Immunochemical Techniques medical laboratory

This topic is for medical laboratory students

1. Unit 3: Immunochemical Techniques: Introduction, Principle and applications of following technologies: o Enzyme linked immunosorbent assay (ELISA) o Radioimmunoassay (RIA) o Chemiluminescence immunoassay (CLIA) o Electrochemiluminescent immunoassay (ECLIA)

2. Enzyme linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay (ELISA) is a plate-based technique used to detect and quantify soluble substances like proteins .  It relies on the highly specific interaction between antigens and antibodies .  In an ELISA, the target antigen is immobilized on a solid surface, either directly or through a capture antibody .  Enzyme-conjugated antibodies are then used to detect and quantify the antigen, with the enzyme producing a measurable colored product upon substrate addition. 

3. Principle Antigens and antibodies react specifically to form the Ag- Ab complex. Antibodies can be linked or attached to enzymes. The enzyme-linked antibodies can modify the specific substrates used to produce a color change within the preparation . The enzyme activity is measured with a colorimeter in a specific wavelength of light to determine the magnitude of the infection in the patient . Types of ELISA Direct ELISA Indirect ELISA Sandwich ELISA Competitive ELISA

4. Applications It is one of the most sensitive and effective methods for the detection of different viral, bacterial, and fungal infections. Screening test for HIV infection. Different EISA test kits are available for the detection of dengue fever, TB, and Hepatitis B infections. Pregnancy test kits based on ELISA are also available. Qualitative and quantitative estimation of various proteins, hormones, toxins, etc. Also used in the detection of different food allergens.

5. Radioimmunoassay (RIA) Radioimmunoassay is one of the sensitive immunoassay techniques which helps in the determination of  antigens  or  antibodies  in a sample with the use of radioisotopes . It is an in vitro type of  antigen-antibody interaction . When radioisotopes instead of enzymes are used as labels to be conjugated with antigens or antibodies, the technique of detection of the antigen-antibody complex is called radioimmunoassay (RIA). Radioimmunoassay (RIA) is an  in vitro  assay that measures the presence of an antigen with very high sensitivity.  The classical RIA methods are based on the principle of competitive binding. In this method, an unlabeled antigen competes with a radiolabeled antigen for binding to an antibody with the appropriate specificity. Thus, when mixtures of radiolabeled and unlabeled antigen are incubated with the corresponding antibody, the amount of free (not bound to antibody) radiolabeled antigen is directly proportional to the quantity of unlabeled antigen in the mixture.

6. Principle Antigens and antibodies bind specifically to form the Ag- Ab complex. The antigen can be labeled or conjugated with radioisotopes. The unlabeled antigens from the sample compete with radiolabeled antigens to bind on paratopes of specific antibodies . The unlabeled antigens replace labeled antigens that are already linked with the antibodies. The unlabeled antigens when bind with antibodies, increases the amount of free radiolabeled antigens in the solution . Hence the concentration of free labeled antigens is directly proportional to the bound unlabeled antigens. It involves a combination of three principles. 1. An immune reaction i.e.  antigen , antibody binding. 2. A competitive binding or competitive displacement reaction. (It gives specificity) 3. Measurement of radio emission. (It gives sensitivity)

7. Immune Reaction When a foreign biological substance enters the body’s bloodstream through a non-oral route, the body recognizes the specific chemistry on the surface of the foreign substance as antigen and produces specific antibodies against the antigen so as nullify the effects and keep the body safe. The antibodies are produced by the body’s immune system so, it is an immune reaction. Here the antibodies or antigens bind and move due to chemical influence. This is different from the principle of electrophoresis where proteins are separated due to charge.

8. Competitive binding or competitive displacement reaction This is a phenomenon wherein when there are two antigens that can bind to the same antibody, the antigen with more concentration binds extensively with the limited antibody displacing others . So here in the experiment, a radiolabelled antigen is allowed to bind to a high-affinity antibody. Then when the patient serum is added to unlabeled antigens it starts binding to the antibody displacing the labeled antigen.

9. Measurement of radio emission Once the incubation is over, then washings are done to remove any unbound antigens. Then radio emission of the antigen-antibody complex is taken, and the gamma rays from the  radiolabeled antigen are measured. The target antigen is labeled radioactively and bound to its specific antibodies (a limited and known amount of the specific antibody has to be added). A sample, for e.g. blood serum, is added in order to initiate a competitive reaction of the labeled antigens from the preparation, and the unlabeled antigens from the serum sample, with the specific antibodies. The competition for the antibodies will release a certain amount of labeled antigen. This amount is proportional to the ratio of labeled to an unlabeled antigen. 

10. A binding curve can then be generated which allows the amount of antigen in the patient’s serum to be derived. That means as the concentration of unlabeled antigen is increased, more of it binds to the antibody, displacing the labeled variant. The bound antigens are then separated from the unbound ones, and the radioactivity of the free antigens remaining in the supernatant is measured. Antigen-antibody complexes are precipitated either by crosslinking with a second antibody or by means of the addition of reagents that promote the precipitation of antigen-antibody complexes. Counting radioactivity in the precipitates allows the determination of the amount of radiolabeled antigen precipitated with the antibody. A standard curve is constructed by plotting the percentage of antibody-bound radiolabeled antigen against known concentrations of a standardized unlabeled antigen, and the concentrations of antigen in patient samples are extrapolated from that curve. The  extremely high sensitivity  of RIA is its  major advantage .

11. Applications It was first used for the detection of peptide hormones. Detection of different viral antigens Detection of many hormones and drugs Detection of Hepatitis B surface antigens Detection of mycotoxins Detection of the early stage of cancer

12. Chemiluminescence immunoassay (CLIA) Chemiluminescence Immunoassay (CLIA) is a highly sensitive and specific laboratory technique used to detect and quantify specific substances like hormones, antibodies, and proteins in biological samples.  It utilizes the emission of light during a chemical reaction ( chemiluminescence ) to determine the concentration of the target analyte .  CLIA combines the sensitivity of chemiluminescence with the specificity of immunoassays, making it a powerful tool in clinical diagnostics. 

13. P rinciple Chemiluminescence Immunoassay (CLIA) is an analytical technique that combines the principles of immunoassays with chemiluminescence to detect and quantify specific substances in a sample.  It works by using antibodies or antigens labeled with a chemiluminescent compound to bind to the target analyte .  When a substrate is added, it reacts with the label, producing light that is then measured.  The intensity of the light is directly proportional to the amount of the analyte present in the sample. 

14. A pplications 1. Clinical Diagnosis : Infectious Diseases: CLIA is used to detect pathogens like viruses and bacteria by identifying specific antigens or antibodies in patient samples. Hormone Levels: It accurately measures hormone imbalances, including reproductive, thyroid, and adrenal hormones. Tumor Markers: CLIA plays a crucial role in screening and monitoring tumor markers, aiding in early cancer detection and management. Autoimmune Diseases: CLIA helps diagnose and monitor autoimmune disorders by detecting relevant antibodies. Cardiac Markers: CLIA is used to detect cardiac markers like troponin , aiding in the diagnosis of heart attacks. 

15. 2. Food Safety: CLIA helps detect foodborne pathogens and toxins, ensuring food safety and preventing outbreaks. It can be used to detect allergens in food products.  3. Environmental Monitoring: CLIA is employed to detect pollutants and contaminants in environmental samples, such as water and soil.  4. Pharmaceutical Analysis: CLIA is used in drug discovery and development for analyzing drug concentrations and metabolites. It helps monitor drug levels in patients to ensure therapeutic efficacy and safety.  5. Other Applications: Research: CLIA is a valuable tool in life science research for various applications, including protein analysis and gene expression studies. Forensic Science: CLIA can be used in forensic investigations for detecting biological evidence. 

16. Electrochemiluminescent immunoassay (ECLIA) Electrochemiluminescence immunoassay (ECLIA) is a highly sensitive and widely used technique for quantifying specific substances, particularly in clinical diagnostics.  It combines the principles of immunoassays with electrochemiluminescence , where light is emitted from a chemical reaction triggered by an electrical stimulus.  This method offers advantages like high sensitivity, speed, and the ability to control the light-emitting reaction, making it suitable for various applications, including detecting hormones, proteins, and other biomarkers. 

17. P rinciple It combines principles of electrochemistry and chemiluminescence to detect and quantify specific substances ( analytes ) in a sample.  It works by using antibodies to bind to the target antigen, triggering a light-emitting reaction when voltage is applied.  The intensity of the light is directly proportional to the amount of analyte present in the sample .

18. Application 1. Clinical Diagnostics : Endocrinology: ECLIA is a valuable tool for diagnosing and monitoring hormonal disorders, such as thyroid dysfunction, diabetes, and reproductive hormone imbalances.  Oncology: It plays a crucial role in detecting and monitoring cancer biomarkers, including prostate-specific antigen (PSA) for prostate cancer and other tumor markers.  Infectious Disease: ECLIA is used to detect antibodies and antigens related to various infectious agents, such as HIV, hepatitis viruses (A, B, and C), and tuberculosis.  Cardiovascular Disease: ECLIA can be used to measure cardiac markers like troponin , which are crucial for diagnosing and managing heart attacks and other cardiac events. 

19. 2. Other Applications: Biosensors: The high sensitivity and miniaturization capabilities of ECLIA make it suitable for developing biosensors for point-of-care testing and other applications. Drug Discovery and Development: ECLIA can be used to monitor drug levels and assess the efficacy of new drug candidates in preclinical and clinical studies. Food Safety: ECLIA can be adapted for detecting foodborne pathogens and allergens, ensuring food safety. Environmental Monitoring: ECLIA can be used to detect pollutants and contaminants in environmental samples.