Immunoassays PDF

DR JIMOH AK DEPT OF CHEMICAL PATHOLOGY IMMUNOASSAY COLLEGE OF MEDICINE AND HEALTH SCIENCES, S ABUAD  INTRODUCTION During most infectious diseases, antibodies and immune cells specific for the infecting agent (antigen) are produced. In other non-inf...

DR JIMOH AK DEPT OF CHEMICAL PATHOLOGY IMMUNOASSAY COLLEGE OF MEDICINE AND HEALTH SCIENCES, S ABUAD  INTRODUCTION During most infectious diseases, antibodies and immune cells specific for the infecting agent (antigen) are produced. In other non-infectious conditions, antibodies or immune cells are also formed. These can be detected and measured to aid in diagnosis and management of the condition. An antigen is defined as any substance when introduced into the body, stimulates the production of an antibody with which it reacts specifically and in a manner that reaction is observable. Antibodies are specific substances in the bodies of the vertebrate secreted in the tissue fluids from the lymphoid cells that have been stimulated by foreign substances (antigens) with which they react specifically. The interaction of the antigen and antibody is a chemical reaction and is specific. Immunoassay is a term used to describe assays, or tests, using immunological reagents such as antigens and antibodies.  Principle of Immunological Techniques When antigens and corresponding antibodies are allowed to react in gels or other mediums, they will diffuse toward one another, and at the point in which they meet in optimal proportions, they will form a visible precipitate. Antigen (soluble) + Antibody (soluble) → Ag-Ab complex (insoluble) Some Immunological Test in Immunology Unit of Clinical Chemistry Laboratory 1. Agglutination reaction 5. Immunoflourescence 2. Heamagglutination test 6. Enzyme Linked 3. Complement fixation test Immunosorbent Assay 4. Precipitation reaction (ELISA) (i) Oudin tube method 7. Radio immunoassay (RIA) (ii) Capillary tube method (iii) Ochterlony (Double radial diffusion) (iv) Macini (single radial diffusion) (v)Immunoelectrophoresis  IMMUNOASSAYS An immunoassay (IA) is a biochemical test that measures the presence or concentration of a macromolecule or a small molecule in a solution through the use of an antibody (usually) or an antigen (sometimes) It uses the principle of immunological method by the binding of antibodies to antigens Immunoassays come in many different formats and variations. Immunoassays may be run in multiple steps with reagents being added and washed away or separated at different points in the assay. Multi-step assays are often called separation immunoassays or heterogeneous immunoassays. Some immunoassays can be carried out simply by mixing the reagents and samples and making a physical measurement. Such assays are called homogeneous immunoassays, or less frequently non-separation immunoassays.  Principle of Immunoassay Immunoassays rely on the ability of an antibody to recognize and bind a specific macromolecule in a mixture of macromolecules. In immunology the particular macromolecule bound by an antibody is referred to as an antigen and the area on an antigen to which the antibody binds is called an epitope. The degree of binding is an important consideration in an immunoassay. The binding of an Ab to an Ag is directly related to the affinity and avidity of the Ab for the epitope, as well as the concentration of the Ab and epitope. An immunoassay may use an antigen to detect for the presence of antibodies, which recognize that antigen, in a solution.  Principle of Immunoassay In addition to the binding of an antibody to its antigen, the other key feature of all immunoassays is a means to produce a measurable signal in response to the binding. This involve chemically linking antibodies or antigens with some kind of detectable label. A large number of labels exist which allow for detection through different means. Many labels are detectable because they either emit radiation, produce a color change in a solution, fluoresce under light, or can be induced to emit light. Labels Immunoassays employ a variety of different labels to allow for detection of antibodies and antigens. Labels are typically chemically linked or conjugated to the desired antibody or antigen. Enzymes-The most popular labels use in immunoassays is enzymes. Immunoassays which employ enzymes are referred to as enzyme immunoassays (EIAs), of which enzyme-linked immunosorbent assays (ELISAs) and enzyme multiplied immunoassay technique (EMIT) are the most common type. Enzymes used in ELISAs include Horseradish peroxidase (HRP) Alkaline phosphatase (AP) Glucose oxidase These enzymes allow for detection by producing an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light or chemiluminescence. Radioactive isotopes-Radioactive isotopes can be incorporated into immunoassay reagents to produce a radioimmunoassay (RIA). Radioactivity emitted by bound antibody-antigen complexes can be easily detected using conventional methods. DNA reporters-A newer approach to immunoassays involves combining real-time quantitative polymerase chain reaction (RT qPCR) and traditional immunoassay techniques. Called real-time immunoquantitative PCR (iqPCR) the label used in these assays is a DNA probe Fluorogenic reporters-Fluorogenic reporters like phycoerythrin are used in a number of modern immunoassays. Protein microarrays are a type of immunoassay that often employ fluorogenic reporters. Electrochemiluminescent tags-Some labels work via electrochemiluminescence (ECL), in which the label LABEL IMMUNOASSAY AND DETECTION METHOD Label-free Immunoassays While some kind of label is generally employed in immunoassays, there are certain kinds of assays which do not rely on labels, but instead employ detection methods that do not require the modification or labeling the components of the assay. Surface plasmon resonance is an example of technique that can detect binding between an unlabeled antibody and antigens. Another demonstrated labeless immunoassay involves measuring the change in resistance on an electrode as antigens bind to it. Another method is the thin-film interference that uses a thin glass rod as a sensing probe to immunocomplex Classifications of Immunoassays Competitive, homogeneous immunoassays- unlabelled analyte in a sample competes with labelled analyte to bind an antibody. The amount of labelled, unbound analyte is then measured. In theory, the more analyte in the sample, the more labelled analyte gets displaced and then measured; hence, the amount of labelled, unbound analyte is proportional to the amount of analyte in the sample. Examples includes: Fluorescence polarization immunoassay (FPIA), Enzyme multiplied immunoassay technique (EMIT) Competitive, heterogeneous immunoassays- unlabelled analyte in a sample competes with labelled analyte to bind an antibody. In the heterogeneous assays, the labelled, unbound analyte is separated or washed away, and the remaining labelled, bound analyte is measured. Examples of Immunoassays Enzyme Immunoassay (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA) The Enzyme Immunoassay (EIA) or Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most widely used immunoassay techniques in the field of life sciences. This versatile and reliable method utilizes the specific binding of antibodies to target molecules to detect and quantify various analytes in biological samples. Principle of EIA/ELISA: At the core of EIA/ELISA principle is the antibody-antigen reaction, and visualization of the same which form the basis of this immunoassay technique. Steps of EIA/ELISA Coating: The first step involves immobilizing a capture antibody onto a solid phase, such as a microplate well. This antibody specifically binds to the target analyte. Blocking: To prevent nonspecific binding, the surface is then blocked with a protein, such as bovine serum albumin or casein. Sample addition: The biological sample containing the target analyte is added to the well and allowed to interact with the capture antibody. Incubation: the reactants are incubated Washing: Excess or unbound materials are washed away to remove any unwanted substances. Detection: A detection antibody, labeled with an enzyme such as alkaline phosphatase or horseradish peroxidase, is added. This antibody specifically recognizes a different epitope on the target analyte. Substrate addition: A substrate that undergoes a color change or a chemical reaction in the presence of the enzyme is added. Signal measurement: The signal generated by the enzyme-substrate reaction is measured using spectrophotometry or a similar method. The intensity of the signal is directly proportional to the amount of the target analyte present in the sample.  Different EIA/ELISA canFormats of EIA/ELISA be performed in different formats, depending on the specific requirements of the assay. Some of the commonly used formats include: Direct ELISA: In this format, the antigen is directly immobilized onto the solid phase, and the detection antibody binds directly to the target analyte. Indirect ELISA: Here, the antigen is coated onto the solid phase, and a primary antibody specific to the target analyte Competitive ELISA: Competitive ELISA is to use a labeled antigen and an unlabeled antigen to compete for binding to a limited amount of specific antibodies. Sandwich ELISA: A type of enzyme-linked immunosorbent assay that uses two specific antibodies to detect and measure the amount of a specific antigen in a sample. Applications of EIA/ELISA EIA/ELISA is one of the most well-established immunoassay formats and finds applications in a diverse range of fields. It is commonly used in clinical diagnostics to detect the presence of antigens or antibodies in biological samples which aids the physicians in their work. ELISA is employed for disease diagnosis, including infectious diseases, autoimmune disorders, and even pregnancy tests. Researchers also utilize ELISA for protein quantification, studying immune responses, and monitoring therapeutic drug levels.  Radioimmunoassay (RIA) is (RIA) Radioimmunoassay a type of immunoassay that utilizes a radioactive isotope to measure the concentration of target analytes in a biological sample. This technique was first developed by Rosalyn Sussman Yalow and Solomon Berson in the 1950s and has since become a widely used method in the field of life sciences. The principle of this immunoassay. RIA involves the use of a radiolabeled antigen or antibody to detect and quantify the presence of a specific target molecule in a sample. The radiolabel is usually a radioactive isotope, such as iodine-125 (^125I) or tritium (^3H), which emits radiation that can be detected using a specialized instrument. The assay is based on the principle of competitive binding, where the labeled antigen competes with the unlabeled antigen in the sample for binding to a limited number of specific antibody sites. The amount of radioactivity bound to the antibody is inversely proportional to the concentration of the target molecule in the sample. By measuring the radioactivity, the concentration of the target analyte can be determined. Advantages of RIA High sensitivity: RIA can detect very low concentrations of analytes, making it a valuable tool for biomedical research and diagnostics. High specificity: The antibody-antigen reaction in RIA is highly specific, allowing for accurate detection and quantification of target molecules. Wide range of applications: RIA has been used in various fields, including clinical diagnostics, endocrinology, pharmacology, and environmental testing. Limitations of RIA Radioactive materials: The use of radioactive isotopes in RIA requires special handling and disposal procedures to ensure safety. Time-consuming: RIA assays typically have longer incubation times compared to other immunoassay methods, which may limit its use in situations requiring rapid results. Cost: The use of radioisotopes and specialized equipment can make RIA more expensive compared to other immunoassay techniques. Applications of Radioimmunoassay (RIA) RIA is a sensitive immunoassay technique that employs a radioactive isotope as a label to quantify target molecules. This type of immunoassay has been particularly useful in studying hormones, vitamins, and drugs, among other substances. RIA finds applications in clinical laboratories for measuring hormone levels in patient samples and assessing endocrine disorders. It is also utilized in research settings to investigate the pharmacokinetics of drugs and molecules involved in the immune response.  Fluoroimmunoassay (FIA) Fluoroimmunoassay (FIA) is a powerful technique widely used in the field of immunoassays for the detection and quantification of various target analytes. This technique utilizes the principle of fluorescence, where the emission of light is triggered by the excitation of certain molecules. In Fluoroimmunoassay, a fluorescent label is attached to either the target analyte or the detection antibody. When these labeled molecules come into contact with each other in the presence of the biological sample, they form an antibody-antigen complex. This complex is then measured using a fluorometer that detects the emitted fluorescence. Advantages of Fluoroimmunoassay Fluoroimmunoassay offers several advantages that make it a preferred choice in certain applications: High Sensitivity: Fluorescence detection is highly sensitive, allowing for the detection of even small amounts of the target molecule in a sample. High Specificity: The antibody-antigen reaction in Fluoroimmunoassay provides high specificity, enabling accurate identification and quantification of the target analyte. Wide Dynamic Range: Fluoroimmunoassay can cover a broad range of target analyte concentrations, making it suitable for various assay applications. Multiplexing Capability: This technique allows for the simultaneous detection and quantification of multiple targets in a single assay, saving time and resources. Label Variety: Fluoroimmunoassays can be performed using a variety of different labels, such as fluorescent dyes or quantum dots, providing Applications of Fluoroimmunoassay Fluoroimmunoassays have found extensive applications in various fields, including: Clinical Diagnostics: It is used in the detection of infectious diseases, cancer biomarkers, hormone levels, and other analytes in patient samples. Drug Discovery and Development: Fluoroimmunoassays play a crucial role in drug screening, pharmacokinetic studies, and monitoring drug levels in biological matrices. Environmental Monitoring: It can be employed to measure pollutants, toxins, or contaminants in environmental samples. Food Safety: Fluoroimmunoassay has found use in assessment of food samples to ascertain their safety. Chemiluminescence Immunoassay (CLIA) Chemiluminescence immunoassay (CLIA) is a highly sensitive and widely used technique in immunoassay analysis. It utilizes the emission of light resulting from a chemical reaction to detect and quantify the presence of a target analyte in a biological sample. This method offers several advantages over other types of immunoassays, making it a popular choice in various applications. Principle of Chemiluminescence Immunoassay In a CLIA, the detection of the target molecule is based on the antibody- antigen reaction, similar to other immunoassay techniques. However, in CLIA, the binding event triggers a chemical reaction that produces light emission. This light emission is captured and measured by specialized instruments. Main Steps of Chemiluminescence Immunoassay Capture Antibody: A specific antibody is immobilized onto the surface of a solid phase, such as microplates or beads. This antibody is designed to bind to the target analyte present in the biological sample. Antigen-Antibody Complex Formation: The sample, containing the target analyte, is added to the solid phase. If the target analyte is present in the sample, it will bind to the capture antibody, forming an antigen-antibody complex. Light Production: Next, a detection reagent is added, which typically consists of an enzyme conjugated to an antibody. This enzyme catalyzes a chemical reaction that leads to the production of light. Light Detection: The emitted light is measured by a highly sensitive photomultiplier tube or a charge-coupled device. The intensity of the emitted light is directly proportional to the amount of target analyte present in the sample. Advantages of Chemiluminescence Immunoassay (CLIA) High Sensitivity: CLIA offers exceptional sensitivity, allowing for the detection of even low concentrations of analytes. This makes it an ideal choice for applications requiring high sensitivity, such as the detection of infectious diseases or biomarkers in early disease diagnosis. High Specificity: By utilizing the specificity of antibody-antigen interactions, CLIA can accurately detect and quantify a target analyte in the presence of other interfering substances. This ensures reliable and specific results. Wide Dynamic Range: CLIA can measure a broad range of analyte concentrations, providing flexibility in experimental design and sample analysis. Applications of Chemiluminescence Immunoassay (CLIA) CLIA utilizes a chemical reaction to generate light emission, allowing the detection and quantification of target analytes. This technique offers excellent sensitivity and wide dynamic range, making it suitable for a diverse range of applications. CLIA is widely used in clinical laboratories for diagnosing infectious diseases, monitoring drug levels, and detecting tumor markers. Researchers also utilize CLIA in fields such as drug discovery  Factors When selectingInfluencing the Choice the appropriate immunoassay of application, for a specific several factors come into play. It is crucial to consider these factors to Immunoassays ensure accurate and reliable results. Here are the key considerations that influence the choice of immunoassays: Target Analyte The nature of the target analyte plays a vital role in the selection of an immunoassay. Different immunoassays are designed to detect specific types of molecules, such as proteins, nucleic acids, small molecules, or infectious agents. It is crucial to choose an immunoassay that can effectively recognize and measure the target molecule of interest. Sensitivity and Specificity The desired sensitivity and specificity of the assay also impact the choice of immunoassay. Some applications require high sensitivity to detect low levels of analytes, while others prioritize high specificity to minimize false-positive or false-negative results. Each immunoassay has its own characteristics in terms of sensitivity and specificity, and selecting the appropriate assay is essential to meet the Factors Influencing the Choice of Immunoassays Sample Type and Matrix The type of biological sample being analyzed is an important consideration. Different immunoassays may have varying compatibility with different sample types, such as serum, plasma, urine, or saliva. Additionally, the sample matrix, such as tissue homogenates or cell lysates, may require specific sample preparation techniques to achieve optimal assay performance. It is crucial to select an immunoassay that is suitable for the sample type and matrix under investigation. Throughput and Automation The desired throughput and level of automation can influence the choice of immunoassay. Some immunoassays are designed for high-throughput analysis and can process a large number of samples rapidly, making them ideal for clinical laboratories or high-volume research settings. On the other hand, some immunoassays are better suited for low-volume applications, where manual or semi-automated procedures are sufficient. Factors Influencing the Choice of Immunoassays Time and Cost Consideration of time and cost is important when selecting an immunoassay. Some immunoassays provide quick results, enabling rapid analysis and decision-making, while others may require longer incubation periods. Additionally, cost considerations include the price of reagents and consumables, as well as the need for specialized equipment or platforms. A balance between assay performance and cost-effectiveness should be considered while choosing the appropriate immunoassay. Experts to Run Immunoassays Immunoassays are tests that require sound technical expertise to run and interpret the results. For that reason, expertise that is available remains an important factor to be considered when choosing types of immunoassays to be run in your laboratory. What would be the point of choosing to buy reagents for the most complex immunoassays if there is no one with expertise for the same in your laboratory? But the good thing is that people can always be trained and learn if they have the requisite educational qualifications of working in the laboratory.  THANK YOU FOR LISTENI NG 2025-01-08 College of Medicine, Ekiti State University, Nigeria