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Ensayo inmunoabsorbente ligado a enzimas
En 1971, Peter Perlman y Eva Engvall desarrollaron un ensayo inmunoabsorbente ligado a enzimas (ELISA o EIA). ELISA se diferencia del Western Blot en…
El ensayo de inmunoabsorción ligado a enzimas o ELISA es un inmunoensayo marcado con amplias aplicaciones en la detección y cuantificación de analitos como anticuerpos, péptidos, glicoproteínas, hormonas y virus.
Los anticuerpos de detección que se unen al antígeno o a la molécula diana se conjugan con enzimas específicas que, tras la adición de sustrato, producen señales medibles.
Dependiendo de los métodos de captura y detección, existen cuatro tipos principales de ELISA.
El ELISA directo utiliza un anticuerpo primario conjugado con enzimas para detectar el antígeno de la muestra recubierto en la placa.
Mientras que para el ELISA indirecto, el antígeno unido captura el anticuerpo primario de la muestra, al que se une el anticuerpo secundario conjugado.
El ELISA sándwich involucra dos anticuerpos, uno recubierto en la placa para capturar el antígeno de la muestra, mientras que el otro anticuerpo conjugado con enzimas se une al epítopo de antígeno específico que forma el sándwich.
En el ELISA competitivo, el antígeno y el anticuerpo primario se preincuban y se añaden a la placa que está recubierta con el mismo antígeno.
Cuanto mayor es la concentración de antígeno de la muestra, más complejos antígeno-anticuerpo se forman, dejando menos anticuerpos libres para unirse al antígeno competidor de la placa, generando así señales más débiles.
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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.