University:
Franklin University
Course:
SCIE-254 | Health & Human Disease
Academic year:

2021
Views:

56

Pages:

42
Author:

LunarLullaby

Antigen-Antibody Interactions Scope of immunology lab    Many infectious and non-infectious disease are currently diagnosed based on immunological methods. Most of them are based on Ag or Ab detection. Understanding the application of this interaction is crucial for a medical practitioner. Flash back to high school   Remember blood grouping. Using a set of specific antibodies you were able to determine ABO and Rh group. Is agglutination applicable for all cases?     RBCs are large particles and their agglutination results in a visible phenomenon. Nowadays, Corona virus is the hot topic. Can we use inactivated virus for detection of antibodies against this virus? Addition of Abs to small particles (e.g. virus) would not result visible reaction by naked eye and an indicator system will be required. A known Ag or Ab helps detecting the unknown one     Specific Abs can help us to detect presence of an Ag and even determine its concentration. Remember ABO and RH grouping. Vice versa is correct, if a known Ag is available. Monoclonal antibodies and recombinant proteins made a revolution in this regard. Recombinant proteins as Antigen Plasmid Design New Vector Plasmids are the most ubiquitous tools in the molecular biologist’s toolbox and are mostly used to study and manipulate genes and express desired proteins. Host Bacteria Yeast Plant Prokaryote Eukaryote Plasmid Type Cloning Expression A century from Magic Bullet concept to fully humanized monoclonal Abs P. Ehrlich’s side chain theory Nobel prize in 1908 Monoclonal Abs Applications: Cell phenotyping  Immunodiagnosis  Tumor detection  Immunotherapy  Functional analysis  Humanization of monoclonal Abs Mouse Humanized Chimaeric Human General structure of Ab classes or isotypes J chain SC Differences between Igs Ab-mediated effector functions Lets see how this knowledge can help us for better diagnosis of the disease and clinical practice. A very common infectious case study    A 25-year-old man, living in a rural area, suffers from 38.5°C fever, myalgia, joint and low back pain. History of consumption of fresh unpasteurized milk within 3 weeks ago. Suspicious to acute brucellosis. How can Immunology lab help in diagnosis?   Consider the course (acute, chronic) of the disease Select a precise serology test for diagnosis and screening. – – – – Rapid wright, Tube Wright Coomb’s Wright, 2ME-Wright Rose Bengal Wright Brucellae ELISA (IgG and IgM) Other divisions in Immunology lab This idea could be applied in diagnosis of other situations such as: Cancer Allergy Autoimmunity Infectious diseases Hormone disorders Thyroid dysfunctions Maternal care of the neonates Reproduction and infertility Immunodeficiency ……… Factors playing role in Ag-Ab interactions   When we are talking about serologic reactions, we are mainly talking about Ag and Ab molecules. Thus, understanding the following properties which may affect this interaction is necessary: – Antigen properties and concentration – Antibody properties and concentration – The ratio of Ag to Ab – The environmental factors Antigen characteristics • Based on size and shape: Particulate, Soluble, Colloidal • Based on the location: Intracellular or extracellular • Based on thymus dependence: Thymus dependent (e.g. proteins) or independent (e.g. polysacharids): affects Ab class Antibody characteristics    Class and subclass: Difference in capability of agglutination, precipitation, complement fixation, transition from mother to fetus The phase of disease: Acute, Sub-acute, Chronic Specificity, Affinity, Avidity, Cross-reactivity The dynamic interaction of Ag-Ab occurs in a couple of steps Primary reaction is invisible 2. Secondary reaction could be detected by naked eye, if large complexes developed. 3. It may result in biological consequences such as complement fixation and degradation of pathogen and phagocytosis. 1. Main features of Ag & Ab interaction     This interaction is through noncovalent bonds, thus it is reversible. Based on lock/key or protein/receptor or enzyme/substrate concept Specific interaction between a paratope and an epitope Immune complexes form in high concentrations Ag & Ab concentration affects immune complex size Relative molarity and number of epitopes and Ag binding sites affect the size of complexes Prozone Equivalence Postzone Maximal binding requires  Antigen accessibility  Appropriate concentration of Ag and Ab  Presence of equivalent number of multivalent sites  Complementarity: high affinity and avidity  Proximity: shaking and agitating, incubation at appropriate temperature  Suitable environment: buffer, salt, pH  Reducing agents may affect protein structure Binding is through non-covalent forces Affinity The strength of the reaction between a single antigenic determinant (epitope) with a single binding site of antibody (paratope) is determined by affinity constant. Avidity  Avidity is the strength of the binding of several Abs (paratopes) with several epitopes on a single immunogen resulting a more tight interaction due to bonus effect.  Overall capacity of an antiserum to bind with a multivalent Ag containing several determinants. Cross-reaction  Ab raised against an Ag, may react with similar Ags (epitopes), but with lower affinity.  The strength of Ab raised against its own Ag is stronger, but the bonds involved in crossreactions are weak.  The sera from patients with Brucellosis may show cross-reactivity with Salemonella Ags. Classification of Ag-Ab reactions Primary: small complexes Labeled immunoassays (Lattice formation not required) Secondary: larger complexes (Lattice formation is required) Tertiary: biological phenomenon (Functionality of Ag-Ab reaction) Primary reactions  Used for detection of lower amounts of Ag or Ab.  Labeled methods Application of a detector system such as enzyme, fluorescent or luminescent,….. molecule. Competitive or non-competitive. Automated systems are mostly based on primary reactions.    Non-Competitive ELISA for antibody detection Immobilize Ag on a solid phase  Incubate with sample (containing Ab)  Add conjugated or labeled anti-Ig  Amount of labeled Ab bound is proportional to amount of Ab in the sample  Non-Competitive ELISA for antibody detection Ab in Patient’s sample Labeled Anti-Ig Ag Immobilized Ag Ag Ag Ag Ag Ag Ag Ag Ag Ag Non-Competitive ELISA for antigen detection Immobilize Ab on a solid phase  Incubate with sample (containing Ag)  Add conjugated or labeled antibody  Amount of bound labeled Ab is proportional to the amount of Ag in the sample  Non-Competitive ELISA for antigen detection Labeled Ab Ag Ag in Patient’s sample Immobilized Ab Competitive ELISA for Ag detection Instrumentation in Immunology lab Siemens and Cobas systems Secondary serologic reactions based on Ag size and type  Agglutination of particulate Ags Secondary serologic reactions based on Ag size and type Agglutination of particulate Ags  Precipitation of soluble Ags  Flocculation of colloidal Ags   In all of above reactions, high concentrations of Ag and Ab is required for reaction Tertiary serologic reactions Summary of serologic tests  In primary reactions low amounts of Ag & Ab is detected using an indicator  In secondary reactions high concentrations of Ag and Ab results a visible consequence  In the tertiary reactions a biological phenomenon such as RBC lysis is observed. Summary of serology application   Immunology labs apply high technology instrumentations to measure minimal concentration of Ag or Ab molecules, helpful for diagnosis of many immunological and nonimmunological diseases. Production of specific monoclonal or polyclonal Abs as well as recombinant Ags are necessary for immunoassay development

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