAF, 550/570nm for FA3, 650/670nm for FA5, 679/702nm for FAA).

Hint: Quality of bottom reading (3a) may vary between microplate readers. For first time users we suggest to perform the washing step and follow protocol 3b.

Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signals of the 0pM and the 180pM PERIOSTIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with appropriate dilution using sample diluent (FD).

4)      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 0pM 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).

Intra-assay

4 samples of known concentrations were tested 4 times within 1 assay run. CVs ranged from 3.5-7.5%

Inter-assay

4 samples of known concentrations were tested in duplicates within 3 different assay runs. CVs ranged from 9-13%

Spike/Recovery

The recovery of Periostin was evaluated by adding known amounts of human recombinant Periostin to 4 different human serum samples.

Mean recovery was 82% (71-100%).

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 N. 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. FIANOSTICS does not take responsibility for functionality of the assay in non-human samples.

Metal Enhanced Fluorescence (MEF) offers the possibility to increase the analytical sensitivity of systems based on fluorescence detection dramatically. MEF is based on the fact that excitation light interacts with the electrons of metal nano structures thus generating very high electromagnetic fields (Localized Surface Plasmons, LSPs) Therefore, such structures are also called “plasmonic structures” and the combination of (e.g. polymeric) support and structure is known as “plasmonic substrate”. These LSPs lead to an increase in emission output of fluorescent molecules (e.g. fluorescently labeled antibodies) when bound to surfaces with suitable nano metal structures that enhances the signal dramatically. FIANOSTICS has developed a new plasmonic enhanced immunoassay platform in cooperation with Sony DADC BioSciences (now STRATEC Consumables since July 1st 2016), that allows up to 300 fold gains of sensitivity. This platform is fully compatible to standard laboratory methodology using 96 well microtiter plate format and assays based on this technology can be run on any standard fluorescence microplate reader. Its unique features enable fluorescence immunoassays with highest sensitivity and without washing steps.

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 signaling sequence, followed by an 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 and especially in adult connective tissues submitted to mechanical stress such as bone, tendons, heart valves, skin and the periodontal ligaments. Further, it is widely 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, thus stabilizing the bone matrix. Next to its role in developing, maintaining and repairing of tissue, Periostin plays a vital role in tumorigenesis by interacting with various cell-surface receptors and signaling pathways e.g. resulting in inactivation of integrin- mediated signaling, leading to promoting cell adhesion and motility which is of relevance for tumor progression and metastasis.

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 stabilizing 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 pathways, 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 tumors.

Asthma / COPD / Allergies

A variety of studies have shown the value of Periostin as a biomarker for research, monitoring and subject stratification for respiratory tract diseases.

Tumor Progression & Metastasis

The use of Periostin as a research marker for widely spread cancers (e.g. mamma- or colon-carcinoma) has been demonstrated. Periostin can be used as stand-alone marker or in combination with established markers to improve research.

Osteoarthritis

Periostin seems to enhance inflammatory processes in osteoarthritic tissue, thus contributing to the progression of the disease.

Bone Fracture Healing

Eriostin serum levels change in presence of bone fractures and during their healing. Therefore, Periostin levels can be used for monitoring healing of fractures; however it has to be taken into account when interpreting serum levels for other diseases.