Assay for quantitative determination of human Aggrecan PG.
Sensitivity: 0.9ng/mL
Range: ~10-250ng/mL
Incubation Time: 3h 25min
Samples: Synovial fluid, serum and culture media
Principle of the test:The Aggrecan, Human, BioAssay™ ELISA Kit is a solid phase ELISA performed on microtiter plate. The assay is based on an oligoclonal system in which a blend of monoclonal antibodies (MAbs) directed against distinct epitopes of PG are used. Antibody-producing cells are immortilized using the myeloma cell fusion method of Kohler and Milstein. A hybridoma cell is produced which secretes specific homogeneous antibodies. The use of a number of distinct MAbs avoids hyperspecificity and allows highly sensitive assays with extended standard range and short incubation time. Standards or samples containing PG react with capture monoclonal antibodies (MAbs 1) coated on the microtiter well and with a monoclonal antibody (MAb 2) labeled with horseradish peroxidase (HRP). After an incubation period allowing the formation of a sandwich; coated MAbs 1-PG-MAb 2-HRP, the microtiter plate is washed to remove unbound enzyme labeled antibodies. Bound enzyme-labeled antibodies are measured through a chromogenic reaction. Chromogenic Solution TMB+H2O2) is added and incubated. The reaction is stopped with the addition of Stop Solution (HCl) and the microtiter plate is then read at the appropriate wavelength. The amount of substrate turnover is determined colorimetrically by measuring the absorbance which is proportional to the PG concentration. A standard curve is plotted and PG concentrations in a sample is determined by interpolation from the standard curve. The use of the EASIA Reader linearity up to 3OD units) and a sophisticated data reduction method (polychromatic data reduction) result in high sensitivity in the low range and in an extended standard range.
Kit Components:A1059-52A: Microtiter Plate 1x96 wells*A1059-52B: Standard 0ng 1x500ul*A1059-52C: Standard 15.7ng 1x500ul *A1059-52D: Standard 28.8ng 1x500ul *A1059-52E: Standard 51.0ng 1x500ul *A1059-52F: Standard 89.0ng 1x500ul *A1059-52G: Standard 197.0ng 1x500ul A1059-52H: Diluent Buffer 2x30mlA1059-52J: Incubation buffer 1x11mlA1059-52K: PG Mab (HRP) 1x5.5ml A1059-52L: Conjugate Buffer 2x11ml*A1059-52M: Control 1 1x108±36ng/ml *A1059-52N: Control 2 1x78±23g/ml *A1059-52P: Control 3 1x38±11ng/ml A1059-52Q: Wash Buffer Concentrate 1x10 mlA1059-52R: Chromogenic Solution TMB (Tetramethylbenzidine) 1x25mlA1059-52S: Stop Solution 1x25ml
Storage and Stability:Store *Standards and controls powder at 4°C, liquid aliquots for 2 months at -20°C and 6 months at -70°C. Store other components at 4°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Immunoassay Control:Ready for use Standard Aggrecan purified from human articular cartilage (A1D1D1 fraction chromatographed on Sepharose CL 2B, material eluted at 0.1 < Kd < 0.7)
Controls come with two parameter vials (one high, one low) and are for human markers only
The control set consists of a human aggregan protein, purified from articular cartillage in two different concentrations and provided as lyophilized powder in two different concentrations.
