L9: ELISA.

HIV

Usually detected by antibodies.

Detectable 23-90 days.

Lyme disease

Masks as an autoimmune disorder.

Antibody based diagnosis.

Caught early- antibiotics for months. Not caught early -Lyme disease develops.

Squamous cell carcinoma. (SCC-Ag)

Caught by antibody detection

Varicella-zoster virus.

Caught by antibody detection. ELISA.

Member of the herpesvirus family that causes two distinct diseases: primary chickenpox and reactivated shingles.

Zika virus.

Children born with small brain if pregnant women have it.

Caught by antibody detection (IgM)

Principle of ELISA.

• Principle is the specificity of antibodies for antigen.

• Detected by labelled antibody.

• Unbound antibody removed by washing

• Colour change

• Colour proportional to amount of antigen (Ag) present.

Direct ELISA

• Antigen bound to solid surface and detected directly by enzyme labelled Antibody.

Advantage: Quick assay as less steps. Less errors as there’s less steps.

Disadvantage: Less flexible: antisera or antibodies raised against different antigens have to be labelled. Less specific: may get higher background noise. No amplification of signal.

Example of Direct ELISA.

Mycoplasma Bovis - causes clinical mastitis in dairy herds.

Antigen: P48 -lipoprotein on surface of Mycoplasma bovis.

Enables rapid surveillance in herds.

Quick response - isolation.

Indirect ELISA.

• Antigen (eg: HIV antigen) bound to solid surface and detected by specific primary Ab.

• Bound Ab then detected by secondary labelled Ab which is specific for the 1st antibody.

Advantages: -Same Ab conjugate used in the detection of several antigens provided. -More specific - two antibodies.

Disadvantage: slower: more steps.

Sandwich ELISA mechanism

• Constant dilution of capture Ab is coated on the solid phase, unabsorbed Ab is washed away.

• Ag (range of dilutions) is added)

• The second antibody can be -Produced in the same or a different species. -Capture and second Abs shouldn’t recognise the same site, competition.

Sandwich ELISA details.

• Most commercially available ELISAs are based on the sandwich format.

• Most sensitive ELISA. (2 to 5 fold)

• Requires relatively large amounts of pure or semi-pure capture antibody.

• Absorbance (OD- Optical Density) increases as Antigen increases.

Competitive ELISA.

Competition: 2 reactants are trying to bind to a third.

• Fixed amount of unlabelled antibody.

• Unlabelled standard or test added.

• With fixed amount of labelled antigen.

• Competition between labelled and unlabelled for sites.

Sandwich and indirect ELISA difference

Sandwich ELISA is designed to detect and quantify antigens.

Indirect ELISA is designed to detect and quantify antibodies present.

Non-specific binding in ELISA.

• Enzyme labelled Ag or Ab binds specifically to immobilised antigen but also non-specifically to the plate.

• This can be minimised by inclusion of non-ionic detergent such as Triton X-100 or Tween 20.

• They don’t interfere with the Ag-Ab interaction, but prevent formation of new hydrophobic interactions without disturbing already formed hydrophobic bonds.

Triton X-100 or Tween 20.

Prevent formation of new hydrophobic interactions without disturbing already formed hydrophobic bonds.

Immobilisation of Ag or Ab; Plastic carriers: Polystyrene and PVC.

• Proteins adsorb passively to polystyrene (eg: 96-well microtitre plates)

• Hydrophobic interactions between non-polar protein substructures and non-polar plastic matrix.

• Physical adsorption

Blocking and Washing.

Blocking: Fill non-specific binding site.

• Normal serum, BSA (bovine serum albumin), casein, gelatin, detergents (Tween 20, Triton-X), Non-fat mild powder, dextran sulphate.

Washing: separate free from bound reagents

• Three times for each well.

• Detergents can be added (0.05% Tween)

Examples of ELISA in diagnosis.

1. Detection of tumour markers in serum.

2. Diagnosis of infectious disease,. - Measure Ab to the pathogen (serology). - Measure pathogen proteins in body fluids.

3. Hormones

4. Inflammatory disease and inflammation

Human chorionic gonadotrophin (hCG) is detected by the immunoassay, ELISA.

Pregnancy tests detect hCG in urine or blood using antibodies that specifically recognise the hormone.

(ELISA can detect lots of steroids, proteins and derivatives of tyrosine like adrenaline, NA and DA)

TSH, LH, hCG, FSH all share

the same alpha subunit.

So for ELISA to detect difference between these hormones, antibodies need to be specific to the B subunit of these hormones.

Prostate cancer.

• Most common malignant tumour, 2nd leading cause of male cancer mortality, lung cancer is the 1st.

• PSA = tumour marker. Serum levels correlate with tumour mass. Indicates stable or a progressive disease (with ELISA).

• Monitoring of PSA is also important to evaluate response to treatment.

HIV 1 and 2.

Diagnosis - detection of viral specific antibodies.

ELISA with recombinant envelope antigens used to measure specific antibodies.

Commonly used to screen blood donations - screening assays configured to give few false negatives therefore false positives a problem.

Sensitivity

Proportion of positives correctly identified.

High sensitivity = few false negatives.

Specificity

Proportion of negatives correctly identified.

High specificity = few false positives.

Abbott alinity.

Detects IgG antibodies against the SARS-CoV-2 nucleocapsid (N) protein.

Performance

• Positive predictive value: 84%

• Negative predictive value: 100%

Covid-19 rapid antigen test kit.

• Use antibodies to detect Sars-Cov-2 nuceloprotein

• Two bars - positive result.

• False negatives are an issue (65%)