Antibody based diagnosis, methods, application and limitation

Antibody based diagnosis refers to diagnostic methods that utilize antibodies to detect the presence of specific pathogens (such as viruses, bacteria, or fungi) or abnormal biomarkers (such as proteins or other molecules) in a patient’s biological samples (e.g., blood, urine, saliva, or tissue).

These diagnostic techniques utilize the specific binding capabilities of antibodies to their target antigens, making them powerful tools for detecting a wide range of diseases and conditions.

Antibody-based diagnostic tests are commonly used in clinical diagnostics, research, and in fields like immunology, oncology, and infectious disease monitoring.

Figure : Representation of and antibody based diagnosis in the first (A) reaction it shows presence of antigen of target pathogen and in the B slide shows reaction when no pathogen present ( means no enzymatic reaction and no bands in the slide)

Before we go further in this topic we need to have basic understanding of the following terms-

Antibodies and Antigens:

Antibodies also known as immunoglobulins are proteins produced by the immune system, specifically B lymphocytes (a type of white blood cell). They recognize and bind to antigens, which are foreign molecules (like pathogens or abnormal proteins) that trigger an immune response.

The specificity of antibodies for their antigens is the basis for antibody-based diagnostic tests, as the interaction between antibodies and antigens is highly selective.

Figure- An antigen (triangle) and its specific antibody (Y shape) molecule

Principles of Antibody-Based Diagnostics:

The diagnostic tests typically use antibodies that have been custom-designed or selected to recognize specific antigens associated with a disease or condition.

These antibodies are linked to a detection system (e.g., a color change, fluorescence, or enzyme activity) to signal the presence of the target antigen in the sample.

Table of Contents

Types of Antibodies Used Antibody based diagnosis

Monoclonal Antibodies (mAbs): These antibodies are derived from a single clone of B cells and are highly specific for a single epitope (a particular region of the antigen). They are widely used in diagnostic tests due to their precision.

Polyclonal Antibodies: These are a mixture of antibodies from different B cell clones, and they can recognize multiple epitopes on the same antigen. They are used less frequently in diagnostics but are still used for some applications.

Chimeric or Humanized Antibodies: These antibodies combine parts from different species (e.g., human and mouse) to reduce immune reactions in human patients.

Figure – Showing different types of based on source of antibody

Common Types of Antibody Based Diagnosis Tests

Enzyme-Linked Immunosorbent Assay (ELISA) for Antibody based diagnosis

ELISA is one of the most widely used antibody-based diagnostic techniques. It is used to detect and quantify soluble substances such as proteins, hormones, or antibodies in blood or other fluids.

In a typical ELISA test, an antigen is immobilized on a solid surface (like a plate), and a sample is added. If antibodies against the antigen are present in the sample, they will bind to the antigen. A secondary antibody, usually conjugated to an enzyme, is then added to bind to the primary antibody. The enzyme produces a color change when a substrate is added, indicating the presence of the target antigen.

e.g. Detecting HIV, hepatitis, pregnancy (hCG), allergens, and various infections.

Western Blot for Antibody based diagnosis

Western blotting is a method used to identify specific proteins in a sample by separating proteins based on their size using gel electrophoresis, transferring them onto a membrane, and then detecting them with antibodies.

After proteins are transferred to the membrane, primary antibodies that bind to the target protein are applied. Secondary antibodies conjugated to a detectable label (such as an enzyme or a fluorescence tag) are used to visualize the protein bands.

Used for confirming the presence of specific proteins or antibodies, such as in HIV diagnosis or autoimmune disease testing.

Immunohistochemistry (IHC) for Antibody based diagnosis

IHC is a method used to detect specific antigens in tissue samples by using antibodies labeled with a visible marker (like a dye or enzyme).

Tissue sections are exposed to antibodies against the target antigen. The antibody binding is visualized under a microscope, indicating the presence and localization of the antigen in the tissue.

Commonly used in pathology to detect specific markers in cancer tissues, as well as for diagnosing infections or autoimmune diseases.

Rapid Diagnostic Tests (RDTs) for Antibody based diagnosis

Rapid tests are designed to provide quick, on-site results, often without the need for laboratory equipment. These tests use antibody-antigen interactions to produce visible results, typically in the form of a color change on a test strip.

The sample (e.g., blood, urine) is applied to a test strip that contains immobilized antibodies. If the target antigen is present in the sample, it will bind to the antibodies and produce a visible signal.

Pregnancy tests (hCG), malaria (Plasmodium antigens), influenza, COVID-19 (SARS-CoV-2 antigen or antibody), HIV, and other infectious diseases.

Immunofluorescence (IF):

Immunofluorescence uses antibodies labeled with fluorescent dyes to detect the presence of target antigens in cells or tissues.

The primary antibody binds to the antigen in the sample, and the secondary antibody is tagged with a fluorescent dye. When exposed to specific wavelengths of light, the fluorescent tag emits light, which can be detected under a fluorescence microscope.

Used for detecting pathogens (e.g., viruses or bacteria) in clinical samples, autoimmune diseases, and cellular research.

Advantages of Antibody Based Diagnostics

High Sensitivity and Specificity: Antibodies can be highly specific to their target antigens, allowing for accurate detection of even low levels of pathogens or disease markers. This is crucial for detecting early stages of disease.

Non-invasive or Minimally Invasive: Many antibody-based tests can be performed using simple biological samples, such as blood, urine, or saliva, avoiding the need for invasive procedures like biopsies.

Rapid Results: Rapid diagnostic tests, such as lateral flow assays or ELISA, can produce results quickly, often within minutes to hours, making them valuable for urgent diagnoses.

Portable and Easy to Use: Many antibody-based tests are easy to perform in non-laboratory settings (e.g., at a doctor’s office or in remote areas), providing point-of-care diagnostic solutions.

Cost-Effective: Some antibody-based tests (especially rapid tests) are relatively inexpensive, making them accessible for widespread use, especially in resource-limited settings.

Limitations of Antibody-Based Diagnostics

Cross-reactivity: Antibodies may sometimes cross-react with non-target antigens, leading to false positives or false negatives. This can reduce the reliability of some tests.

Sensitivity Issues: Some antibody-based tests may not be sensitive enough to detect low levels of antigens or may fail to detect infections or diseases at early stages.

Need for Proper Sample Collection: Accurate results depend on the quality and type of sample collected. Improper sample collection or handling can lead to inaccurate or invalid results.

Limited to Detecting Known Antigens: Antibody-based tests can only detect what they are designed to recognize. If a novel pathogen or a new biomarker emerges, new tests need to be developed to identify it.

Laboratory Equipment: Some antibody-based tests, like ELISA or Western blot, require specialized laboratory equipment and trained personnel, limiting their use in low-resource or field settings.

Applications in Disease Diagnosis

Infectious Diseases: – Antibody-based diagnostics are widely used for detecting infections caused by viruses, bacteria, fungi, and parasites. Examples include tests for HIV, hepatitis, malaria, COVID-19, syphilis, influenza, and tuberculosis.

Cancer Diagnosis: Certain antibodies can detect tumor markers that are present in cancerous tissues. For example, HER2 antibodies are used in breast cancer diagnosis, and PSA (prostate-specific antigen) tests are used to detect prostate cancer.

Autoimmune Diseases– Tests like antibody screening for lupus (e.g., antinuclear antibodies, or ANA) help diagnose autoimmune diseases where the immune system attacks the body’s own tissues.

Pregnancy Tests: Pregnancy tests detect the presence of the human chorionic gonadotropin (hCG) hormone, which is produced after implantation of the fertilized egg in the uterus.

Allergy Testing: Antibody-based tests are used to identify specific allergens causing allergic reactions by measuring IgE antibodies against known allergens.