Download biomedica product listFluoBolt™ WNT3A FIA | FIA-1705
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Method
Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well plate format
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Sample type
Serum, Plasma
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Sample volume
20 µl / well
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Assay time
overnight / room temperature (18 – 26°C)
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Sensitivity
LOD (0 pmol/l + 3 SD): 51 pmol/l; LLOQ: 175 pmol/l
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Standard range
0 – 2800 pmol/l (= 0 – 110,320 pg/ml)
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Conversion factor
1 ng/ml = 25 pg/ml (MW: 39.4 kDa)
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Cross-reactivity
Human WNT3A shares around 100-97% aa sequence with primates, 96-95% bears, 96% whales and 96% mice. Cross-reactivity of this assay with other species than human has not been tested.
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Detection target
This assay detects human endogenous and recombinant WNT3A.
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Regulatory status
CE marked – for IVD use in the EU
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Detection range
51 – 2,800 pmol/l (2,009 – 110,320 pg/ml)
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Precision
In-between-run (n=4): ≤ 10 % CV
Within-run (n=4): ≤ 11 % CV
Product Overview
The FluoBolt™ WNT3A immunoassay is an o.n. Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well plate format for the quantitative determination of WNT3A in serum and plasma. The assay employs human based serum standards to ensure the measurement of biologically reliable data.
Principle Of The Assay
The FluoBolt™ WNT3A immunoassay is an o.n. Metal Enhanced Direct Sandwich Fluorescence Immunoassay in 96-well plate format for the quantitative determination of WNT3A in serum and plasma samples.
Figure explaining the principle of metal enhanced fluorescence:
In a first step, standard/sample/control and detection antibody (fluorescent labeled anti-WNT3A) are pipetted into the wells of the microtiter plate, which are pre-coated with anti-WNT3A antibody. WNT3A 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 WNT3A in the sample is determined directly from the dose response curve.
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 (Localised 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.
Typical Standard Curve
The figure below shows a typical standard curve for the FluoBolt™-WNT3A ELISA. The immunoassay is calibrated against recombinant full length WNT3A:
Kit Components
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Contents |
Description |
Quantity |
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GM |
Anti-human WNT3A antibody, pre-coated MEF microtiter plate, packed in vacuum sealed aluminum bag |
1 x 96 well |
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WP |
20X wash buffer concentrate, natural cap |
1 x 25 ml |
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GAF, GA3, GA5, GAA |
Anti-human WNT3A antibody, labeled with FITC, Cy3, Cy5 or AlexaFluor680, black flask |
1 x 2.5 ml |
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GS |
Standards 1-6, (400, 200, 100, 50, 25, 0 pmol/l), white caps, lyophilized |
6 vials, 0.25 ml |
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GCA/B |
Control A and B, yellow cap, lyophilized (for |
2 vials, 0.25 ml |
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GD |
Sample diluent, natural cap, ready to use |
1 x 10 ml |
Storage instructions: All reagents of the FluoBolt™-WNT3A immunoassay kit are stable at 4°C until the expiry date stated on the label of each reagent.
Sample Collection & Storage
Collect venous blood samples by using standardized blood collection tubes for serum or plasma. We recommend performing plasma or 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 plasma or serum samples should be measured as soon as possible. Since WNT3A is not stable at room temperature, samples should not be stored at room temperature for longer periods (> 1 hour). For longer storage aliquot samples and store at -25°C or lower. Do not freeze-thaw samples more than 5 times.
Lipemic or hemolyzed samples may give erroneous results. Samples should be mixed well before assaying.
Reagent Preparation
Add 250 μl of distilled or deionized water to the lyophilized GS (Standards) and GC (Controls). Leave at room temperature (18-26°C) for at least 15 min but maximum 30 min before use in the assay. Since WNT3A is not stable at room temperature, reconstituted standards and controls should not be stored at room temperature for longer periods (> 1 hour). Reconstituted GS and GC are stable at -25°C or lower until expiry date stated on the label. Reconstituted GS and GC can undergo up to 5 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. 25 ml of WP + 475 ml 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.
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 (GAA) since 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 antibody.
Mark positions for DS/ Sample/ DC (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 25 μl of labeled detection antibody (GA) 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, cover tightly with the delivered adhesive film and incubate over night at room temperature (18-26°C) in the dark. |
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4. |
The next day read the plate either without any further processing (4a) or after performing a washing step (4b) using a fluorescent microplate reader. |
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4a. |
If your reader allows bottom reading, read the plate without any further processing at the Ex/Em wavelength fitting to the delivered antibody (495/518 nm for GAF, 550/570 nm for GA3, 650/670 nm for GA5, 679/702 nm for GAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0 pmol/l and the 2800 pmol/l WNT3A standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (GD). |
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4b. |
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 antibody (495/518 nm for GAF, 550/570nm for GA3, 650/670 nm for GA5, 679/702 nm |
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! |
Hint: Quality of bottom reading (4a) may vary between microplate readers. For first time users we suggest performing the washing step and follow protocol (4b). |
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6. |
Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signals of the 0 pmol/l and the 2800 pmol/l WNT3A standard. Samples with signals exceeding the signal of the highest standard must be re-run with appropriate dilution using sample diluent (GD). |
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7. |
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 months at the plate surface, depending on signal intensity achieved. |
Calculation Of Results
Subtract the fluorescence intensity of the 0 pmol/l 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
WNT3A is a secreted glycoprotein and belongs to the WNT family. Members of this family can interact with cell membrane receptors, thus playing a role in autocrine regulations and paracrine signaling. WNT3A is expressed in placenta at moderate levels, as well as in lung, spleen and prostate at low levels. The canonical sequence of WNT3A consists of 352 amino acids (aa) and has a mass of 39.365 kDa. It is rich in cysteine and forms many disulfide bonds from cysteine residues. At aa 87 and aa 298 glycosylation appears, since N-Acetylglucosamine is covalently attached to asparagine. At position aa 209 WNT3A is covalently lipidated at a conserved serine residue resulting in strong hydrophobic properties of the molecule. Therefore, in its physiological form it constitutes a soluble complex with afamin, which functions as a carrier for hydrophobic molecules in body fluids and is essential for the activity and solubility of WNT3A.
WNT3A plays important roles in cell growth and differentiation, embryonic development, neural development, immune regulation, bone formation and carcinogenesis. Therefore, research has investigated association of
elevated expression of WNT3A with prostate, breast or hepatocellular cancer.
Literature
Single step, direct fluorescence immunoassays based on metal enhanced fluorescence (MEF-FIA) applicable as microplate-, array-, multiplexing- or point of care-format.
Hawa G et al., Anal Biochem. 2018;549:39-44.
Wnt3a: Functions and Implications in Cancer.
Sha H et al., Chin J Cancer. 2015;34(12):554-62.
Elevated levels of Wnt3a and low levels of Dickkopf-1 in serum are associated with syndesmophyte formation in ankylosing spondylitis.
Klingberg E et al., Ann Rheum Dis. 2012;71:A64.
Expression of Wnt3a in hepatocellular carcinoma and its effects on cell cycle and metastasis.
Caijie L et al., Int J Oncol. 2017;51(4):1135-1145.
Wnt3a signaling within bone inhibits multiple myeloma bone disease and tumor growth.
Qiang YW et al., Blood. 2008;112(2):374-382.
The Importance of WNT Pathways for Bone Metabolism and Their Regulation by Implant Topography.
Galli C et al., Eur Cell Mater. 2012;24:46-59.
Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.
Mihara E et al., eLife. 2016;5:e11621.
Wnt3a involved in the mechanical loading on improvement of bone remodeling and angiogenesis in a postmenopausal osteoporosis mouse model.
Li X et al. , FASEB J. 2019 Aug;33(8):8913-8924
Repair effect of Wnt3a protein on the contused adult rat spinal cord.
Yin ZS et al. Neurol Res. 2008 Jun;30(5):480-6.
All FluoBolt™ IMMUNOASSAYs are validated according to sensitivity, specificity, precision, accuracy, dilution linearity, sample stability, expected values in blood donor collections and sample matrix .
Calibration
The FluoBolt™-WNT3A immunoassay is calibrated against recombinant human WNT3A protein.
Detection Limit & Sensitivity
To determine the sensitivity of the FluoBolt™-WNT3A 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 WNT3A, with a confidence level of 99%. It is defined as the mean back calculated concentration of standard 1 (0 pmol/l of WNT3A, 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 WNT3A, 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 FluoBolt™-WNT3A immunoassay:
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LOD |
51 pmol/l |
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LLOQ |
175 pmol/l |
Precision
The precision of an FluoBolt™-WNT3A 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 WNT3A immunoassay lots (in-between-run precision or reproducibility).
Within-Run Precision
Within-run precision was tested by measuring the same 4 samples 3 times within one FluoBolt™-WNT3A 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. CVs ranged from 3 - 10%.
In-Between-Run Precision
In-between-run precision was assessed by measuring the same 4 samples 3 times within multiple FluoBolt™-WNT3A immunoassay lots. The measurements were carried out by one operator. CVs ranged from 7 - 11%.
Accuracy
The accuracy of an FluoBolt™-WNT3A immunoassay is defined as the precision with which it can recover samples of known concentrations.
The recovery of WNT3A in serum was evaluated by adding known amounts of human recombinant WNT3A to 4 different human serum samples. Mean recovery was 87%.
Mean recovery in plasma was significantly lower than in serum:
Citrate-plasma: 45% (n=6)
EDTA-Plasma: 58% (n=8)
Dilution Linearity
Tests of dilution linearity and parallelism ensure that both endogenous and recombinant samples containing WNT3A 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.
3 human serum samples were spiked with recombinant WNT3A and diluted 1+1 and 1+2 with the sample diluent (HD) supplied with the kit. Mean linearity was 74%.
Specificity
Analyte Specificity:
This assay detects human WNT3A.
Species Specificity:
Human WNT3A shares around 100-97% aa sequence with primates (e.g. orangutan or chimpanzee), 96-95% bears (e.g. grizzly bear or giant panda), 96% whales (e.g. beluga whale or dolphins) and 96% mice. Reactivity of this assay with other species than human has not been tested. So, using this assay for WNT3A measurements in serum or plasma of species with high sequence homology may be possible but must be evaluated by the user. Biomedica & FIANOSTICS do not take responsibility for functionality of the assay in non-human samples.
