Serological testing for COVID-19, pregnancy test, HIV test, vitamin D deficiency test, autoimmune disease test, cancer biomarker testing, and many others running on the list!
Rejoice! Technology had made all this possible following just a simple and rapid routine laboratory protocol. Have you ever wondered and thought of the technology standing behind these tests? Yes, you are right, the answer is Immunoassays!
Immunoassays had an impeccable contribution to the progress of the medical, pharmaceutical, and research fields. Their applications are vast and of great importance; especially in clinical diagnostics, drug discovery/monitoring, food testing, quality control, or biological research. Monoclonal antibody technology is the main scaffold standing behind these assays.
In brief, immunoassays are bio-analytical methods that rely on antibody technology to detect quantitatively or qualitatively a specific analyte of interest. Whether you are an expert in the field or the first time to encounter this world; No worries! Today, we will get you covered and we will showcase the whole story in a smooth, simple, and exciting way! Let’s dive in…
What are immunoassays?
Immunoassays outcome is to detect/quantify a specific analyte in a sample. The latter is usually a biomarker that gives us an indication about a health condition related to infectious diseases, hormonal imbalances, metabolic diseases, and many others. High sensitivity means an extreme affinity between an analyte and an antibody. The high specificity of an antibody to its target makes the immunoassay highly selective.
Types of Immunoassays:
Five types of immunoassays are commonly distinguished (1), they differ mainly by the type of reporter (label) used.
- Radioimmunoassay (RIA): a radioisotope is attached to an antigen-antibody complex.
- Enzyme Immunoassay (EIA) or Enzyme-Linked Immunosorbent Assays (ELISA): the antibody is conjugated to an enzyme that mediates a color change.
- Fluoroimmunoassay (FIA): antibodies are labeled with a fluorescent probe.
- Chemiluminescent Immunoassay (CLIA): based on a chemical reaction that releases light (luminescent).
- Counting Immunoassay (CIA): coating polystyrene beads with many antibodies.
Refer to references (1, 2) for a detailed description of each method. Today, we will highlight a few of these techniques that have won great popularity.
1. ELISA immunoassay
ELISA is the most common type of immunoassays. It is a rapid method commonly used as a diagnostic tool to detect and measure the level of biomarkers in the blood. This immunoassay has high specificity and sensitivity.
It can be non-competitive (indirect, direct, sandwich) or competitive. In the current COVID-19 pandemic, many of us have heard of ELISA kits used in detecting IgG or IgM immmunoglobulin in the serum of a suspected individual.
2. Flow cytometry
Flow cytometry is also considered a powerful application of immunoassays. It is a pillar in multiple medical disciplines and biological research. This technique is used to sort and examine different live cells based on their physical and chemical characteristics. In fact, cells have different fluorescence and scattering profiles. This method relies on primary antibodies that are conjugated to different fluorochromes. Each of these antibodies targets a specific cell surface antigen. Instrumentation has rapidly evolved in the last years.
It is a method used for isolating and concentrating a specific protein out of a solution. This is achieved by using an antibody that specifically binds to the protein of interest. The antigen/antibody complex is pulled out of the sample using a secondary antibody (an immobilized antibody against the primary antibody) or using protein A/G coated agarose beads. Finally, the antigen is eluted and further analyzed.
Monoclonal antibody technology
Monoclonal antibodies were initially produced by ‘Hybridoma Technology’. This pioneering technique has revolutionized the field of research, diagnostics, and therapeutics.
George Köhler and Cesar Milstein were the two scientists behind the development of hybridoma technology in 1975. Their breakthrough was awarded the Nobel Prize in physiology or medicine (4).
In this method, the monoclonal antibodies are derived from a specific single cell called Hybridoma. The latter is obtained after the fusion of a B lymphocyte that secretes a specific antibody but does not divide indefinitely with a myeloma tumor cell.
What is an antibody?
An antibody is a large glycoprotein that is expressed on the surface of a B-cell and is secreted by a differentiated B lymphocyte (plasma cell) after encountering a foreign antigen. The part of the antigen that interacts with the antibody is called an epitope. This is a key event of our humoral adaptive immune response against foreign invaders.
The antibody structure
- An antibody is formed of two light chains and two heavy chains. Heavy and light chains or heavy chains bind together by disulfide bonds.
- Two sites that bind antigens are located on the top of the Y-shaped structure.
- Fab region varies among different antibodies and is antigen-specific.
- Fc region is constant and determines the different antibody isotypes (5 classes in mammals: IgG, IgM, IgA, IgE, and IgD).
- IgM predominates in primary infection. IgG is the most common circulating antibody and it predominates in secondary infection.
The antibody-antigen binding mechanism
Each antibody recognizes a small sequence on the surface of an antigen called epitope or antigen determinant. The interactions are generally weak and non-covalent. Agglutination is the name of the process by which an antigen and an antibody combine. You have probably heard of the word ‘agglutination’ in blood group testing.
The structure of B-cell-bound immunoglobulin is shown on the left. A molecular representation of the antibody-antigen interaction is shown on the right (6)
Development of monoclonal antibody technology
Monoclonal antibodies can be produced in a lab on a large scale. The Hybridoma technique was the most popular for antibody production and you can refer to (7) for the complete details of the method.
New technologies were added later as alternatives for Hybridoma technology.
Polyclonal antibody versus a monoclonal antibody
Antibodies can be monoclonal or polyclonal.
- Monoclonal antibodies: homogenous population of antibodies that are secreted by the same type of B cell clone.
- Polyclonal antibodies: a heterogeneous population of antibodies that are secreted by different B cell lineages during an immune response.
- Monoclonal antibodies have high specificity and affinity for a single epitope on an antigen. In addition, they are characterized by low background noise. They can be produced in large quantities for therapeutic/diagnostic use.
- Polyclonal antibodies are produced by live animals and they have an affinity toward a specific antigen but each recognizes a different epitope on the same antigen. They are quick to produce and cost-effective compared to monoclonal antibodies. However, a risk of batch variability and cross-reactivity exists. Polyclonal antibodies are usually used to detect a native protein and are also used in general research applications.
Insights into current and future developments
- Technological progress had a great impact on the development of immunoassays. Advancement in the photonic field led to the development of new generation label-free immunoassay.
- Technology led as well to the automation of immunoassays. For example, the development of continuous-flow immunoassays.
- Multiplex immunoassay technologies have been also developed [Luminex xMAP technology (LMX) and the Meso Scale Discovery (MSD)].
Future challenges are focused on developing less labor-dependent work and time-saving protocols. Improvement of antibody performance (for instance, sensitivity) is also a key strategy for better results. The artificial intelligence (AI) and the development of bioinformatics tools will be also critical in data analysis. Their outcomes in other domains are already outstanding!
- Amino N, et al. [Various types of immunoassay]. Nihon Rinsho. 1995 Sep; 53(9):2107-11.Japanese.PMID: 7474366.
- Cox KL, et al. Immunoassay Methods. 2012 May 1 [Updated 2019 Jul 8]. Bookshelf URL: https://www.ncbi.nlm.nih.gov/books
- Maarten Bloemen. Immunomagnetic separation of bacteria by iron oxide nanoparticles, Thesis, May 2015.
- Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497, 1975.
- Leavy, O. The birth of monoclonal antibodies. Nat Immunol 17, S13, 2016.
- Jose L Sanchez-Trincado el al, Fundamentals and methods for T- and B- cell Epitope prediction, Journal of immunology research, 2017.
- Jane Zveiter Moraes, Hybridoma technology: is it still useful? Current Research in Immunology, Volume 2, 2021.