Antibody Basics: Part 11 - Diagnostics - ELISA, Western blot, fluorescence

Welcome back to Antibody Basics by Biointron, Part 11. In this episode we’ll talk about diagnostics such as ELISA, Western blot, and immunofluorescence.

Antibody-Based Diagnostics

Antibodies are produced quickly in response to infections, making them valuable for diagnostic purposes. They recognize and bind to specific epitopes on antigens - specificity and high affinity make them ideal for detecting the presence of pathogens, biomarkers, or other molecules indicative of disease. As infections progress, antibodies undergo processes such as affinity maturation and class/isotype switching. These changes in antibody distribution also allow for differentiation between acute and chronic infections. They are often used in enzyme-linked immunosorbent assays (ELISA), Western blots, and immunofluorescence.

ELISA

Enzyme-linked immunosorbent assay (ELISA) is a heterogeneous enzyme immunoassay technique used in clinical analyses. ELISA is a plate-based assay for detecting and quantifying antibodies or antigens in a sample. The target molecule (antigen or antibody) is immobilized on a solid surface. A specific antibody labeled with an enzyme is used for detection. The enzyme cleaves a substrate, producing a colored or chemiluminescent signal proportional to the target molecule's concentration

Direct ELISA: The antigen is directly attached to the microplate well, and a labeled primary antibody binds to it. Straightforward and quick due to fewer steps, which also reduces the risk of non-specific binding. Limited sensitivity because of no signal amplification.

Indirect ELISA: The antigen is immobilized on the well, followed by binding of an unlabeled primary antibody. A labeled secondary antibody then binds to the primary antibody. This format is more sensitive due to signal amplification from the secondary antibody. Various primary antibodies can be used but it may have higher background noise.

Sandwich ELISA: A capture antibody is immobilized on the well to bind the target antigen from a sample. A labeled detection antibody then binds to the captured antigen, forming a “sandwich.” This method is highly sensitive and specific because it involves multiple binding events. It is suitable for complex samples like blood or serum.

Competitive ELISA: This is used when sample antigen competes with a labeled antigen for binding to a specific antibody, either attached to the well or in solution. The amount of labeled antigen bound inversely correlates with the concentration of sample antigen. This method can detect small antigens or in samples with low antigen concentration.

Western blots

Western blotting is a protein separation and identification technique. Proteins are separated by size using gel electrophoresis. Separated proteins are transferred to a membrane for probing. Specific antibodies (primary and secondary) are used to detect proteins of interest. Detection can be achieved using chemiluminescence, fluorescence, or colorimetric methods.

Uses:

• Investigate protein abundance

• Kinase activity

• Cellular localization

• Protein–protein interactions

• Monitoring post-translational modifications (cleavage, phosphorylation)

• Ubiquitinylation

• Glycosylation

• Methylation

• SUMOylation 

Immunofluorescence

Immunofluorescence is a technique that allows visualization of many components in a given tissue or cell type. Fluorescence-based immunoassays offer high sensitivity and real-time detection. Antibodies are conjugated to fluorescent molecules (fluorophores), allowing for sensitive detection of antigen-antibody interactions. After fixation and antigen retrieval, either Direct (Primary) or Indirect (Secondary) IF is employed. Although direct is quicker, the indirect method is more widely used for its high sensitivity, signal amplification, and ability to detect several targets in the same sample.

Multiple detection formats exist:

• Homogeneous assays: Antigen and antibody are mixed in solution with a fluorophore that changes properties upon binding.

• Heterogeneous assays: Similar to ELISA, but with a fluorescently labeled secondary antibody.

• Fluorescence resonance energy transfer: Energy transfer between donor and acceptor fluorophores upon antigen-antibody binding.

Direct IF: The fluorophore label is conjugated directly to the primary antibody that will be reacting with the target epitope.

Indirect IF: A primary antibody binds to the target epitope, then a fluorophore-tagged secondary antibody recognizes and binds to the primary antibody.

Choosing the Right Tool

Example: SARS-CoV-2 Serological Testing

• Commonly used immune-based tests contain SARS-CoV-2-specific recombinant antigens immobilized onto nitrocellulose membranes. 

• Mouse anti-human IgM and IgG antibodies conjugated with colored latex beads are immobilized on conjugate pads. 

• The test sample contacts the membrane and colored antibodies form latex conjugate complexes.

• This complex immobilized on the membrane is captured by the SARS-CoV-2-specific recombinant antigen.

• If SARS-CoV-2-specific IgG/IgM are present in the sample, this leads to a colored band.

Technology: Antibody-Based Proteomics

Personalization of cancer therapy requires the identification of biomarkers for stratification of patients and monitoring of responses to targeted therapies. The generation and validation of specific antibodies offers a HTP mechanism for exploration of the proteome and a logical approach for fast-tracking the translation of identified biomarkers. Multiple approaches exist which capitalize on the inherent specificity and sensitivity of antibodies as affinity reagents. Integrating data from HTP screening methods, such as DNA microarrays, mass spectrometry and functional genomic screens, with antibody-based proteomics, is an opportunity to identify new cancer biomarkers.