Antibodies are small protective proteins produced by our immune system in response to disease or the presence of an antigen. It is also known as immunoglobulin.
In mammals, five isotypes of antibodies are found, which include IgA, IgG, IgD, IgE, and IgM.
For decades, these molecules have helped biomedical and life science researchers understand many diseases and develop targeted therapies. In addition, research-grade antibodies are fundamental in the research labs for detection studies.
The assays that primarily involve the use of antibodies include western blotting, ELISA, and other immunoassays. In some assays, such as western blotting, two types of antibodies—primary and secondary antibodies—are used to detect the target protein with high specificity.
Primary antibodies bind specific antigens, such as peptides, proteins, or other small molecules. Whereas, secondary antibodies carry fluorophore or radioactive labels and bind to the primary antibody.
After the primary antibody binds via its V region to the target protein, the labeled secondary antibody binds to the C region of the primary antibody using its V region.
A range of conjugates fluorescent dyes, such as fluorescein isothiocyanate (FITC) and Alexa Fluor dyes; or enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) are available, designed for specific lab workflow.
In this article, we are going to elaborate on the primary antibodies, including their functions, uses in lab workflows, and the industries that extensively use them.
Based on the means of antibody production, the primary antibodies are categorized into two groups: Monoclonal antibodies and Polyclonal antibodies.
Monoclonal antibodies recognize only a single antigen and bind to a specific epitope, reducing cross-reactivity. One example of such antibodies are epitope-tag antibodies.
Monoclonal antibodies are derived from a single cell. An antigen is injected into a host animal to initiate an immune response. Then, cells (mostly spleen cells) are isolated from these organisms and cultured with the malignant myeloma cells in lab conditions to form hybrid antibody-expressing cells, called hybridomas.
Hybridomas have B-cell properties, which enable them to synthesize and secrete genetically homogeneous antibodies targeted against a single epitope of an antigen (called monoclonal antibodies).
Various monoclonal antibodies are available, named after their isolation from a particular host, such as rabbit monoclonal antibody, mouse monoclonal antibody, and rat monoclonal antibody.
Polyclonal antibodies bind to multiple epitopes of a single antigen. These antibodies are produced from different lineages of B-cells of an animal. Thus, they are heterogeneous and bind to more than one epitope of an antigen.
The polyclonal antibodies are produced by injecting an antigen into a mammal, such as a rabbit, mouse, or goat. As a result, a specific antibody (mainly IgG) is produced against the antigen, which is purified from the animals’ serum.
Primary antibodies are commercially custom-prepared based on the workflow they are suitable for or to achieve specific experimental targets. Here’s an example of two such antibodies:
Antibodies conjugated to a range of fluorophores are available to cater to specific flow cytometry workflow requirements. They are suitable to identify cells and detect activation states, proliferation, cytokine release, and homing profiles.
Recombinant antibodies contain identical antibody chains that target the same epitope. They are obtained from a specific animal’s monoclonal antibody-producing cell lines. They provide superior lot-to-lot reproducibility, sensitivity, and specificity than a standard antibody.
Primary antibodies are essential to detect biomarkers for several diseases, such as Parkinson’s, diabetes, cancer, and Alzheimer’s disease. Additionally, they are also useful in studying the multi-drug resistance (MDR) and absorption, distribution, metabolism, and excretion (ADME) of therapeutic agents.
These studies are done using many lab workflows, some of which are given below:
Western blot is a routine technique used in labs to identify proteins from a crude mixture or sample of tissue homogenates and detect protein modifications. In this process, proteins are blotted on a membrane followed by their incubation with primary antibodies.
Then, excess proteins are washed away and the membrane is incubated with secondary antibodies for protein detection.
ELISA stands for enzyme-linked immunosorbent assay. It’s an analytical biochemical assay, which is used to detect the presence of a ligand in a biological sample using antibodies.
In this method, a solution containing antigen to be tested is added to the wells of a microtiter plate. Then, a primary antibody conjugated with an enzyme is added, which binds to the antigen. Followed by this, the substrate for the conjugated enzyme is added to the plate which helps to evaluate the presence of antigens or specific antibodies.
Immunological techniques are types of experimental procedures that use the antigen-antibody interaction to measure, induce, and characterize immune responses. The types of immunological techniques include immunohistochemistry (IHC), immunofluorescence, immunoprecipitation, and immunocytochemistry (ICC).
These techniques involve the use of primary and secondary antibodies to analyze the concentration of the target protein in the given sample, localization of a protein of interest, and study immune response.
Primary antibodies have tremendous applications in research, therapeutic, and diagnostic areas. It serves a crucial role in the detection and analytical studies performed in the labs.
Primary antibodies play an important role in identifying disease markers for many diseases such as Parkinson’s, cancer, Hepatitis-C (HCV), and Alzheimer’s disease.
During COVID-19 (a disease caused by the SARS-CoV-2 virus) pandemic, these molecules were extensively used to diagnose the disease, develop vaccines, and discover potential drugs for its eradication.
Research labs performing a range of workflows, such as western blotting, immunological assays, and ELISA considerably require the availability of primary antibodies.
They help in the interpretation of and validation of workflow results, and epigenetic research, analyze cell-signaling pathways and facilitate the expression and purification of recombinant proteins.
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Primary antibodies are one of the essential reagents used in research and diagnostic laboratories. They help in workflows like western blotting, immunohistochemistry, immunoprecipitation, immunocytochemistry, and ELISA to study proteins, including their localization, concentration, and purification.
Based on the means of production, antibodies are of two types: monoclonal antibodies that bind to a single epitope of an antigen, and polyclonal antibodies that bind to multiple epitopes of a single antigen.
Premium quality antibodies are crucial to carrying out the workflows to ensure excellent performance and reproducibility, resulting in high-quality data. Moreover, the high-quality reagents paired with high-throughput equipment also help to achieve high specificity, reliability, and reproducibility in an assay to enhance the data quality.
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