FluoBolt™ Asporin

Additional material supplied with the kit

• 2 self-adhesive plastic films
• Protocol sheet
• Instruction manual for use
• Desiccant bag

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

Asporin contains a calcium binding site. 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 Asporin analysis. 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 ES. Add 250μl of distilled or deionized water to the lyophilized ES (Standards) and EC (Controls). Leave at room temperature (18-26°C) for 20min. Reconstituted ES and EC are stable at -25°C or lower until expiry date stated on the label. Reconstituted ES and EC 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. We recommend to run samples and standards in duplicates. 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.

Seal all wells that will not be used in the following assay run with the accompanying adhesive film (cut to fit!).

In standard format, the kit is delivered with an AlexaFluor680 labeled detection antibody (EAA) 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. 670/720nm). 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 50μl of the selected fluorescence labeled detection antibody (EAF or EA3 or EA5 or EAA) to all wells required. Swirl gently.

2)      Add 10μl 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.

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 EAF, 550/570nm for EA3, 650/670nm for EA5, 679/702nm for EAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0pM and the 400pM ASPORIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (ED).

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 EAF, 550/570nm for EA3, 650/670nm for EA5, 679/702nm for EAA).

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 400pM ASPORIN standard. Samples with signals exceeding the signal of the highest standard must be re-run with appropriate dilution using sample diluent (ED).

4)      Store the plate with desiccant 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. The assay was evaluated with 4PL algorithm. Different curve fitting methods need to be evaluated by the user.

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

5 samples of known concentrations were tested 3 times within 1 assay run. CVs ranged from 3-7%.

Inter-assay

2 samples of known concentrations were tested in duplicates within 3 different assay run. CVs ranged from 8-11%.

Spike/Recovery

The recovery of Asporin was evaluated by adding known amounts of human recombinant Asporin to 4 different human serum samples. Mean recovery was 129%.

Linearity

4 human serum samples were spiked with recombinant Asporin and diluted 1+2 and 1+9 with the sample diluent (ED) supplied with the kit. Mean linearity was 87%.

Analyte Specificity

This assay detects human Asporin. Addition of recombinant TGF-β1 to the standards supplied with this kit did not reduce signal intensity.

Species Specificity

Human Asporin shares around 99% aa sequence identity with higher apes (e.g. gorilla or chimpanzee) but only 90% with rat/mouse and 87% with bovine/ equine Asporin. Cross reactivity of this assay with other species than human has not been tested. So using this assay for those 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.

The quantification of serum biomarkers plays an important role in medical science. Very often their concentrations in human blood are very low, which creates a permanent need for raising the sensitivity of the assay methods used.

Metal-enhanced fluorescence (MEF) is a promising technology to deliver the required highly sensitive tests. It is well established, that MEF results from localized surface plasmons (LSPs) generated by the interaction of the excitation light with nanometer-sized noble metal structures, that dramatically increases the quantum yield of fluorescent molecules thus leading to highly sensitive detection systems (e.g. for fluorescent immunoassays).

In Fig. 1 a typical MEF measurement setup is sketched.

Fig. 1 Schematic representation of metal-enhanced fluorescence and measurement setup. The fluorescence of the near surface fluorophore is drastically enhanced in contrast to the fluorophore in the bulk solution.

Design and manufacturing reproducibility of the required metal structures is the key for a) optimizing the enhancement effect and b) reliability of the assay system they are used for.

This is why commercial application of this technology has failed so far.

STRATEC Consumables (former Sony DADC BioSciences) and FIANOSTICS successfully solved these problems by using highly reproducible nano-structuring technologies (patent pending), originally developed for Blu-Ray and DVD manufacturing. The manufactured MEF substrates were successfully used in the detection of fluorescence labelled antibodies and its application in fluorescence labelled immunoassays (MEF-FIAs) for biomarkers in human serum was demonstrated. This new MEF platform is compatible to any given assay format (e.g. microtiter plate, microfluidic chips, arrays or lateral flow devices).

Manufacturing method

The master structures were manufactured by laser lithography, copied into a Ni stamp by electroforming and replicated in a disc format by a vario-thermal injection compression process. For the polymer a medical grade of cycle olefin was used. The disc were selectively coated with Ag-spots by a DC sputtering process and protected by a surface adhesive film to avoid degradation of the Ag layer. Subsequently slide in microscope format were milled out and laser-welded onto 96-well microtiter plates.

Design of MEF substrate

The MEF substrate are polymer slides with a nanometer sized hexagonal arrays of well-like structures on the surface. The wells typically have diameters below 0.5μm, an aspect ratio of about 2 and a micron pitch. They are coated homogenously with several nanometer of Ag.

By adjusting the geometry of the wells-array and the thickness of the Ag-layer the MEF effect can be optimized.

Fig. 2 (a) SEM image of an uncoated MEF substrate; (b) AFM measurement of the MEF substrate coated with several nanometer of silver

Manufacturing method

The master structures were manufactured by laser lithography, copied into a Ni stamp by electroforming and replicated in a disc format by a vario-thermal injection compression process. For the polymer a medical grade of cycle olefin was used. The disc were selectively coated with Ag-spots by a DC sputtering process and protected by a surface adhesive film to avoid degradation of the Ag layer. Subsequently slide in microscope format were milled out and laser-welded onto 96-well microtiter plates.

Overview

Application

To demonstrate the functionality of the MEF substrate, Cy5 labeled antibody (goat anti-rabbit) were adsorbed to the MEF substrate and measured with a commercial fluorescence microplate reader. The measurements were done in bottom configuration, meaning that excitation and emission through the bottom of the plate. Depending on the fluorophore, antibody concentration in the sub-picomolar range can be clearly detected (see Fig. 3).

Fig. 3 Enhanced fluorescence signal of a Cy5 labeled antibody adsorbed a MEF substrate

Summary and Outlook

The functionality of the MEF substrates could be successfully demonstrated in fluorescence labelled immunoassays for biomarkers in human serum. An enhancement of several orders of magnitudes could be achieved, allowing the quantification of biomolecules down to low picomolar and sub-picomolar concentrations. Preliminary tests have shown, that the MEF substrates can be also used for other fields application like SERS (Surface Enhanced Raman Spectroscopy).

Asporin, also known as periodontal ligament-associated protein 1 (PLAP1) is a dimeric secreted extracellular matrix protein, which belongs to the small leucine-rich proteoglycan (SLRP) family. It consist out of 380 amino acids and has a highly conserved pro peptide sequence which contains a series of leucine rich repeats and are flanked by two cysteine residues in the C Terminal region. Further it has four cysteine residues that form disulphide bonds as well as aspartic acid repeats in the N-Terminal region.

High levels of Asporin can be found in aorta, uterus and osteoarthritic articular cartilage. Further, moderate levels of aspirin expression can be found in small intestine, heart liver, bladder, ovary, stomach, the adrenal-, thyroid-, and mammary gland. Lower levels of expression can be seen in the trachea, bone marrow and lung. Asporin is known to negatively regulate PDL differentiation and mineralisation as well as it inhibits BMP-dependent activation of SMAD proteins. Further, it directly binds to TGFb-1 subsequently, binds to collagen by way of its LRR domain. Through its interaction with TGFb-1, Asporin negatively regulates chondrogenesis in the articular cartilage by blocking the TGF-beta/receptor interaction on the cell surface and inhibiting canonical TGF-beta/Smad signalling. Moreover, it has the ability to bind calcium, giving it regulatory properties in osteoblast driven mineralisation and regulates FGF2 through direct and indirect interactions. Next to its regulatory properties in terms of cartilage and bone homeostasis, Asporin expression has also been linked to cancer invasion and progression. However, its value as biomarker remains to be established yet.

Asporin is a 380 amino acid long, secreted dimeric extracellular matrix protein, which functions as a direct binding partner for TGFβ-1 and, like Decorin and Biglycan, belongs to the der Small Leucine- Rich Proteins (SLRP) class I family. They are characterised by a high degree of homology at the genetic and molecular level and the presence of leucine- rich repeats (LRRs), which are flanked by disulfides formed by cysteine residues. Unlike other members of the SLRP class II family, Asporin does not contain a glycosaminoglycan binding site, but it has several unique aspartic acid repeats in the N-Terminal region. The main biological function of Asporin most likely consists in the regulation of TGF-β1 activity to which it binds directly.

Research Applications

Osteoarthritis (OA)

Lumbar Disc Disorder (LDD)

Tumor Progression Studies