Week 10 (INCOMPLETE)

Immunoassays are _ tests used to detect the ___ of a specific _ using ___

biochemical, presence or concentration, chemical, antibody-antigen reactions

Immunoassays rely on…

specificity and affinity of antibody to their specific antigen

Labelled immunoassays

Use detectable labels to quantify antigen-antibody interactions

Labelled immunoassays can detect analytes present in…

low concentrations

Clinical immunology requires… assays, making label-based methods…

robust high-throughput validated, the preferred choice

Label-free vs label-based immunoassays

Signal

Molecule that will react as part of the assay; labelled material that measures amount of antigen present

Commonly used signal in ELISA

Horseradish peroxidase (HRP) enzyme

What does HRP do in ELISAs?

Catalyses oxidation reactions to generate colorimetric signal for Ag-Ab detection

ELISA

Enzyme-Linked Immunosorbent Assay

ELISA is used to…

assess presence of antigen or antibody in a given sample + its quantification

ELISA (1): An enzyme _ with an antibody reacts with a __ to generate a ___

conjugated, colourless substrate, coloured reaction product

ELISA (2): The colour intensity is _ to the amount of __, which in turn is relatedto the levels of __ in an optimised ELISA procedure. The intensity of colour change is read with a…

proportional, enzyme activity, target analyte, spectrophotometre

Types of ELISA

Direct, indirect, sandwich, competitive

Direct ELISA

Enzyme-labelled primary antibody binds to immobilised target (antigen), linked enzyme reacts with substrate to produce visible signal

Indirect ELISA

Primary antibody and enzyme-labelled secondary antibody used: primary bind to immobilised antigen, secondary binds to primary, enzyme reacts with substrate, produces measurable visible signal

Sandwich ELISA

Antigen of interest binds to immobilised capture antibody, primary detection antibody binds to antigen, enzyme-labelled secondary detection antibody binds to primary, enzyme reacts with substrate

Competitive inhibition assay

Unlabeled primary antibody incubated with sample containing antigen, Ag-Ab complex forms, antibody excessive compared to antigen

Quantitative ELISA purpose

Measures exact concentration of antigen or antibody in the sample

Quantitative ELISA interpretation

Uses standard curve to determine precise concentrations

Quantitiative ELISA applications

Hormone levels, cytokins, viral loads

Qualitative ELISA purpose

Detects presence or absence of antigen/antibody

Qualitative ELISA interpretation

Reported as positive or negative

Qualitative ELISA applications

Disease diagnosis, pregnancy tests

Semi-Quantitative ELISA purpose

Measures relative amount of antigen/antibody

Semi-quantitative ELISA interpretation

Signal intensity compared to reference or control

Semi-quantitative ELISA applications

Antibody titers, vaccine response studies

Hook effect

In antigen-antibody reactions when concentration of one component is extremely high = lack of proper complex formation, results in false-negative or falsely low results

The hook effect is most commonly seen in…

sandwich immunoassays

Hook effect remedy

Serial dilution

Direct ELISA principle

Detects antigen directly using a labelled primary antibody

Direct ELISA advantages

Faster (fewer steps), less cross-reactivity

Direct ELISA disadvantages

Lower sensitivity, no signal amplification

Indirect ELISA principle

Antigens used to capture unlabelled primary antibody, labelled secondary antibody used as a detection antibody

Indirect ELISA advantages

Higher sensitivity (signal amplification from secondary Ab), more flexible (one secondary Ab can detect may primary Ab)

Indirect ELISA disadvantages

Longer protocol, higher cross-reactivity risk

Sandwich ELISA principle

Uses capture and detection antibodies to detect antigen (antigen is ‘sandwiched’ between them)

Sandwich ELISA advantages

Highest sensitivity and specificity, good for complex samples (serum, plasma)

Sandwich ELISA disadvantages

Requires two high-affinity antibodies, more expensive and complex

Competitive ELISA principle

Unlabeled antigen competes with labeled antigen for antibody binding

Competitive ELISA advantages

Detecting small molecules (haptens, hormones, toxins), works in complex samples (minimal putrification)

Competitive ELISA disadvantages

Inverse signal relationship (harder interpretation), may require optimisation, low specificity

ELISA: Negative control

Has no analyte

ELISA: Positive control

Contains analyte

ELISA: Standards

Contain analyte of known concentration

ELISA: Substrate blank

Only contains substrate, does not receive any sample or detector antibodies; necessary to evaluate substrate reaction

Blank wells control for any _, or ___ itself to the measured OD

variation, contribution of the plate

Expected values for substrate blank

Quite low, approaching zero