MCAF, Human (Monocyte Chemotactic Activating Factor) BioAssay(TM) ELISA Kit

Monocyte chemotactic and activating factor (MCAF), also known as monocyte chemotactic protein 1 (MCP-1), lymphocyte�derived chemotactic factor (LDCF), and glioma-derived chemotactic factor (GDF), is a recently-identified chemotactic cytokine for monocytes. cDNA cloning and structural analysis has revealed that this 76-amino acid polypeptide with a predicted molecular mass of 8,700 daltons belongs to a family of structurally-related low molecular weight proteins characterized by four conserved cysteine residues designated C-C family or intercrine beta family (1,2,3). This MCAF ELISA is a 2.5 hour solid-phase immunoassay readily applicable to measure MCAF levels in serum, plasma, cell culture supernatant and other biological fluids in the range of 0-1600pg/ml. It has shown no crossreactivity with various other C-C and C-X-C chemokines, IL-8 superfamily proteins. This MCAF ELISA is expected to be effectively used for futher investigation into the relationship between MCAF and various diseases. This MCAF enzyme-linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for MCAF. Standards or samples are then added to the appropriate microtiter plate wells and incubated. MCAF if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound MCAF and other components of the sample. In order to quantify the amount of MCAF present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for MCAF is added to each well to "sandwich" the MCAF immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and a TMB (3,3'5,5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain MCAF and enzyme-conjugated antibody will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm � 2nm. In order to measure the concentration of MCAF in the samples, this kit contains two calibration diluents (Calibrator Diluent 1 for serum/plasma testing and Calibrator Diluent 2 for cell culture supernatant/ urine testing). According to the testing system, the provided standard is diluted (2-fold) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D.) versus MCAF concentration (pg/ml). The concentration of MCAF in the samples is then determined by comparing the O.D. of the samples to the standard curve. Sensitivity: < 4pg/ml Range: 0-1600pg/ml Kit Components: 1. MCAF Microtiter Plate, 1x96 wells 2. MCAF Conjugate, 1x15ml 3. MCAF Standard, 2x1 vial 4. Calibrator Diluent 1, 1x22ml 5. Calibrator Diluent 2, 1x 22ml 6. Wash Buffer (20X), 1x60ml 7. Substrate A, 1x11ml 8. Substrate , 1x11ml 9. Stop Solution, 2N Sulphuric Acid (H2SO4). Storage and Stability: Store components at 4 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.�