BMOL30090 L9 ELISA

see notes

Abs in diagnosis

• HIV (detectable 23-90 days)

• Lyme disease

• squamous cell carcinoma (SSC-Ag)

• Varicella-zoster virus (chick pox and shingles)

• Zika virus (IgM) - first 3 months of pregnancy, small brain, mosquito-bourne

what is ELISA

enzyme linked immunosorbent assay

principle of ELISA

• specificity of Abs for antigen

• detected by labelled Ab

• unbound Ab removed by washing

• colour change

• colour proportional to Ag present

principle of ELISA image

ELISA assay formats

• direct (simplest) and indirect

• sandwich

• competitive or inhibition

Direct ELISA

• Ag bound to solid surface and detected directly by enzyme labelled Ab

• Advantages:

  • quick assay - less steps

  • less steps = less errors

• Disadvantages:

  • less flexible - antisera or Abs raised against different Ags have to be labelled

  • les specific - may get higher backgr. noise

  • no amplification of signal

direct ELISA example

Mycoplasma bovis causes clinical mastitis in dairy herds

• Ag: P48 (lipoprotein on surface of M bovis)

• enables rapid surveillance in herds

• quick response - isolation

indirect ELISA

• extensively used assay format

• Ag (e.g. HIV Ags) bound to solid surface and detected by specific primary Ab

• bound Ab then detected by secondary labelled Ab which is specific for the first Ab

• Advantages:

  • same Ab conjugate used in detection of several Ags provided

  • more specific (2 Abs required)

• Disadvantages:

  • slower (more steps)

indirect ELISA image

sandwich ELISA

• most commercially available ELISAs

• most sensitive assays (2-5 fold)

• constant dilution of Ab is coated on the solid phase, unabsorbed antibody is washed away

• Ag (range of dilutions) added

• the second Ab can be:

  • produced in same or different species

  • capture & second Abs shouldnt recogn. the same site - competition

• requires rel. large amounts of pure/semi-pure capture Ab

• OD (absorbance) increases as [Ag] increases

Sandwich ELISA image

sandwich ELISA curve

OD is absorbance

competitive ELISA

2 reactants try to bind with a third

• fixed amount of unlabelled Ab

• unlabelled standard or test added

• with fixed amount of labelled Ag

• competition between labelled/unlabelled for sites

competitive ELISA image

less competitive = more colour ?

immobilisation of Ag or Ab

plastic carriers: polystyrene or PVC

• proteins adsorb passively to polystyrene

• hydrophobic interactions between nonpolar protein substructures and non-polar plastic matrix

• physical adsorption

• flat bottomed plates

non-specific binding

• enzyme labelled Ag or Ab binds specifically to immobilised Ag but also non-specifically to plate

• minimised by inclusion of non-ionic detergent such as triton X-100 or Tween 20

• do not interfere with Ag-Ab interaction

• prevent formation of new hydrophobic interactions without disturbing already formed hydrophobic bonds

blocking

fill non specific binding site

• normal serum, BSA, casein, gelatin, detergents (Tween 20, Triton X), non-fat mild powder, dextran sulfate

washing

separate free from bound reagents

• 3x for each well

• detergents can be added (0.05% Tween)

blocking and washing image

examples of ELISA in diagnosis

1. detection of tumour markers in serum

2. diagnosis of infectious disease

   1. - measure Ab to the pathogen (serology)

   2. - measure pathogen proteins in body fluids

3. hormones

4. inflam. diseases and inflam.

image: examples of ELISA diagnostics for hormones

distinguish between pituitary hormones

• TSH - hypo/hyperthyroidism

• LH - fertility issues in M/F

• hCG - pregnancy/tumour markers

• FSH - fertility issues in M/F

• growth hormone

• share the same alpha subunit

prostate cancer

• most common malignant tumour (2nd cause of male cancer mortality)

• PSA - tumour marker (false negatives v common)

• serum levels correlate with tumour mass

• indicate stable/progressive disease (ELISA)

• monitoring of PSA also important to evaluate response to treatment

overall survival by PSA status graph

HIV 1 & 2

• window where it is not detectable

• diagnosis - detection of viral specific antibodies

• ELISA with recombinant envelope antigens used to measure specific Abs

• commonly used to screen blood donations - screening assays configured to give few false negatives, therefore false positives are a problem (wont miss any positives by accident, but makes false pos likely)

HIV graph

over-the-counter Ab tests

• ‘point of care’ Ab tests

• pregnancy test kits - detects human chorionic gonadotrophin (hCG)

• COVID-19 antigen & antibody (IgG/IgM) tests

pregnancy test kit

• immobilised Abs against hCG

• labelled Abs bind to hCG (red & mobile)

• carried by flow to immobilised Ab

• coloured band

• no hCG = no interaction with immobilised Ab

pregnancy test image

COVID-19 rapid antigen test kit

• used Abs to detect Sars-Co-v-2 nucleoprotein

• two bars (C and T) = positive result

• C only = control

• false negatives are an issue (65%)

COVID-19 antibody tests

• different formats - ELISAs, multiplex, home test kits

• performance is an issue in many

  • sensitivity - proportion of positives correctly identified

  • specificity - proportion of negatives correctly identified

• e.g. Abbot Alinity

  • detects IgG to nucleocapsid

  • performance - pos predictive value = 84%

  • neg predictive value = 100%

COVID-19 Ab tests image