ELISA: Basics and Key Components

ELISA Basics

• ELISA stands for Enzyme-Linked Immunosorbent Assay and is a laboratory method
• It detects the presence of an antibody or antigen in a patient specimen
• ELISA tests can be qualitative or quantitative
• ELISAs can be performed manually or automated

Key Components

• Antibodies (Ab): Immunoglobulins are gamma globulin proteins in blood used by the immune system to identify and neutralize foreign objects like viruses
• Antigens (Ag): Proteins that, when introduced into the body, stimulate the production of antibodies
• Immunoassay: A lab technique using the binding between an antigen and its corresponding antibody to identify and quantify specific antigens or antibodies in a sample
• Analyte: The specimen being analyzed, which can be either an antibody or an antigen in ELISA tests

Solid Phase

• ELISA utilizes a solid phase component
• Typically a 96-well plate, magnetic microparticles, or beads

Key Materials

• Ag/capture Ab: Antigen or capture antibody is attached to the solid phase.
• Patient specimen: The sample from the patient being tested.
• Conjugate: An enzyme-labeled anti-species detector antibody.
• Substrate: A chemical compound converted by an enzyme to produce a detectable chromogenic or fluorescent signal.
• Blocking buffer: Casein or BSA is used to block nonspecific adsorption of proteins to the solid phase.
• Wash buffer: Used to remove unbound substances.
• Stop solution: Halts the enzyme's action on the substrate, preventing excessively bright colors that a spectrophotometer cannot read.
• Controls: Positive and negative controls ensure the validity of the assay.
• Spectrophotometer: An optical device used to view results by reading specific wavelengths for specific colors produced by the enzyme/substrate combinations used.

Enzyme Labels

• A detection enzyme can be linked directly to the primary antibody
• The primary antibody can be linked indirectly to a secondary antibody
• The enzyme can be linked to a protein like streptavidin if the primary antibody is biotin-labeled
• Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP) are common enzyme labels

ELISA Protocols

• Antigen is coated onto a microplate
• Protein adsorption is allowed, and unoccupied sites are blocked with a neutral protein
• Antibody solution is added to each well
• HRP or AP conjugated secondary antibody is added, and a colorimetric reaction is developed with an appropriate substrate
• Absorbance is read using a spectrophotometer with an appropriate filter
• Relative antigen levels are quantified.

Detection Formats

• Radio-Immunoassay: Uses a radioactively labeled detector antibody, emitting gamma radiation
  • Can be unstable and hazardous
• Chromogenic: Uses a colored reaction product that absorbs light in the visible range
  • The substrate becomes colored when catalyzed by an enzyme, and the optical density is proportional to the analyte amount
• Fluorescent Immunoassay: Involves an enzyme and substrate that produce a fluorescent reaction product when excited by a specific wavelength of light
  • Fluorophores absorb light, raising the energy level and causing fluorescence upon return to the baseline state
  • Mini-Vidas platform is used for this method (biomeriux, France)
  • Offers higher sensitivity and a wider dynamic range compared to chromogenic assays
• Chemiluminescent Microparticle Immunoassay: Employs an enzyme that converts a substrate into a reaction product that emits photons of light
  • Acridinium Ester is conjugated to the antibody or antigen, resulting in chemiluminescence when exposed to hydrogen peroxide and sodium hydroxide
  • Architect is used for this method (Abbott, USA)
  • Higher sensitivity, a wider dynamic range and lower background interference

ELISA Formats

• Direct
• Indirect
• Capture (Sandwich)
• Competition/Inhibition

Direct ELISA

• An antigen-specific antibody is attached to a solid phase
• A patient specimen is added
• Enzyme-labeled antibody (conjugate) is added to the sample
• Chromogenic substrate is added, and the presence of enzyme induces a color change

Indirect ELISA

• An antigen or sample is added to the plate
• Blocking buffer is applied to block remaining protein-binding sites
• A suitable primary antibody is added
• A suitable secondary antibody – HRPO conjugate is then added, it binds to the primary antibody
• TMB substrate is added and converted by HRPO to a detectable form

Direct vs. Indirect Comparison

• Direct ELISA uses a labeled primary antibody that reacts directly with the antigen
• Suited for use with antigen directly immobilized on a solid phase or in a capture assay format
• Not widely used in ELISA but common in immunohistochemical staining of tissues and cells.
• Indirect ELISA uses a labeled secondary antibody
  • Most popular ELISA format: the secondary antibody has specificity for the primary antibody

Advantages of Direct ELISA

• Quick
• Only one antibody is needed
• Eliminates cross-reactivity of the secondary antibody

Disadvantages of Direct ELISA

• Immunoreactivity of the primary antibody altered by labeling with enzymes or tags
• Labeling primary antibodies for each specific ELISA system is time-consuming and expensive
• No flexibility in choice of primary antibody label from one ELISA to another
• Minimal signal amplification is achieved

Advantages of Indirect ELISA

• Wide variety of labeled secondary antibodies commercially available
• Many primary antibodies for one species can use the same labeled secondary antibody detection
• Maximum immunoreactivity of the primary antibody is retained if it is not labelled
• Increased Sensitivity
  • Each primary antibody contains several epitopes
• Various visualization markers can be used with the same primary antibody

Disadvantages of Indirect ELISA

• Cross-reactivity may occur with a secondary antibody, causing a nonspecific signal
• Requires an extra incubation step in ELISA

Capture ELISA

• An antibody specific for IgG or IgM is attached to a solid phase
• Patient specimen with possible high IgM is added
• Specific antigen is added
• Enzyme-labeled antibody (conjugate) is added
• Chromogenic substrate is added, and presence of the enzyme induces a color change

IgM μ-Capture ELISA

• An antibody directed to the human IgM (μ- heavy chain portion) is immobilized on a solid phase instead of using a specific antigen
• Anti-μ antibodies are highly IgM specific
  • They are directed to the conserved region of the μ heavy chain unique to IgM
• IgM in the patient specimen is captured by these anti-μ antibodies
• Competing IgG and non-IgM RF are not captured
• Washing removes all unbound IgG and immune complexes
• Specific antigen and an enzyme-conjugated antibody specific to that antigen are added to detect bound IgM

Non-Specific Reactions

• Mechanisms include variety of ways in which it is non-specific
• Use high-quality solid-phase material and stringent washing techniques
  • Prevent the non-specific binding of immunoreactants to the solid phase
• Use purified or monoclonal antibodies
  • prevent non-specific reactions
• Some specimens yield false positives from non-specific antiglobulin activity directed against other immunoreactants
• Reduce antiglobulin activity by adding a mild reducing agent, such as 20% N-acetyl-cysteine
  • This addresses the presence of IgM rheumatoid factor (RF)

IgM Assay Challenges

• Several factors interfere with the detection of IgM
  • Must be removed or neutralized to ensure accurate results
• Ag-specific IgG from previous or latent infection competes with Ag-specific IgM from a new infection for capture Ag
  • Prevents the formation of a sandwich = false-negative results
• Anti-IgG (Fc portion) may be included to bind IgG and be removed during the wash step

Rheumatoid Factor

• Rheumatoid factor is an autoimmune antibody; predominantly IgM
• IgM targets the Fc region of IgG
• RF crosslinks interfering = false positive
• Removal of IgG prevents false positives
  • It may cause binding of the RF to labeled IgG, yielding in false negatives
• Can use commercial adsorbents to remove the RF
• Modify the assay by using an IgM μ-capture ELISA is an alternative approach

Competitive ELISA

• High specificity can be achieved using two antibodies – the antigen is specifically captured
• Suitable for complex specimens because no protein purification is required prior to measurement
• Direct and indirect detection methods can be used to provide flexibility and sensitivity

Monoclonal vs Polyclonal

• Monoclonal Antibodies recognize a single epitope
  • Allows fine detection and quantification of small differences in antigens
• Polyclonal Antibodies are often used as capture antibodies to pull down as much antigen as possible