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#enzyme-linked immunosorbent assay

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Immunological Applications

ELISA is commonly used for:

Enzyme-linked immunosorbent assay revolutionized immunodiagnostics by converting antigen antibody binding into measurable enzymatic colorimetric signal without radioactivity, originally described by Eva Engvall and Peter Perlmann 1971. Polystyrene microtiter plate with high binding capacity via hydrophobic interaction adsorbs capture antibody coated overnight at alkaline pH 9.6 carbonate buffer 1 to 10 microgram per ml. Non-specific sites blocked with protein bovine serum albumin five percent, casein, or nonfat milk to prevent background adsorption through hydrophobic pockets. Sample containing antigen such as viral capsid HIV p24, cytokine IL-6, or hormone insulin incubated allowing specific capture, unbound material removed by repeated washes with phosphate buffered saline containing 0.05 percent Tween-20 disrupting weak interactions. Detection uses secondary antibody conjugated covalently to horseradish peroxidase or alkaline phosphatase via periodate oxidation forming Schiff base. Addition of chromogenic substrate tetramethylbenzidine TMB with hydrogen peroxide yields oxidation to blue diimine measured after sulfuric acid stopping as yellow product absorbance 450 nm proportional to analyte concentration via four-parameter logistic standard curve with dynamic range three logs.

Ref: Engvall & Perlman Immunochemistry 1971 ELISA; Alberts MBoC ELISA HRP TMB sandwich principle diagnostic.

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Immunological Applications

ELISA is commonly used for:

ELISA pioneered Engvall Perlmann 1971 exploits highly specific high-affinity antigen-antibody interaction KD nanomolar picomolar detecting analytes complex biological fluids serum urine CSF culture supernatant. Principle immobilization one component passive hydrophobic adsorption polystyrene 96-well high-binding blocking nonspecific sites BSA casein preventing background. Indirect sandwich formats most sensitive plate coated capture antibody monoclonal high-affinity against target antigen human chorionic gonadotropin hCG pregnancy marker HIV p24 capsid antigen cytokines IL-6 TNF-alpha allergen-specific IgE incubated specific immune complex formation governed mass action kinetics association rate 10^5 per M per s avidity bivalent enhancing stability. Washing PBS 0.05 percent Tween removing unbound proteins specific complex remains strong noncovalent hydrogen bonds van der Waals. Secondary antibody conjugated HRP enzyme 40 kDa via glutaraldehyde biotin-streptavidin amplification four biotin binding sites affinity 10^-15 M binding Fc primary antibody catalyzes oxidation chromogenic substrate TMB 3,3',5,5' presence H2O2 generating blue radical 650 nm sulfuric acid stop converts yellow diimine 450 nm measured spectrophotometrically microplate reader intensity directly proportional antigen concentration via 4PL standard curve interpolation recombinant standards. Sensitivity pg/mL dynamic range 3 logs high throughput screening hundreds samples daily low cost no radioisotope adaptability automation made ELISA cornerstone diagnostic infectious diseases autoimmunity allergy therapeutic monitoring.

Ref: Engvall and Perlmann 1971 ELISA Original J Immunol; NCBI Bookshelf ELISA Principles; Janeway Immunoassay ELISA Technique Variants Chapter 3.

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