Unit 7: Immunological assays - Subjective Questions

ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based analytical technique designed for detecting and quantifying soluble substances such as antigens, antibodies, proteins, and hormones.

Basic Principle:

• ELISA is based on the specific interaction between an antigen and its corresponding antibody.
• One of the reactants (antigen or antibody) is immobilized onto a solid surface (typically a polystyrene microtiter plate).
• A detection antibody linked to an enzyme binds to the target.
• Addition of a chromogenic substrate produces a measurable colour change catalyzed by the enzyme.
• The intensity of colour is directly or inversely proportional to the amount of analyte present and is measured spectrophotometrically as optical density (OD).

Key features:

• Combines immunological specificity with enzymatic signal amplification.
• Common enzymes used: Horseradish Peroxidase (HRP) and Alkaline Phosphatase (ALP).

There are four major types of ELISA:

1. Direct ELISA:

• Antigen is coated onto the plate.
• An enzyme-labelled primary antibody binds directly to the antigen.
• Simple and fast but less sensitive.

2. Indirect ELISA:

• Antigen is coated onto the plate.
• An unlabelled primary antibody binds to the antigen.
• An enzyme-labelled secondary antibody binds to the primary antibody.
• More sensitive due to signal amplification; commonly used for antibody detection (e.g., HIV screening).

3. Sandwich ELISA:

• A capture antibody is coated on the plate.
• Antigen binds to it, then a detection antibody binds to another epitope of the antigen.
• Highly specific; used for detecting antigens with multiple epitopes.

4. Competitive ELISA:

• Sample antigen competes with a labelled antigen for antibody binding sites.
• Signal is inversely proportional to analyte concentration.
• Used for small molecules (haptens).

The procedure for Indirect ELISA involves the following steps:

1. Coating: Add the antigen to the microtiter plate wells and incubate to allow adsorption onto the surface.
2. Washing: Wash the wells with buffer (e.g., PBS-Tween) to remove unbound antigen.
3. Blocking: Add a blocking agent (e.g., BSA or non-fat milk) to prevent non-specific binding.
4. Primary Antibody Addition: Add the test serum / primary antibody; it binds specifically to the antigen. Wash away unbound antibody.
5. Secondary Antibody Addition: Add an enzyme-conjugated secondary antibody that binds the primary antibody. Wash off excess.
6. Substrate Addition: Add a chromogenic substrate (e.g., TMB). The enzyme converts it into a coloured product.
7. Stopping Reaction: Add a stop solution (e.g., dilute sulphuric acid) to halt the reaction.
8. Measurement: Measure the optical density (OD) using an ELISA plate reader at a specific wavelength.

The colour intensity is proportional to the amount of antibody present in the sample.

Summary: Direct ELISA is quicker but less sensitive, while Indirect ELISA provides higher sensitivity through the use of a labelled secondary antibody.

Sandwich ELISA vs Competitive ELISA:

Conclusion: Sandwich ELISA is preferred for large multivalent antigens, whereas Competitive ELISA is ideal for small molecules that cannot bind two antibodies simultaneously.

Key ELISA components and their roles:

(a) Coating Buffer:

• Usually carbonate-bicarbonate buffer (pH 9.6).
• Helps in immobilizing the antigen/antibody onto the solid surface of the well.

(b) Blocking Agent:

• Common agents: BSA, non-fat dry milk, gelatin.
• Occupies the unbound sites on the plate to prevent non-specific binding and reduce background noise.

(c) Substrate:

• E.g., TMB (3,3',5,5'-Tetramethylbenzidine) for HRP, pNPP for ALP.
• Reacts with the enzyme to produce a coloured product, generating a measurable signal.

(d) Stop Solution:

• E.g., dilute sulphuric acid () for TMB.
• Halts the enzymatic reaction at a fixed time and often intensifies/stabilizes the colour for accurate reading.

Applications of ELISA:

Medical Diagnostics:

• Detection of HIV antibodies (HIV test).
• Diagnosis of Hepatitis B and C.
• Detection of Dengue, COVID-19, and other infectious diseases.
• Pregnancy testing (detection of hCG hormone).

Immunology & Research:

• Quantification of cytokines, hormones, and antibodies.
• Monitoring immune response after vaccination.

Food Industry:

• Detection of allergens, toxins, and pathogens in food.
• Detection of pesticide residues.

Pharmaceutical & Biotechnology:

• Drug monitoring and detection.
• Quality control of biopharmaceutical products.

Environmental Testing:

• Detection of pollutants and contaminants in water and soil.

ELISA is valued for its specificity, sensitivity, and high-throughput capability.

Washing Step:

• Involves rinsing wells with buffer such as PBS-Tween 20.
• Removes unbound reagents (antigens, antibodies, conjugates) after each incubation.
• Ensures that only specifically bound molecules remain.

Effect if omitted:

• Unbound enzyme-conjugates remain, producing high background and false-positive results.

Blocking Step:

• Uses inert proteins (BSA, milk, gelatin) to cover unoccupied binding sites on the plate.
• Prevents non-specific adsorption of detection antibodies.

Effect if omitted:

• Detection antibodies bind non-specifically to the plate surface, causing elevated background signal and reduced signal-to-noise ratio, lowering assay accuracy.

Conclusion: Both steps are essential for specificity, accuracy, and reliability of ELISA results.

Enzymes used in ELISA act as labels/reporters that generate a detectable signal.

1. Horseradish Peroxidase (HRP):

• Substrates: TMB (blue → yellow after stopping), OPD, ABTS.
• Widely used due to high turnover rate and stability.

2. Alkaline Phosphatase (ALP):

• Substrate: pNPP (p-Nitrophenyl phosphate) → yellow product.
• Produces a stable, linear signal.

3. Beta-galactosidase:

• Substrate: ONPG.
• Used less commonly.

Function:

• The enzyme catalyzes conversion of a colourless substrate into a coloured product.
• The colour intensity correlates with the amount of bound antibody/antigen.
• Measured spectrophotometrically as optical density (OD).

The enzyme-substrate system provides signal amplification, greatly enhancing ELISA sensitivity.

Quantitative ELISA determines the concentration of an unknown analyte using a standard curve.

Procedure:

1. Prepare a series of standards with known concentrations of the analyte.
2. Run the standards and unknown samples together in the ELISA.
3. Measure the optical density (OD) of each well using a plate reader.

Standard Curve Construction:

• Plot OD values (Y-axis) against known concentrations (X-axis).
• The relationship is often linear in a certain range or sigmoidal (4-parameter logistic).

Determining Unknown Concentration:

• The OD of the unknown sample is located on the curve.
• The corresponding concentration is interpolated.

For a simple linear relationship:

where is concentration, is slope, and is intercept. Rearranging:

This allows accurate estimation of analyte concentration in the sample.

Antigen:

• A foreign molecule (protein, polysaccharide, etc.) that can trigger an immune response and specifically bind to an antibody.
• The specific region recognized by the antibody is called the epitope.

Antibody (Immunoglobulin):

• A Y-shaped glycoprotein produced by B-lymphocytes/plasma cells.
• Binds specifically to antigens at its antigen-binding site (paratope).

Nature of Antigen-Antibody Interaction:

• Highly specific — like a lock-and-key fit between epitope and paratope.
• Involves non-covalent bonds:
  • Hydrogen bonds
  • Electrostatic (ionic) interactions
  • Van der Waals forces
  • Hydrophobic interactions
• The strength of binding is defined by affinity (single site) and avidity (overall strength).
• The interaction is reversible but generally stable under physiological conditions.

This specificity forms the basis of immunoassays like ELISA.

Advantages of ELISA:

• High specificity due to antigen-antibody interaction.
• High sensitivity — can detect very low analyte concentrations (pg/mL range).
• Quantitative results using standard curves.
• High throughput — many samples tested simultaneously in microtiter plates.
• Safe — no radioactive materials (unlike RIA).
• Cost-effective and reagents are stable.
• Adaptable to automation.

Limitations of ELISA:

• Requires specific antibodies which may be expensive to produce.
• Cross-reactivity can cause false results.
• Instability of enzymes may affect reproducibility.
• Only measures one analyte at a time (traditional format).
• Requires careful washing and technique; errors cause high background.
• Semi-quantitative unless properly calibrated.

Conclusion: Despite limitations, ELISA remains a gold-standard immunoassay for diagnostics and research.

Principle:
In Sandwich ELISA, the antigen is captured between two antibodies — a capture antibody and a detection antibody — hence the name 'sandwich'. It is used for antigens with at least two epitopes.

Steps:

1. Coating: Coat the plate with a specific capture antibody.
2. Blocking: Block unoccupied sites with BSA/milk.
3. Sample Addition: Add the sample; the antigen binds to the capture antibody.
4. Washing: Remove unbound substances.
5. Detection Antibody: Add a second (detection) antibody that binds another epitope of the antigen.
6. Enzyme Conjugate: Add enzyme-linked antibody (or the detection antibody is already labelled).
7. Substrate Addition: Add substrate → colour develops.
8. Measurement: Measure OD, which is directly proportional to antigen concentration.

Diagram (description):
Plate → Capture Antibody → Antigen → Detection Antibody → Enzyme → Substrate → Colour

Advantage: Very high specificity and sensitivity, ideal for complex samples.

Conjugate in ELISA:

• A conjugate is an antibody chemically linked to an enzyme (e.g., antibody-HRP).
• It acts as the detection molecule producing a measurable colour signal.

Preparation of Enzyme-Antibody Conjugates:

• Glutaraldehyde method: Cross-links enzyme and antibody via amino groups.
• Periodate oxidation method: Used mainly for HRP, oxidizes carbohydrate groups to form aldehydes that bind antibody amino groups.
• Maleimide/thiol coupling: Uses sulfhydryl groups for site-specific conjugation.

Importance:

• Provides the enzymatic signal needed for detection.
• Determines the sensitivity and specificity of the assay.
• Must retain both enzyme activity and antibody binding capacity.
• Enables signal amplification — a single enzyme converts many substrate molecules.

A well-prepared conjugate is critical for accurate and reproducible ELISA results.

Optical Density (OD) / Absorbance:

• OD is a measure of the amount of light absorbed by the coloured product in each well.
• Measured using an ELISA plate reader (spectrophotometer) at a specific wavelength (e.g., 450 nm for TMB).

Relation to Concentration:

• Governed by Beer-Lambert Law:
  
  where:

  • = absorbance (OD)
  • = molar absorptivity
  • = concentration
  • = path length

• In Sandwich/Indirect ELISA: OD is directly proportional to analyte concentration.

• In Competitive ELISA: OD is inversely proportional to analyte concentration.

Interpretation:

• Higher OD → more coloured product → more bound analyte (in direct formats).
• A cut-off OD value distinguishes positive from negative samples in qualitative tests.

OD readings, when compared to a standard curve, allow quantification of the target molecule.

Conclusion: ELISA has largely replaced RIA in most laboratories due to its safety, cost-effectiveness, and ease of use, while offering comparable sensitivity.

False-Positive Result:

• A positive signal obtained even when the target analyte is absent.

Causes:

• Insufficient washing leaving unbound conjugates.
• Inadequate blocking → non-specific binding.
• Cross-reactivity of antibodies.
• Contamination of wells or reagents.

False-Negative Result:

• No signal despite the target analyte being present.

Causes:

• Degraded antibodies/enzymes.
• Improper incubation time or temperature.
• Excessive washing removing bound complexes.
• Low analyte concentration below detection limit.

Minimization Strategies:

• Use appropriate blocking agents and optimized washing.
• Include positive and negative controls.
• Use fresh, validated reagents.
• Maintain standardized protocols and proper incubation conditions.
• Use highly specific antibodies to reduce cross-reactivity.

Proper controls and technique are essential for reliable results.

Principle of Competitive ELISA:

• Based on the competition between the sample antigen and a labelled (reference) antigen for a limited number of antibody binding sites.

Working:

1. Plate is coated with a specific antibody.
2. The sample antigen and a fixed amount of enzyme-labelled antigen are added together.
3. Both compete to bind the antibody.
4. More sample antigen → less labelled antigen binds → less colour.
5. Less sample antigen → more labelled antigen binds → more colour.

Signal Relationship:

• Inverse relationship: Colour intensity (OD) is inversely proportional to sample antigen concentration.

Applications:

• Detection of small molecules (haptens) with single epitopes.
• Measuring drugs, hormones (e.g., cortisol, thyroxine).
• Detection of pesticides and small toxins.
• Useful when antigen is too small for the sandwich format.

It offers high specificity for small analytes where two antibodies cannot bind simultaneously.

Materials Required:

• Microtiter plate (96-well polystyrene plate) — solid phase.
• Antigens / Antibodies — specific to the target.
• Enzyme-conjugated antibodies (HRP/ALP conjugates).
• Coating buffer (carbonate-bicarbonate, pH 9.6).
• Washing buffer (PBS with Tween-20).
• Blocking agent (BSA, skimmed milk).
• Substrate solution (TMB, pNPP).
• Stop solution ().
• Standards and controls.

Instruments/Equipment:

• ELISA plate reader (microplate spectrophotometer) — measures OD.
• Micropipettes and multichannel pipettes — accurate dispensing.
• Plate washer (manual or automated).
• Incubator — controlled temperature incubation.
• Vortex mixer / plate shaker.
• Refrigerator (2–8 °C) — reagent storage.

Proper handling and calibration of these instruments ensure accurate and reproducible ELISA results.

ELISA in HIV Diagnosis:
ELISA is the primary screening test for detecting HIV antibodies in a patient's serum.

Type Used: Indirect ELISA

• Because it detects antibodies produced against HIV antigens in the patient's blood.

Procedure:

1. HIV antigens (recombinant/synthetic) are coated onto the microtiter plate.
2. Patient's serum is added — if HIV antibodies are present, they bind to the antigens.
3. Wash to remove unbound antibodies.
4. Add enzyme-labelled anti-human antibody (secondary antibody) that binds the patient's antibodies.
5. Add substrate → colour develops if HIV antibodies are present.
6. Measure OD; compare with cut-off value.

Interpretation:

• OD ≥ cut-off → Reactive (positive) → confirmatory test (e.g., Western Blot) required.
• OD < cut-off → Non-reactive (negative).

Why Indirect ELISA:

• High sensitivity ensures very few infected cases are missed (low false negatives), making it ideal for screening.

Note: A positive ELISA is always confirmed by a more specific test before diagnosis.