32.15
Dosage Immuno-enzymatique
En 1971, Peter Perlman et Eva Engvall ont développé un test immuno-enzymatique (ELISA ou EIA). ELISA diffère du Western blot en ce sens que les tests…
Le test immuno-enzymatique ou ELISA est un test immunologique marqué avec de nombreuses applications dans la détection et la quantification d’analytes tels que les anticorps, les peptides, les glycoprotéines, les hormones et les virus.
Les anticorps de détection qui se lient à l’antigène ou à la molécule cible sont conjugués à des enzymes spécifiques qui, lors de l’ajout de substrat, produisent des signaux mesurables.
Selon les méthodes de capture et de détection, il existe quatre principaux types d’ELISA.
L’ELISA direct utilise un anticorps primaire conjugué à une enzyme pour détecter l’antigène de l’échantillon recouvert sur la plaque.
Alors que pour l’ELISA indirect, l’antigène attaché capture l’anticorps primaire de l’échantillon, auquel l’anticorps secondaire conjugué se lie.
L’ELISA sandwich implique deux anticorps, l’un enrobé sur la plaque pour capturer l’antigène de l’échantillon, tandis que l’autre anticorps conjugué à une enzyme se lie à l’antigène-épitope spécifique formant le sandwich.
Dans l’ELISA compétitif, l’antigène et l’anticorps primaire sont pré-incubés et ajoutés à la plaque recouverte du même antigène.
Plus la concentration d’antigène de l’échantillon est élevée, plus il y a de complexes antigène-anticorps formés, ce qui laisse moins d’anticorps libres à lier à l’antigène concurrent de la plaque, générant ainsi des signaux plus faibles.
View the full transcript and gain access to JoVE Core videos
Q1: What is an enzyme-linked immunosorbent assay and what can it detect?
ELISA is a labeled immunoassay that detects and quantifies analytes including antibodies, peptides, glycoproteins, hormones, and viruses. Detection antibodies bind to target molecules and are conjugated with specific enzymes that produce measurable signals upon substrate addition. This versatile technique is widely used in diagnostics, pregnancy testing, food allergen detection, and cancer biomarker identification.
Q2: How does direct ELISA differ from indirect ELISA?
Direct ELISA uses a single enzyme-conjugated primary antibody to detect sample antigen coated on the plate. Indirect ELISA adds a secondary antibody for detection, making it more sensitive and flexible. The secondary antibody quantifies antigen-specific antibody presence by measuring color intensity from the conjugated enzyme-chromogen reaction.
Q3: When should sandwich ELISA be used instead of other ELISA types?
Sandwich ELISA is more specific and sensitive than direct and indirect formats, making it ideal for detecting large proteins with multiple epitopes. Two antibodies work together: one captures the sample antigen on the plate, while an enzyme-conjugated antibody binds to a specific epitope, forming a sandwich. The measured absorbance directly correlates to captured antigen amount.
Q4: What is the principle behind competitive ELISA?
In competitive ELISA, sample antigen and primary antibody are pre-incubated before adding to plate-coated antigen. Higher sample antigen concentration produces more antigen-antibody complexes, leaving fewer free antibodies to bind plate antigen, generating weaker signals. Signal intensity becomes inversely correlated to sample antigen concentration, allowing analyte quantification.
Q5: What enzymes and substrates are commonly used in ELISA?
Alkaline phosphatase and horseradish peroxidase are the most widely used enzymes in ELISA because appropriate substrates are readily available. The substrate is typically a chromogen, a colorless molecule converted into a colored end product by enzyme activity. Some ELISAs use fluorogens, nonfluorescent molecules converted to fluorescent forms, creating fluorescent enzyme immunoassays detectable by microscope or spectrophotometer.
Q6: Why is sandwich ELISA preferred for detecting small proteins?
Competitive ELISA is actually ideal for small protein detection because small molecules lack multiple epitopes needed for sandwich ELISA's two-antibody binding. Competitive ELISA allows crude samples to be used directly and offers highly flexible experimental setup. The assay's signal interference method enables accurate concentration determination for small analytes.
Q7: How does ELISA differ from Western blotting as a detection method?
ELISA assays are conducted in microtiter plates or in vivo, whereas Western blotting uses an absorbent membrane. Both techniques use antibodies conjugated to enzymes, but ELISA's microtiter plate format allows simultaneous processing of multiple samples. ELISA's enzyme-substrate reaction produces colored or fluorescent signals that can be visualized or quantified directly.