![]() ![]() Direct detection, while not widely used in ELISA, is quite common for immunohistochemical staining of tissues and cells. Direct detection can be performed with an antigen that is directly immobilized on the assay plate or with the capture assay format. ![]() The direct detection method uses a primary antibody labeled with a reporter enzyme or a tag that reacts directly with the antigen. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer, or luminometer). A large selection of substrates is available commercially for performing ELISA with an HRP or AP conjugate. Other enzymes have been used as well these include β-galactosidase, acetylcholinesterase, and catalase. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Signal measurement-detection of the signal generated via the direct or secondary tag on the specific antibody.Probing/detection-incubation with antigen-specific antibodies that affinity-bind to the antigens.Plate blocking-addition of irrelevant protein or other molecule to cover all unsaturated surface-binding sites of the microplate wells.Coating/capture-direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.This ability to use high-affinity antibodies and wash away non-specific bound materials makes ELISA a powerful tool for measuring specific analytes within a crude preparation.Īlthough many variants of ELISA have been developed and used in different situations, they all depend on the same basic elements: Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from non-bound material during the assay. It is this binding and immobilization of reagents that makes ELISAs easy to design and perform. ELISAs are typically performed in 96-well or 384-well polystyrene plates, which passively bind antibodies and proteins. Originally described by Engvall and Perlmann (1971), the method enables analysis of protein samples immobilized in microplate wells using specific antibodies. The enzyme linked immunosorbent assay (ELISA) is a powerful method for detecting and quantifying a specific protein in a complex mixture. You can also visit our ELISA builder tool, answer a series of questions, and be presented with recommendations on which components will work best for your unique ELISA needs.ĮLISA builder tool Search ELISA kits Explore ELISA protocols Explore ELISA reagents The below article will guide you through decisions and options for building an ELISA. The most crucial element of an ELISA is a highly specific antibody-antigen interaction. Detection is accomplished by measuring the activity of the reporter enzyme via incubation with the appropriate substrate to produce a measurable product. In an ELISA, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then complexed with an antibody that is linked to a reporter enzyme. Other names, such as enzyme immunoassay (EIA), are also used to describe the same technology. Detecting Low Abundance Proteins in Western BlottingĮLISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying soluble substances such as peptides, proteins, antibodies, and hormones.Overview of Post-Translational Modification.Carbonyl-Reactive Crosslinker Chemistry.Sulfhydryl-Reactive Crosslinker Chemistry.Polyethylene Glycol (PEG) and Pegylation of Proteins.Overview of Crosslinking and Protein Modification.Metabolic Labeling and Chemoselective Ligation.Sample Preparation for Mass Spectrometry.Methods for Detecting Protein–RNA Interactions.Methods for Detecting Protein-DNA Interactions.Overview of Protein-Nucleic Acid Interactions.Label Transfer Protein Interaction Analysis.Crosslinking Protein Interaction Analysis.Overview of Protein–Protein Interaction Analysis.Immunofluorescence Method for IHC Detection.Avidin-Biotin Complex Method for IHC Detection.Immunohistochemistry vs Immunocytochemistry.Spike and Recovery and Linearity of Dilution Assessmentįactors Affecting Signal Generation in ELISA Chromogenic Western Blotting Substrates.Blocking Buffers for Western Blot and ELISA.Protein-Protein Variation of Protein Assays.Desalting and Gel Filtration Chromatography.Overview of dialysis, desalting, buffer exchange and protein concentration.Overview of the Immunoprecipitation (IP) Technique.His-tagged Proteins–Production and Purification.GST-tagged Proteins–Production and Purification.Covalent Immobilization of Affinity Ligands.Overview of Protease and Phosphatase Inhibition for Protein Preparation.Overview of Cell Fractionation and Organelle Isolation.Detergents for Cell Lysis and Protein Extraction. ![]()
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