Introduction
All vertebrates possess an advanced immune system, with mammals having the most complex and highly specialized form. The immune system provides protection against infections and foreign substances through coordinated actions of specialized cells and molecules.
The main cells responsible for immune defense are B-lymphocytes and T-lymphocytes. When foreign substances (called antigens) enter the body, B-cells release antibodies — proteins that specifically recognize and bind to these antigens. This antigen–antibody interaction forms the basis for various diagnostic tests known as Immunochemical Techniques.
Immunochemical techniques rely on the selective, reversible, and non-covalent binding of antigens by antibodies. These reactions are used to detect or quantify antigens or antibodies in biological samples.
What Are Immunochemical Techniques?
Immunochemistry is a branch of immunology that studies the chemical aspects of immune reactions, particularly the interactions between antigens and antibodies. These techniques are powerful tools in diagnostic biochemistry and clinical laboratories, as they can detect even minute amounts of biological molecules that change during disease states such as cancer or infection.
Common Immunochemical Techniques:
- Immunoassay
- Radioimmunoassay (RIA)
- Enzyme-Linked Immunosorbent Assay (ELISA)
- Immunoprecipitation
- Immunoelectrophoresis
- Immunofluorescence
- Immunohistochemistry
Each of these techniques exploits the high specificity of the antigen–antibody reaction for diagnostic or research purposes.
Radioimmunoassay (RIA)
Radioimmunoassay (RIA) is one of the earliest and most sensitive immunochemical techniques. It was developed in 1959 by Solomon Berson and Rosalyn Yalow (who later won the Nobel Prize) for the measurement of insulin in human serum.
Principle:
RIA combines the principles of radioactivity and immunological reactions. It is based on a competitive binding reaction between radioactively labelled and unlabelled antigens for a limited number of antibody binding sites.
The process involves:
- The antibody is prepared by injecting the antigen (e.g., insulin) into an animal such as a rabbit or goat.
- The patient’s sample (containing unlabelled antigen) is mixed with a known quantity of radioactively labelled antigen (commonly using 131I).
- Both labelled and unlabelled antigens compete to bind to the specific antibody.
- As the concentration of unlabelled antigen increases, fewer labelled antigen–antibody (Ag*–Ab) complexes form.
- The bound antigen–antibody complex is separated, and its radioactivity is measured.
The amount of radioactivity detected is inversely proportional to the concentration of the unlabelled antigen in the sample. A standard curve prepared with known antigen concentrations allows quantification of the unknown sample.
Applications of RIA:
- Detection and quantification of hormones (e.g., insulin, thyroxine, cortisol).
- Measurement of peptides, vitamins, and drugs present in very low concentrations (nanogram or picogram levels).
- Used in the diagnosis of hormonal disorders, cancers, and monitoring therapeutic drug levels.
- Biomedical research for studying antigen–antibody interactions.
RIA has now been largely replaced by non-radioactive methods like ELISA due to safety and environmental concerns associated with radioisotopes.
Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA (Enzyme-Linked Immunosorbent Assay) is a non-radioactive alternative to RIA. It uses enzymes instead of radioactive isotopes to detect antigen–antibody interactions. ELISA is equally sensitive and safer, as it avoids radiation hazards.
Principle:
ELISA is based on the specific binding between an antigen and its corresponding antibody. The enzyme linked to either the antigen or antibody reacts with a substrate to produce a measurable coloured product, indicating the presence of the target molecule.
Steps in ELISA:
- Coating: The antibody specific to the target protein (antigen) is attached to a solid surface, usually polystyrene microplates.
- Binding: The biological sample containing the antigen is added, allowing the antigen to bind to the immobilized antibody.
- Detection: A second enzyme-linked antibody specific to the antigen is added. This forms a sandwich complex (antibody–antigen–enzyme-linked antibody).
- Washing: Unbound components are washed away.
- Substrate Reaction: A suitable substrate (chromogenic reagent) is added. The enzyme (commonly peroxidase, alkaline phosphatase, or amylase) acts on it to produce a coloured product.
- Measurement: The intensity of the colour developed is directly proportional to the amount of antigen present in the sample.
The colour intensity is measured using a spectrophotometer or ELISA reader.
Applications of ELISA:
- Detection of small quantities of proteins, hormones, and antibodies.
- Commonly used for the pregnancy test, detecting human chorionic gonadotropin (hCG) in urine.
- Diagnosis of AIDS by detecting antibodies against the HIV virus.
- Used in screening tests for hepatitis, COVID-19, and other infectious diseases.
- Applied in food industry for allergen and toxin detection.
Comparison Between RIA and ELISA
| Feature | RIA | ELISA |
|---|---|---|
| Detection Method | Uses radioisotopes (e.g., 131I) | Uses enzyme-linked colour reactions |
| Safety | Radiation hazard present | Safe and non-radioactive |
| Sensitivity | Very high (picogram range) | High (nanogram range) |
| Equipment Required | Gamma counter | ELISA reader/spectrophotometer |
| Applications | Hormones, drugs, vitamins | Antibodies, antigens, hormones |
Detailed Notes:
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