ELISA testing

What does ELISA stand for?

Enzyme Linked Immunosorbent Assay

What are two important points about ELISA?

Uses the specificity of antibodies to detect a molecule of interest in an aqueous sample through adsorption to a solid surface; Uses an enzyme reaction to generate a detectable signal (often a colour change) that relates to the amount of target molecule present

What are the important components of an ELISA?

Solid phase; Antibody binding and specificity; Purified standards of known concentration; Amplification steps increasing the signal; Signal generation and detection; Plenty of washing

What are the main types of ELISA?

Direct, Indirect, Sandwich, Competitive, ELISPOT, Multiplex and array technology, Lateral flow test (LFA)

Where is the conjugated antibody located in direct and indirect ELISA?

In direct ELISA, the conjugated antibody binds directly to the target antigen; in indirect ELISA, a secondary conjugated antibody binds to the primary antibody

In a sandwich ELISA, is it direct or indirect?

Indirect

In a competitive ELISA, how does antigen presence affect the signal?

If antigen is present, plenty of antibody binds in solution and little remains to bind to the solid phase, producing a LOW signal; if antigen is absent, antibodies bind to the solid phase, producing a HIGH signal

What happens in the solution phase of a competitive ELISA?

Antibodies bind to free antigen in solution

What happens in the bound solid phase of a competitive ELISA?

Antibodies bind to antigen on the solid surface when no antigen is present in solution

What is ELISPOT used for?

Measuring immune responses at the single-cell level, especially cytokine production

Who are two references for ELISPOT?

Kalyuzhny AE (2005) Handbook of ELISpot; Slota M (2011) ELISPOT for measuring human immune responses to vaccines

What is multiplex ELISA?

An assay that allows simultaneous detection of multiple analytes such as cytokines

Which references discuss multiplex ELISA?

Multiplex analysis of cytokines | British Society for Immunology; Tighe (2015) ELISA in the multiplex era. Proteomics-Clinical Applications 9, p406–422

What is a lateral flow test (LFA)?

A simple device for detecting the presence or absence of a target analyte without specialized equipment, commonly used in point-of-care testing

Who described LFA in literature?

Koczula (2016) Lateral Flow Tests. Essays in Biochemistry 60, p111–120

What are general uses of ELISA?

Detection of disease (e.g. H. influenzae capsule, COVID); Detection of allergens (e.g. Der P1–house dust mite); Detection of antibodies (e.g. IgM, IgA, IgG to COVID); Detection of biological molecules in research (e.g. IL-8, cytokines)

What are specific uses of ELISA?

Detection of disease-specific antibodies or molecules: HIV, COVID LFA, Lyme disease (Borrelia burgdorferi antibodies), Toxoplasmosis (Toxoplasma gondii antibodies), Varicella-zoster virus, Zika virus IgM (MAC-ELISA), Pregnancy testing (hCG)

Which enzyme systems are mainly used in ELISA?

Alkaline phosphatase (AP) and Horseradish peroxidase (HRP)

What substrate and colour change are associated with alkaline phosphatase in ELISA?

Substrate: p-nitrophenyl phosphate (pNPP); Product: p-nitrophenol (YELLOW)

What substrate and colour change are associated with horseradish peroxidase in ELISA?

Substrate: Tetramethylbenzidine (TMB); Product: BLUE when oxidised, converts to YELLOW upon acidification

At what wavelength is colour change detected in ELISA?

OD450 nm

What are the main steps in an indirect sandwich ELISA for IL-8?

Step 1: Add standards and samples (with block); Step 2: Add biotinylated anti–IL-8 polyclonal antibody; Step 3: Add streptavidin–HRP; Step 4: Add TMB peroxide substrate and measure colour change at OD450 nm

Why are amplification steps important in ELISA?

They increase the signal strength, improving sensitivity and detection limits

Why are washing steps critical in ELISA?

To remove unbound components, reduce background noise, and ensure accurate signal detection

What colour change indicates a positive HRP reaction in ELISA?

A blue colour (which can convert to yellow upon acidification)

What colour change indicates a positive alkaline phosphatase reaction in ELISA?

A yellow colour due to p-nitrophenol formation

What are examples of analytes detected by ELISA in research?

Cytokines such as IL-8, immune markers, and other biological molecules

What are examples of diseases detected using ELISA-based tests?

HIV, COVID, Lyme disease, Toxoplasmosis, Varicella-zoster, Zika virus, and pregnancy via hCG detection