Human MCP 1 ELISA Kit is a sandwich Enzyme-Linked Immunosorbent Assay (sELISA) designed for the in vitro quantitative determination of human MCP 1 in serum, plasma, and cell culture supernatant.
Assay Type
Sandwich (quantitative)
Principle of Assay
Human MCP 1 ELISA Kit (A334837) employs a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of human MCP 1 in serum, plasma, and cell culture supernatant. A 96-well microtiter plate is provided pre-coated with an antibody specific for MCP 1. Standards and test samples are added to the wells, allowing MCP 1 to bind to the immobilized capture antibody. A prepared antibody mixture is added and incubated to form antibody–antigen complexes. After incubation, unbound material is removed by washing, and TMB substrate is added to develop colour through an HRP-catalysed reaction. The reaction is stopped with acidic stop solution, producing a yellow endpoint signal. The intensity of the colour is proportional to the amount of MCP 1 captured in each well. The concentration of MCP 1 is determined by measuring absorbance at 450 nm and interpolating from the standard curve.
Components
This ELISA Kit contains 10X 600 µL Capture Antibodies, 10X 600 µL Detector Antibodies, 2 Lyophilized Recombinant Protein Vials, 40 mL Assay Diluent #11, 10X 20 mL Wash Buffer, 12 mL TMB Solution, 12 mL Stop Solution, Pre-Coated 96-Well H-Microplate (12 × 8 well strips), and 2 Plate Seals.
A representative standard curve for the Human MCP-1 ELISA showing optical density (O.D.) at 450 nm plotted against MCP-1 concentrations (pg/mL) in Assay Diluent #11 on a logarithmic scale. The curve demonstrates a strong linear relationship across the tested concentration range, confirming the assay’s accuracy and sensitivity for MCP-1 quantification.
Human MCP-1 concentrations were determined in undiluted, 2×, 4×, and 8× diluted human serum and plasma samples. Serum samples exhibited higher MCP-1 concentrations compared to plasma, with consistent detection across all dilutions, indicating reliable assay performance and minimal matrix interference.
Human MCP-1 levels were quantified in undiluted and serially diluted (2×, 4×, 8×, and 16×) PBMC stimulated supernatants. The results show consistent MCP-1 concentrations across dilutions, indicating strong assay linearity and minimal dilutional effects within the tested range.
Human MCP-1 levels were assessed in undiluted and serially diluted (2×, 4×, 8×, 16×, and 32×) PBMC unstimulated supernatants. MCP-1 concentrations remained relatively consistent across dilutions, suggesting reliable assay performance and minimal interference from matrix effects in unstimulated samples.
Percentage recovery of Human MCP-1 was evaluated in serum (red) and cell culture media (blue) samples at various dilutions (25%-1%). Both sample types demonstrated recoveries within acceptable assay performance ranges (approximately 95-115%), indicating minimal matrix interference and consistent assay accuracy across dilution factors.
Percentage recovery of Human MCP-1 was assessed in plasma at serial dilutions ranging from 10% to 0.3%. The recovery values remained consistent (approximately 95-105%) across all dilutions, indicating high assay accuracy and minimal matrix interference in plasma samples.
Schematic overview of the 60 Minute ELISA workflow illustrating antigen capture and colorimetric signal development. The target antigen is captured on the solid phase by an immobilized capture antibody while an HRP conjugated detection antibody binds simultaneously, forming a ternary immune complex during a 45 minute incubation. After washing to remove unbound components, tetramethylbenzidine (TMB) substrate is added. The horseradish peroxidase enzyme catalyzes oxidation of TMB, generating a blue reaction product that reflects the amount of bound antigen. Addition of stop solution terminates the reaction and converts the signal to a stable yellow color, which is measured spectrophotometrically after a 10 minute incubation. The entire assay is completed within approximately 60 minutes.
