Immunochemistry Notes.pdf

Immunochemistry  Definition and Scope Immunochemistry is a specialized field that focuses on the chemistry of the immune system. It encompasses the study of various chemical components involved in immune responses, including antibodies (immunoglobulins), antigens, cytokines, chemo...

Immunochemistry  Definition and Scope Immunochemistry is a specialized field that focuses on the chemistry of the immune system. It encompasses the study of various chemical components involved in immune responses, including antibodies (immunoglobulins), antigens, cytokines, chemokines, and other related molecules. The discipline investigates how these components interact with one another and their roles in both normal immune function and disease processes.  Key Components The primary components studied in immunochemistry include: 1.Antibodies/Immunoglobulins: Proteins produced by B cells that specifically bind to antigens. 2.Antigens: Substances that provoke an immune response; they can be proteins, polysaccharides, or other molecules found on pathogens or foreign substances. 3.Cytokines/Chemokines: Signaling proteins that mediate and regulate immunity, inflammation, and hematopoiesis. 4.Epitopes: Specific parts of an antigen recognized by antibodies.  Techniques in Immunochemistry Various techniques have been developed within immunochemistry to analyze these components and their interactions. Some of the most common methods include: 1.Enzyme-Linked Immunosorbent Assay (ELISA): A plate-based assay technique designed for detecting and quantifying proteins, hormones, antibodies, or antigens. 2.Immunoblotting (Western Blot): A method used to detect specific proteins in a sample using gel electrophoresis followed by transfer to a membrane and probing with antibodies. 3.Immunohistochemistry (IHC): A technique used to visualize the presence and location of specific antigens in tissue sections using labeled antibodies. 4.Immunofluorescence: Similar to IHC but uses fluorescently labeled antibodies to detect antigens under a fluorescence microscope.  Applications of Immunochemistry Immunochemistry has significant applications across various fields: 1.Medical Diagnostics: Techniques like IHC are crucial for diagnosing diseases such as cancer by identifying specific markers within tissue samples. 2.Research: Immunochemical methods are employed in studies ranging from virology to molecular evolution, helping researchers understand disease mechanisms at a molecular level. 3.Therapeutics Development: By understanding how immune components interact with pathogens or diseased cells, new drug treatments can be developed.  Historical Context The field has evolved significantly since its inception. One notable early example is the Wasserman test for syphilis. Svante Arrhenius was instrumental in laying foundational concepts for immunochemistry through his publication “Immunochemistry” in 1907. In summary, immunochemistry is an essential discipline that combines principles of chemistry and biology to explore the immune system’s components and their functions. It provides critical insights into disease mechanisms and aids in developing diagnostic tools and therapeutic strategies.   IMMUNE RESPONSE : The immune response is a complex physiological reaction that occurs within an organism to defend against harmful invaders such as bacteria, viruses, fungi, and other foreign substances. This response is essential for maintaining health and preventing infections. The immune system can be broadly categorized into two main types: 1. Innate immunity 2. Acquired (adaptive) immunity. 1.Innate Immunity : Innate immunity is the body’s first line of defense against pathogens. It is non- specific and responds quickly to any foreign invader without prior exposure. Key components of innate immunity include: A. Physical Barriers: These include the skin and mucous membranes that act as barriers to prevent pathogens from entering the body. B. Cellular Components: Various white blood cells, such as neutrophils and macrophages, play crucial roles in identifying and destroying pathogens through processes like phagocytosis. C. Chemical Factors: The body produces various proteins and chemicals, including cytokines and complement proteins, which help mediate the immune response. When a pathogen breaches these barriers, pattern recognition receptors (PRRs) on immune cells recognize specific structures known as pathogen-associated molecular patterns (PAMPs). This recognition triggers a cascade of immune responses aimed at eliminating the threat. 2.Acquired Immunity: Acquired immunity develops over time through exposure to specific antigens. It involves a more sophisticated response that includes: A. Lymphocytes: B cells and T cells are central players in acquired immunity. B cells produce antibodies that target specific antigens, while T cells directly attack infected or cancerous cells. B. Memory Cells: After an initial exposure to an antigen, some B and T cells become memory cells. These cells remain in the body long-term and enable a faster and more effective response upon subsequent exposures to the same antigen. Vaccination is a method used to stimulate acquired immunity by introducing a harmless form of an antigen into the body, allowing it to develop memory without causing disease.  Inflammation : Inflammation is another critical aspect of the immune response. It occurs when tissues are damaged or infected. The inflammatory process involves: 1.Release of chemicals like histamine that increase blood flow to the affected area. 2.Recruitment of additional immune cells to help fight off infection or heal tissue damage. 3.Symptoms such as redness, heat, swelling, and pain are common indicators of inflammation.   Immune System Disorders: Disorders can arise when the immune system malfunctions. These may include: A. Autoimmune Diseases: Conditions where the immune system mistakenly attacks healthy tissues (e.g., rheumatoid arthritis). B. Immunodeficiency Disorders: Situations where the immune response is inadequate (e.g., HIV/AIDS). C.Allergies: An exaggerated response to harmless substances (e.g., pollen). In summary, the immune response is vital for protecting against infections and diseases through its innate and acquired mechanisms. Understanding how these systems work together helps in developing treatments for various immunological disorders.  STRUCTURE AND CLASSIFICATION OF IMMUNOGLOBULINS : Immunoglobulins (Ig), also known as antibodies, are glycoprotein molecules produced by plasma cells (a type of B cell) in response to an antigen. They play a crucial role in the immune system by identifying and neutralizing pathogens such as bacteria and viruses. The structure and classification of immunoglobulins are essential for understanding their function in the immune response.  Structure of Immunoglobulins : Immunoglobulins share a common basic structure consisting of four polypeptide chains: two identical heavy chains and two identical light chains. These chains are linked together by disulfide bonds, forming a Y-shaped molecule. The key components of immunoglobulin structure include: A. Heavy Chains: Each immunoglobulin has one of five types of heavy chains: gamma (γ), alpha (α), mu (μ), delta (δ), or epsilon (ε). The type of heavy chain determines the class of the immunoglobulin. B. Light Chains: There are two types of light chains: kappa (κ) and lambda (λ). Each immunoglobulin contains either two kappa or two lambda light chains. C. Variable Region: The tips of the Y-shaped structure contain variable regions that differ among antibodies. This variability allows for the specific binding to different antigens. The variable region is composed of both heavy and light chain segments. D. Constant Region: The stem of the Y-shaped molecule contains constant regions that determine the antibody’s class and its effector functions, such as complement activation or binding to Fc receptors on immune cells. E. Hinge Region: Some classes have a hinge region that provides flexibility, allowing the antibody to bind effectively to antigens that may be spaced apart. F. Glycosylation Sites: Immunoglobulins also have carbohydrate moieties attached at specific sites, which can influence their stability, distribution, and interaction with other molecules in the immune system.  Classification of Immunoglobulins Immunoglobulins are classified into five main classes based on their heavy chain type: 1. IgG (Immunoglobulin G):  Structure: Monomer  Function: Provides the majority of antibody-based immunity against invading pathogens; can cross the placenta.  Subclasses: IgG1, IgG2, IgG3, IgG4 2. IgA (Immunoglobulin A):  Structure: Exists as a monomer in serum but often forms dimers in secretions.  Function: Found in mucosal areas such as the gut, respiratory tract, and urogenital tract; plays a critical role in mucosal immunity.  Subclasses: IgA1 and IgA2 3. IgM (Immunoglobulin M):  Structure: Pentamer   Function: First antibody produced during an initial immune response; effective at forming complexes with antigens due to its multiple binding sites. 4. IgE (Immunoglobulin E):  Structure: Monomer  Function: Involved in allergic reactions and responses to parasitic infections; binds strongly to allergens and triggers histamine release from mast cells. 5. IgD (Immunoglobulin D):  Structure: Monomer  Function: Primarily found on the surface of immature B cells; its exact function is less understood but is believed to play a role in B cell activation. Each class has distinct roles within the immune system, contributing to various aspects of pathogen recognition, neutralization, opsonization, and activation of other immune components. In summary, immunoglobulins are vital components of the adaptive immune system characterized by their unique structures tailored for specific functions against diverse pathogens.  Mechanism of Antibody Production : Antibody production is a complex process that involves several key steps and components of the immune system. The primary cells responsible for producing antibodies are B lymphocytes, which are a type of white blood cell. Below is a detailed breakdown of the mechanism of antibody production: 1. Antigen Recognition : When a foreign substance, known as an antigen (which can be a pathogen like bacteria or viruses), enters the body, it is recognized by the immune system. Antigens have specific regions called epitopes that are recognized by B cells. 2. Activation of B Cells: Upon encountering an antigen, B cells undergo activation. This activation can occur directly when B cells bind to the antigen through their surface immunoglobulin receptors or indirectly with the help of T helper cells. T helper cells provide necessary signals through cytokines and direct cell-to-cell contact. 3. Clonal Expansion: Once activated, B cells proliferate and differentiate into two main types of cells: plasma cells and memory B cells. Plasma cells are responsible for producing large quantities of antibodies specific to the encountered antigen, while memory B cells remain in the body to provide long-term immunity against future infections by the same antigen. 4. Antibody Secretion: Plasma cells secrete antibodies into the bloodstream and lymphatic system. Each antibody produced is specific to the particular epitope on the antigen that triggered its production. 5. Antibody Structure: Antibodies are Y-shaped glycoproteins composed of four polypeptide chains: two heavy chains and two light chains. The tips of the “Y” contain variable regions that determine specificity for binding to antigens, while the stem region (constant region) determines the class or isotype of the antibody (e.g., IgA, IgG). 6. Affinity Maturation : During an immune response, some activated B cells undergo somatic hypermutation in their variable regions, leading to mutations that may increase their affinity for the antigen. This process allows for selection of B cells that produce higher-affinity antibodies over time. 7. Isotype Switching: B cells can also undergo class switching, where they change from producing one type of antibody (e.g., IgM) to another (e.g., IgG). This switch enables different functional properties suited for various stages of an immune response. 8. Memory Formation: After clearance of an infection, most plasma cells die off; however, some persist as memory B cells which can quickly respond upon re-exposure to the same antigen by rapidly differentiating into plasma cells and producing antibodies again. In summary, antibody production involves recognition of antigens by B lymphocytes, activation and clonal expansion of these B cells into plasma and memory B cells, secretion of specific antibodies into circulation, affinity maturation through somatic hypermutation, class switching for functional diversity, and formation of memory for long-term immunity.  Antigens: HLA Typing Human leukocyte antigen (HLA) typing, also known as tissue typing, is a critical blood test that identifies specific proteins called antigens present on the surface of cells. These antigens play a vital role in the immune system’s ability to distinguish between self and non-self entities. The presence of these antigens is essential for various medical procedures, particularly organ and stem cell transplants. Understanding Antigens and Their Role Antigens are markers on cells that help the immune system recognize foreign substances such as bacteria or viruses. In the context of transplantation, the body recognizes transplanted organs or tissues as foreign if they possess different antigens than those found in the recipient’s body. This recognition can trigger an immune response where white blood cells attack the transplanted organ, leading to rejection. HLA Typing Process : 1. Sample Collection: HLA typing begins with collecting a sample of blood from the patient. This can be done by inserting a needle into a vein in the arm or through a buccal swab (a swab taken from inside the cheek). No special preparation is needed for either method. 2. Identifying Antigen Patterns: The test identifies specific patterns of antigens inherited from both parents. Each individual has their unique combination of HLA antigens, except for identical twins who share the same pattern. For example, an individual’s HLA type may look like this: A2, A30; B8, B70; DR3, DR8. 3. Matching for Transplantation: The primary purpose of HLA typing is to find suitable matches for organ or stem cell transplants. The more closely matched the donor’s and recipient’s HLA types are, the higher the likelihood of transplant success. Most successful matches occur among close family members due to shared genetic material. 4. Probability of Matching: Siblings have a 25% chance of being an exact match for all six key antigens (A, B, and DR), while there is a 50% chance they will share three antigens and a 25% chance they will share none. Finding an exact match with an unrelated donor is significantly rarer—approximately one in 100,000. 5. Crossmatching: Before transplantation occurs, crossmatching tests are performed to determine if the recipient has pre-existing antibodies against potential donors’ antigens. If antibodies are present against a donor’s antigens, it indicates incompatibility and suggests that receiving an organ from that donor could lead to severe complications. 6. Importance in Transplant Success: While matching antigens is crucial for minimizing rejection risk during transplants, it is important to note that even patients with perfect matches can experience rejection due to other unidentified factors influencing immune responses. In summary, HLA typing serves as an essential tool in modern medicine for ensuring compatibility between donors and recipients during transplants by identifying specific antigen patterns that influence transplant success rates.  Free Radicals and Antioxidants : What are Free Radicals? Free radicals are highly reactive molecules that contain unpaired electrons. They can be formed through various processes, including normal metabolic reactions in the body, exposure to environmental factors such as pollution, radiation, and cigarette smoke. The most common type of free radicals produced in living organisms are reactive oxygen species (ROS), which include superoxide anions, hydroxyl radicals, and hydrogen peroxide. These free radicals can cause oxidative damage to cellular components such as DNA, proteins, and lipids. The formation of free radicals is a natural part of many biological processes. For example, during cellular respiration in mitochondria, electrons can escape from the electron transport chain and react with oxygen to form superoxide. Additionally, when the immune system fights off pathogens, it generates free radicals as part of its defense mechanism. However, when there is an imbalance between the production of free radicals and the body’s ability to neutralize them with antioxidants, oxidative stress occurs. What are Antioxidants? Antioxidants are substances that can prevent or slow down the damage caused by free radicals. They work by neutralizing these reactive molecules through various mechanisms. Some antioxidants donate electrons to free radicals without becoming unstable themselves; this process effectively “quenching” the reactivity of free radicals. There are two main types of antioxidants: 1. endogenous (produced by the body) 2. exogenous (obtained from the diet) Endogenous antioxidants include enzymes like superoxide dismutase (SOD), catalase, and glutathione peroxidase. Exogenous antioxidants come from dietary sources such as vitamins C and E, selenium, flavonoids found in fruits and vegetables, carotenoids like beta-carotene found in carrots and sweet potatoes, and other phytochemicals present in plant-based foods. The Role of Antioxidants in Health Antioxidants play a crucial role in maintaining health by protecting cells from oxidative damage that may lead to chronic diseases such as cancer, cardiovascular diseases, neurodegenerative disorders, and aging-related conditions. Diets rich in colorful fruits and vegetables have been associated with lower risks of these diseases due to their high antioxidant content. However, while dietary antioxidants have shown benefits in observational studies linking them to reduced disease risk, randomized controlled trials on antioxidant supplements have often failed to demonstrate significant protective effects against chronic diseases. This discrepancy suggests that consuming antioxidants through whole foods may be more beneficial than taking isolated supplements. In summary,  Free radicals are unstable molecules that can cause cellular damage.  Antioxidants neutralize free radicals and protect against oxidative stress.  A balanced intake of dietary antioxidants is essential for optimal health. The relationship between free radicals and antioxidants is complex but vital for understanding how our bodies maintain health amidst constant oxidative challenges.  Specialized Proteins: Collagen, Elastin, Keratin, Myosin, Lens Protein 1. Collagen : Collagen is the most abundant protein in mammals and serves as a critical structural component in various connective tissues. It comprises about 30% of the total protein content in the human body and is found in skin, tendons, ligaments, cartilage, and bones. The structure of collagen is characterized by a triple helix formation made up of three polypeptide chains that are coiled around each other. This unique structure provides tensile strength and flexibility to tissues. Collagen is rich in glycine and proline amino acids, which are essential for its stability and function. The synthesis of collagen involves post-translational modifications such as hydroxylation of proline and lysine residues, which are crucial for forming stable triple helices. 2. Elastin : Elastin is a highly elastic protein that allows tissues to resume their shape after stretching or contracting. It is primarily found in connective tissues such as skin, lungs, arteries, and ligaments. The primary structure of elastin consists of repeating units rich in glycine and proline. Elastin molecules are cross-linked through desmosine bonds formed between lysine residues, contributing to its elasticity. This property enables blood vessels to expand and contract with blood flow while maintaining structural integrity. 3. Keratin : Keratin refers to a family of fibrous proteins that serve as key structural components in hair, nails, skin, feathers, horns, and hooves. There are two main types of keratin: alpha-keratins (found in mammals) and beta-keratins (found in reptiles). Alpha-keratins primarily consist of alpha-helices that can form coiled structures held together by disulfide bonds; this contributes to their strength and resilience. Beta-keratins are composed mainly of beta-sheets providing additional structural support. Keratin’s insolubility in water makes it effective for protective roles against environmental damage. 4. Myosin : Myosin is a motor protein essential for muscle contraction and movement within cells. It interacts with actin filaments to facilitate muscle contraction through ATP hydrolysis- driven conformational changes. Myosin exists in several isoforms that vary based on tissue type (e.g., skeletal muscle myosin vs cardiac myosin). The myosin molecule has a head region that binds to actin filaments and a tail region that interacts with other myosin molecules or cellular structures. 5. Lens Protein : Lens proteins primarily refer to crystallins found in the lens of the eye. These proteins maintain transparency and refractive properties necessary for proper vision. Crystallins are categorized into three main classes: alpha-, beta-, and gamma-crystallins. They possess unique structural features allowing them to remain soluble at high concentrations without aggregating—a crucial characteristic for maintaining lens clarity over time. In summary,  Collagen provides tensile strength.  Elastin offers elasticity.  Keratin serves as a protective structural component.  Myosin facilitates movement through muscle contraction.  Lens proteins (crystallins) maintain optical clarity necessary for vision.   Electrophoretic and Quantitative Determination of Immunoglobins - ELISA etc. Investigation and Their Interpretation Introduction to Immunoglobulins Immunoglobulins (Ig), also known as antibodies, are glycoprotein molecules produced by plasma cells (a type of white blood cell) in response to an antigen. They play a crucial role in the immune response by identifying and neutralizing pathogens such as bacteria and viruses. There are five main classes of immunoglobulins in humans: IgG, IgA, IgM, IgE, and IgD, each with distinct functions and characteristics.  Electrophoretic Techniques for Immunoglobulin Analysis Electrophoresis is a laboratory technique used to separate charged particles in a fluid using an electric field. In the context of immunoglobulin determination, serum proteins can be separated based on their size and charge. The most common form used for this purpose is serum protein electrophoresis (SPEP). 1. Sample Preparation: Blood samples are collected from patients, typically through venipuncture. Serum is obtained by allowing the blood to clot and then centrifuging it to remove cells. 2. Gel Electrophoresis: The serum sample is applied to a gel matrix (usually agarose or polyacrylamide). An electric current is applied, causing proteins to migrate through the gel at different rates based on their size and charge. 3. Staining and Visualization: After electrophoresis, the gel is stained with specific dyes (e.g., Coomassie Brilliant Blue or silver stain) that bind to proteins, allowing visualization of distinct bands corresponding to different protein fractions. 4. Interpretation of Results: The resulting pattern is analyzed qualitatively and quantitatively. Each band corresponds to different types of proteins including albumin and various immunoglobulin classes. The relative intensity of these bands can indicate abnormalities in immunoglobulin levels.   Quantitative Determination of Immunoglobulins Quantitative measurement of immunoglobulins can be performed using several methods: 1. Enzyme-Linked Immunosorbent Assay (ELISA): Principle: ELISA is a plate-based assay technique designed for detecting and quantifying proteins such as antibodies or antigens. Procedure: A microplate is coated with specific antibodies that capture the target immunoglobulin from the sample. After washing away unbound substances, a secondary enzyme-linked antibody specific for the target immunoglobulin is added. A substrate for the enzyme is introduced; upon reaction, a color change occurs which can be measured spectrophotometrically. Interpretation: The intensity of color correlates with the concentration of immunoglobulin present in the sample. 2. Nephelometry: This method measures light scattered by particles in solution when illuminated by a laser beam. It provides quantitative data on immunoglobulin concentrations based on standard curves generated from known concentrations. 3. Radial Immunodiffusion (RID): In this technique, serum samples are placed in wells within an agar gel containing specific antibodies against the target immunoglobulin class. As diffusion occurs, a precipitin ring forms whose diameter correlates with concentration. 4.Turbidimetry: Similar to nephelometry but measures the decrease in transmitted light due to scattering caused by immune complexes formed between antibodies and antigens. Clinical Significance The determination of immunoglobulin levels has significant clinical implications: 1. Diagnosis of Immune Disorders: Abnormal levels may indicate conditions such as multiple myeloma (elevated monoclonal Ig), chronic infections (elevated polyclonal Ig), or primary immunodeficiencies (low total Ig). 2. Monitoring Disease Progression or Treatment Response: Regular monitoring can help assess how well treatments are working or if disease states are changing over time. 3. Research Applications: Understanding immune responses in various diseases can lead to better therapeutic strategies. In conclusion, both electrophoretic techniques like SPEP and quantitative assays such as ELISA provide essential tools for analyzing immunoglobulins in clinical settings. These methods not only aid diagnosis but also enhance our understanding of immune function.