FluoBolt-Klotho Fluorescence Immunoassay

Additional Information

Assay Background

α-KLOTHO is a protein primarily expressed in kidney. It can be found either as a membrane bound or a secreted form. The membrane bound form consists of 1012 amino acids (aa), starting with a 56 aa long signalling sequence and followed by two glycosyl hydrolase 1 regions (position 57-506 and 515-953). Both glycosyl hydrolase 1 regions lack one essential Glu active site residue. Thus, it is inactive in vivo as a glycosidase although it belongs to the glycosyl hydrolase 1 family. KLOTHO’s secreted isoform, which predominates over the membrane bound form, consists of 549 amino acids (aa). It is produced by alternative splicing and differs from the membrane bound form by aa 550 to 1012 missing. α-KLOTHO is expressed in kidney, small intestine, placenta and prostate. The soluble peptide can be found in serum and cerebrospinal fluid. It may play a role in the calcium/ phosphorus homeostasis regulation by e.g. inhibiting active vitamin D synthesis. Further, it is also known as an anti-aging-hormone by extending life span by inhibiting insulin/ IGF1 signalling pathway, as experiments in mice showed. KLOTHO is a co-receptor of fibroblast growth factor 23 (FGF-23). Research has investigated association of altered serum KLOTHO levels with chronic kidney disease and failure, renal- and hepatocellular carcinomas, osteoporosis or cardiovascular diseases.

About Metal Enhanced Fluorescence

Metal Enhanced Fluorescence (MEF) using metal nano-structures offers the possibility to increase the analytical sensitivity of systems based on fluorescence detection dramatically. FIANOSTICS has developed a new MEF- immunoassay platform, 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 us to develop high sensitivity, single step with no wash florescence immunoassays for low abundance biomarkers.

Assay Principle

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 running 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!).
    • Add 50 μl of the selected fluorescence labeled detection antibody(HAF or HA3 or HA5 or HAA) to all wells required. Swirl gently.
    • Add 10 μl of standard, control or sample to the wells according to themarked positions on the protocol sheet, swirl gently, cover tightly withthe delivered adhesive film and incubate over night at room temperature(18-26°C) in the dark.
    • a.) 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 HAF, 550/570 nm for HA3, 650/670 nm for HA5, 679/702 nm for HAA). Gain should be set to achieve at least 10000 fluorescence units (F.U.) between the signal of the 0 pmol/l and the 400 pmol/l KLOTHO standard. Samples with signals exceeding the signal of the highest standard must be re-run with an appropriate dilution using sample diluent (HD).
    • b.) 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 HAF, 550/570 nm for HA3, 650/670 nm for HA5, 679/702 nm for HAA).

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

• • Store the plate with the 2 desiccant bags supplied at 4°C (2-8°C) in thealuminum bag. Unused wells are stable until expiry date stated on thelabel. Fluorescence signals of standards, controls and samples remaindetectable for at least two months at the plate surface, depending onsignal intensity achieved.