The Role of Antibodies in Diagnostics

Antibody Structures & Functions: Different Types of Antibodies

Our bodies naturally produce antibodies as part of the adaptive immune response to invading pathogens. However, with advancements in biomedical science and technology, we now can engineer antibodies to bind specific targets for a range of intended applications. 

This involves producing antibodies in a desired animal species or genetically modifying the genetic sequence of antibodies to build customized antibodies that bind an intended target with high precision. These engineered antibodies can be separated into three different categories based on how the antibodies are made.

Monoclonal Antibodies

A single B cell specializes in making large amounts of a unique antibody. That antibody is designed to bind only one region (epitope) on a target molecule (antigen). There are specific antibody production techniques that allow scientists to make large amounts of a single unique antibody that targets a specific epitope on an antigen. 

They do this by either isolating the individual B cell and making clones (hybridoma method) or isolating the genetic sequence of the antibody (antibody phage display and B cell sorting) from the B cell. Either technique allows for the mass production of an antibody from a single B cell clone (i.e., monoclonal). 

Therefore, monoclonal antibodies consist of identical antibody molecules that can exhibit remarkable specificity and affinity for an antigen, underscoring their clinical use.

Polyclonal Antibodies

When the adaptive immune system recognizes a specific threat, like a toxin, numerous B cells collectively produce antibodies that bind the toxin and protect the organism. However, each antibody binds a unique epitope (region) on the toxin (antigen). Together, the antibodies collaboratively bind the whole toxin molecule and neutralize the threat. 

Therefore, a polyclonal antibody is a mixture of antibodies derived from different B cells that are intended to bind to the same target. This makes polyclonal antibodies ideal for diagnostic applications because patients are genetically distinct. Therefore, the intended target (antigen) in a patient population will vary genetically, biochemically, and/or structurally.

Recombinant Antibodies

The DNA sequence of an antibody can be genetically engineered to give the antibody different features. This includes binding specificity to an intended target, improved binding strength, compatibility in humans (humanization), making small fragments of an antibody, or making a different class of antibody with specific immunological features.

Which Type of Antibodies Are Preferred for Therapeutic and Diagnostic Use?

The introduction of monoclonal antibodies in 1975 marked a significant turning point. Before this breakthrough by Köhler and Milstein, immunoassays primarily relied on polyclonal antisera sourced from immunized rabbits. Using serum from immunized rabbits without separating non-specific antibodies or proteins offered several limitations, including:

• Limited supply: The production of antisera from immunized rabbits involves the sacrifice of animals, which may raise ethical concerns and limit the supply of antisera available for use in immunoassays.
• Batch-to-batch variability: Antisera produced in rabbits can exhibit batch-to-batch variability in terms of antibody titer, specificity, and affinity. This variability can impact the reproducibility and reliability of immunoassays conducted using antisera from different batches.
• Cross-reactivity: Antisera from immunized rabbits may exhibit cross-reactivity with antigens other than the intended target, leading to false-positive or nonspecific results in immunoassays. This can be particularly problematic when analyzing complex samples containing multiple antigens.
• High background noise: Antisera from rabbits may contain endogenous antibodies or other proteins that contribute to high background noise in immunoassays, reducing the sensitivity and specificity of detection.
• Interference from immunoglobulins: The presence of immunoglobulins (e.g., IgG, IgM) in rabbit antisera can interfere with detection methods such as enzyme-linked immunosorbent assay (ELISA) or Western blotting, leading to inaccurate quantification or false interpretation of results.
• Limited compatibility: Rabbit antisera is not compatible with certain immunoassay formats or detection systems designed for use with antibodies derived from other species (e.g., mouse, goat). This can limit the flexibility and versatility of immunoassays in experimental settings.
• Immunogenicity: When used in diagnostic or therapeutic applications in humans, antisera from immunized rabbits may elicit immune responses, leading to allergic reactions or other adverse effects.

Therefore, Köhler and Milstein’s pioneering work accelerated the clinical use of antibodies. Their method involved fusing B cells with myeloma cells, resulting in a novel cell hybrid capable of not only producing antibodies but also proliferating rapidly, allowing a single antibody-producing B cell (mono) to be copied into identical B cell clones (clonal) each making the same antibody, hence the term monoclonal antibody. 

This hybridoma cell line technology allowed scientists to produce batches of identical antibodies without the need for repeated animal immunization. This innovative approach ushered a new era in antibody technology, catalyzing advancements in both therapy and diagnostics. 

Monoclonal antibodies quickly gained prominence and have since become indispensable in a vast array of clinical applications, playing pivotal roles in accelerating biomedical research, industry, agricultural sciences, immunotherapies, and immunodiagnostics.

What Is the Role of Antibodies in Diagnostics?

The widespread application of mass-produced monoclonal antibodies in clinical settings has greatly benefited various medical fields, including diagnostics, therapeutics, and targeted drug delivery. These antibodies play a crucial role in diagnosing a wide range of human illnesses, such as cancer, metabolic disorders, hormonal imbalances, infectious diseases like pneumonia, and even infertility. 

What Properties of Antibodies Make Them Useful for Diagnostic Tests?

Antibodies bind target antigens with precision. This makes antibodies ideal for detecting specific molecules produced by pathogens during infectious diseases or biomarkers of chronic diseases like autoimmunity and cancer. But that’s not all.

Diagnostic tests can leverage the binding specificity of our own antibodies to identify antibodies that bind pieces of viruses, bacteria, or parasites, suggesting past exposure to a pathogen and helping determine disease history. Diagnostic antibodies also allow for precise spatial, temporal, and disease burden characteristics, providing information about disease progression.

Among the various substances utilized in diagnostics and medical imaging, antibodies stand out as the sole agents possessing enough sensitivity to identify post-translational modifications (PTMs) of target molecules. This underscores the potential of developing anti-PTM antibodies to detect particular disease-related modifications at the earliest stages, which can provide health professionals with more information needed to make an accurate prognosis.

How Are Diagnostic Antibodies Produced?

Antibody-driven diagnostic methods are highly effective tools for promptly and precisely identifying disease biomarkers. These tests have undergone various adaptations to meet the increasing need for dependable, rapid, and sensitive instruments, particularly for clinical applications.

In response to this demand, numerous diagnostic assays have emerged. Many of these assays utilize antibodies produced via hybridoma technology. Hybridoma technology stands out as a method for producing antibodies for diagnostics because these hybrid cell lines offer a reliable and well-established approach and an unlimited source of MAb, known for efficiently generating high-quality antibodies at a reasonable cost.

The in vivo affinity maturation process saves time by eliminating the need for additional engineering of these reagents before use. Poteogenix’s hybridoma development process for diagnostic applications offers a comprehensive range of application-guaranteed packages, ensuring your antibodies maintain their high affinity, specificity, and stability in your unique assay format and conditions.

Poteogenix’s hybridoma development process for diagnostic applications offers a comprehensive range of application-guaranteed packages, ensuring your antibodies maintain their high affinity, specificity, and stability in your unique assay format and conditions.

Antibody-Based Diagnostics

Many clinical applications rely on antibodies. Antibody-based immunoassays are the most commonly used diagnostic assays. Antibody screening tests can detect the presence of specific antibodies in a patient’s blood cells.

For example, enzyme-linked immunosorbent assays (ELISAs) use antibodies to identify pathogens like HIV or Hepatitis Viruses. Immunohistochemistry (IHC) is often used to diagnose tissue abnormalities in diseases such as cancer. Let’s break it down.

Antigenic Characterization

Antibodies help identify and characterize antigens. For instance, binding to specific epitopes reveals the presence or absence of a pathogen or pathogen components and, depending on the antibody, unique post-translational modifications or antigen variations of a pathogen. This information aids in diagnosis and disease treatment.

Flow Cytometry

Flow cytometry uses fluorescently labeled antibodies to identify and characterize cells in a heterogeneous population. This helps medical professionals reliably diagnose blood disorders, identify cancerous cells, and monitor treatment responses.

Western Blotting

In Western blotting, protein samples are denatured and run through a polyacrylamide gel that separates proteins based on size using a method called gel electrophoresis. The proteins are then removed from the three-dimensional gel and transferred to a two-dimensional membrane. 

The proteins “blotted” onto a membrane (a piece of special paper) are incubated in the presence of an antibody used to detect the protein and measure its relative abundance. This technique is essential for diagnosing diseases like HIV and Lyme disease.

Serological Tests (Blood Tests)

Serological tests detect antibodies in the patient’s serum. These tests help diagnose infections such as dengue fever, hepatitis B, HIV/AIDS, syphilis, and more. For example, since 2012, we have the HIV antibody test for home use. Unlike traditional blood-based tests, this diagnostic tool uses saliva samples, delivering results within around 20 minutes.

Antibodies for Characterization and Diagnosis of Diseases

From rapid diagnostic kits to sophisticated laboratory assays, the applications of antibody-based tests span a wide spectrum of medical specialties. What exactly can be detected using antibody-based tests?

The answer encompasses a wide range of conditions, reflecting the remarkable adaptability and effectiveness of this diagnostic approach.

Infectious Diseases

Specific antibody-based tests rapidly identify the pathogens associated with exposure to disease. Antibodies are central to diagnosing infectious diseases. For instance, detecting antibodies against SARS-CoV-2 helps confirm the disease COVID-19. Unilike polymerase chain reaction (PCR) tests, antibody diagnostics are more reliable resulting in rapid detection of the pathogen.

Autoimmune Diseases

Autoimmune diseases occur when the immune system attacks healthy tissues. Antibody-based tests help diagnose conditions such as rheumatoid arthritis and lupus using purified antigens. These tests are conducted in conjunction with X-rays, various imaging procedures, and biopsies to aid in the diagnosis of autoimmune conditions.

They serve multiple purposes, including assessing the severity of the disorder, tracking disease progression, and evaluating the efficacy of treatments. Autoimmune tests involve immobilizing the targets of autoantibodies and incubating the patient’s serum. If autoantibodies are present, they will bind the autoantigen. 

A second antibody, commonly fused with a detection molecule, is used to bind the conserved region of the human autoantibody to detect and measure its abundance. This includes but is not limited to, detecting autoantibodies like anti-dsDNA, anti-RNP, anti-Smith (or anti-Sm), anti-Sjogren’s SSA and SSB, anti-scleroderma or anti-Scl-70, anti-Jo-1, and anti-CCP, and anti-cardiolipin.

Cancer

Monoclonal antibodies play a diverse role in both the treatment and diagnosis of cancer. Researchers have identified numerous tumor-associated antigens circulating in the bloodstream, which serve as biomarkers for detecting cancer. These biomarkers can also be used to project disease severity and survival rates.

So, antibodies can be used to identify cancer-specific markers (tumor antigens), while therapeutic antibodies such as trastuzumab (Herceptin) target cancer cells directly, aiding in treatment.

Infertility

Purified antigens also play a role in the diagnosis of infertility. Anti-sperm antibodies (ASAs) are antibodies that target sperm antigens. They are considered autoantibodies in the males since they can bind self-antigens on the sperm cell. However, ASAs can also be present in women and are not classified as autoantibodies.

These antibodies can interfere with the sperm’s ability to move (motility), penetrate the cervical mucus, or fertilize an egg, potentially leading to fertility issues without impacting sperm count. Thus, detecting the presence of ASAs is one method used to diagnose infertility. Immunizing either males or females with sperm antigens is being explored as an irreversible contraceptive vaccine. 

While research into contraceptive vaccines has been ongoing for several decades, no contraceptive vaccine has yet been approved for general use. Many challenges remain in the development of effective and safe vaccines, including achieving the desired level of efficacy without causing adverse effects or unintended disruptions to the menstrual cycle or reproductive health.

Allergies

Purified antigens play a crucial role in diagnosing allergic diseases by identifying specific allergens triggering immune responses in individuals. Some people build an adaptive immune response to allergens by producing a specific type of antibody class called immunoglobulin E (IgE).

Diagnostic tests for allergies typically involve measuring the levels of allergen-specific IgE antibodies in the blood or through skin prick tests. Identifying these antigens can help the patient manage allergies effectively, whether through allergen avoidance, medications, or immunotherapy.

Diagnostic Antibodies as the Gateway to Effective Treatment

Antibodies serve as the cornerstone of clinical diagnostic tools, guiding us toward even more accurate, faster diagnoses and personalized treatments. Their versatility and specificity have paved the way for groundbreaking advancements in healthcare, enabling us to confront diseases with unprecedented precision. As we look ahead, we’ll continue to harness the potential of antibodies for a future where disease is detected early and treated effectively.

Experience the next level of antibody development with Proteogenix. Whether you’re looking to produce and develop antibodies or need assistance with engineering and characterizing them, our expert team is here to help, no matter the size or scope of your project.  

With a focus on optimization and validation tailored to your specific needs, our custom antibodies are meticulously crafted to deliver exceptional performance, ensuring accurate and reliable results every time. Let’s work together – book a call today.