CHEM2456 FINAL EXAM REVIEW

Unit 3 - An Introduction to Immunology

• Immunology = study of the body’s defense mechanisms
• Immunity = protection from disease and especially infectious diseases
• Immune systems = the cells and molecules that provide the immune protection
• Immune response = the manner in which the body responds to a foreign agent, e.g., virus, chemical
• Antigen = the foreign agent that elicits an immune response
• Two systems of immunity: Innate and Acquired

The Innate Immunity System

• Includes physical, chemical, and cellular barriers and is always present
• Physical barriers = barrier structures or substances
• Skin covers the vast majority of the body and acts as a physical barrier to prevent entry into the body
• Mucous membranes line the mouth, nose, eyelids, windpipe (trachea), lungs, stomach, intestines, urethra, and urinary bladder
• Mucous membranes produce mucus, a thick, viscous material that can trap foreign bodies and contains lysozyme which is an enzyme that can breakdown bacterial cell walls
• Chemical barriers = conditions that make pathogen survival difficult
• Stomach acid inhibits microbial growth and can kill microbes since most susceptible bacteria exist for less than fifteen minutes at a pH below 3
• Denaturation and hydrolysis of proteins occur at acidic pH
• Tears contain salt and lysozyme help to lyse possible pathogens, under stressful situations, known as lacrimal
• Sweat contains salt, butyric acid and lysozyme. Sweat is acidic and helps to reduce microbial growth, helping to lyse pathogens
• Lysozyme is an enzyme that lyses cell walls of gram-positive bacteria and was named by Alexander Fleming when he was testing mucus from a patient with a head cold on bacterial cultures
• Inflammation helps to localize infection and is characterized by redness, heat and swelling of the injured part
• Complement system = biochemical cascade that helps antibodies to rid pathogens or targets pathogens for destruction by other cells
• Cellular barriers = recruitment of particular types of cells which are the main line of defence in non-specific immunity
• Mast cells reside in connective tissue and mucus membranes, involved with wound healing and defence against pathogens, and also play a role in allergy
• Phagocytes engulf pathogens by extending their plasma membranes around pathogens and includes macrophages, neutrophils and dendritic cells

Phagocytes

• "Cell eaters" engulf or "phagocytose” pathogens by extending their plasma membranes around pathogens
• Macrophages = “large eaters” can move outside of the vascular system and across capillary walls, destroys pathogens through the process of “respiratory burst" (production of reactive oxygen species)
• Neutrophils are also called granulocytes because of granules in their cytoplasm
• Neutrophils produce hydrogen peroxide, oxygen radicals and hypochlorite that kill off pathogens, and adults produce 100 billion neutrophils per day
• Dendritic cells look like neuronal dendrites but are not part of the nervous system
• Dendritic cells are involved with antigen presentation which acts as a bridge between the innate and adaptive immune systems
• Natural killer (NK) cells does not directly attack invading pathogens but destroys compromised host cells such as virus-infected cell or tumour cells
• Natural killer cells induce apoptosis (programmed cell death) of infected cells causing them to destroy themselves from the inside

The Adaptive (acquired) Immunity System

• A system that involves many different types of cells that function if the innate system fails to prevent a foreign cell or molecule from entering the body
• A system that is highly specific and capable of recognizing and eliminating specific antigens
• Can take up to 7 days to become fully effective
• Can provide immunologic memory i.e. “remembers” antigens which is a basis for vaccinations
• Involves hypersomatic mutation that allows a small number of genes to produce extreme numbers of antigen receptors
• Consists of lymphocytes known as B- and T-cells, effector cells and memory cells
• Average healthy adult has around 2 trillion lymphocytes
• B-cells are called B-cells because they mature in the bone marrow
• Mature B-cells can interact with dendritic cells and can differentiate into plasma cells (produce antibodies) or memory cells
• T-cells are called T-cells because they mature in the thymus and involves 4 different types
• Helper T-cells assist with B- and T-cell differentiation and release chemicals that attract macrophages
• Cytotoxic T-cells bind to specific cells and release chemicals that lyse the cells (for example, infected cells)
• Suppressor T-cells regulate other immune cells and helps to turn down an immune response
• Memory T-cells have a similar function to memory B cells and helps if a reinfection occurs

Antibodies

• Antibodies (Ab) are a part of the acquired immune system and target foreign material for destruction by the innate immune systems
• Immunoglobulin G (IgG) is the most common antibody, named IgG due to it being glycosylated (whole Ab is ~150 kDa)
• Is composed of four polypeptide chains: two light chains (subscript L), and two heavy chains (subscript H)
• The polypeptide chains are joined by disulfide bonds
• Each chain has a variable domain and one (light) or three (heavy) domains, and each domain is ~110 amino acids
• Fragment antigen-binding (Fab) = antigen binding fragment, and are the arms of the Y
• Crystallizable Fragment (Fc) = the trunk of the Y
• The variable region recognizes the epitope by non-covalent interactions, that are known as complementarity
• Epitope is the foreign (non-histocompatible (non-self)) material
• Antibodies can be monoclonal (recognize a single specific epitope) or polyclonal (several different antibodies that bind to different epitopes on the same protein)
• The Fc region binds to a Fc receptor in an effector cell, a phagocyte that will engulf the foreign material and neutralize it
• B cells are a cell type that display different types of antibodies (with different variable regions) on their surface
• If a specific epitope is bound by an antibody (or several antibodies) on the B cell receptor, this stimulates the production of more of that type of antibody through signal transduction pathways in the cells
• To raise antibodies against a protein of interest inject an organism with the purified protein, harvest its blood and if the sequence is known it can also be produced recombinantly

Applications of Immunological Molecules

• Antigen-antibody specificity allows for binding of a single type of molecule, which is useful for Quantification (ELISA), Detection (Western blotting, lateral flow assays), and Purification (Affinity chromatography – Unit 2)
• Enzyme-linked immunosorbent assay (ELISA) relies on an antibody that has an enzyme linked to its Fc region (chemical linkage between the enzyme and the antibody)
• Spectrophotometric enzyme activity assay can be used to detect the enzyme and therefore antibody
• Commonly uses horseradish peroxidase which catalyzes the reaction

Types of ELISA

• Direct ELISA involves coating a 96 well plate coated with sample, all proteins in the sample are adsorbed onto the plate surface
• A blocking buffer "blocks" any remaining protein binding sites on the plate to prevent antibodies from binding directly to the plate
• The enzyme-linked antibody is added and incubated to bind the antigen, then the unbound antibodies are washed away
• Enzyme substrates are added, and a plate-reading spectrophotometer detects the emitted light
• As concentration of antigen increases, emitted light increases
• Advantages: fast and simple protocol
• Disadvantages: less specific since you are only using one antibody, and a potential for high background if all proteins from a sample are immobilized in well
• Indirect ELISA involves coating a 96 well plate with sample, all proteins in the sample are adsorbed onto the plate surface
• A blocking buffer "blocks" any remaining protein binding sites on the plate to prevent antibodies from binding directly to the plate
• Primary antibody, which binds the antigen, is bound and then washed a way
• A secondary enzyme-linked antibody is added (epitope is the Fc part of the primary antibody), is bound to the primary antibody, and is washed away
• Enzyme substrates are added, and a plate- reading spectrophotometer detects the emitted light
• As concentration of antigen increases, emitted light increases
• Advantages: amplification using a secondary antibody, and flexibility
• Disadvantages: potential for cross-reactivity caused by secondary antibody
• Reason to perform indirect ELISA: higher sensitivity
• Sandwich ELISA involves a plate coated in capture antibodies
• A blocking buffer blocks any remaining protein binding sites on the plate to prevent antigen from binding to the plate
• Antigen incubation allows the the antigen to be bound
• Antibody incubation can be done as a direct method or indirect
• Enzyme substrates are added, plate-reading spectrophotometer detects the emitted light
• As concentration of antigen increases, emitted light increases
• Advantages: highest specificity and sensitivity, and compatible with complex sample matrices
• Disadvantages: longer protocol, and challenging to develop
• Competitive ELISA involves a 96 well plate coated with reference antigen
• A blocking buffer "blocks" any remaining protein binding sites on the plate to prevent antibodies from binding directly to the plate
• Incubate the sample (which contains the antigens) with limited amount of labelled antibodies
• Add the sample-Ab mixture to the plate (Abs that did not bind to sample will bind to plate instead as the more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")
• Ab bound to sample will wash away, and Ab bound to reference will remain
• Enzyme substrates are added, and plate-reading spectrophotometer detects the emitted light As concentration of antigen in the free sample increases, light detection will decrease
• Advantages: ability to quantitate small molecules
• Disadvantages: less specific since you are only using one antibody, and requires a conjugated antigen

Western Blots

• Used to visualize a single protein of interest in SDS-PAGE
• The steps are to run SDS-PAGE, transfer all proteins to membrane, block, stain with primary Ab (1º Ab) and wash, stain with secondary Ab (2º Ab) and wash, and then detect via horseradish peroxidase (HRP)
• Identification of a specific protein in a complex mixture of proteins, where a protein mixture is separated by polyacrylamide gel electrophoresis, and the protein bands are transferred by electrophoresis (electroblotting) on to a membrane matrix
• Nitrocellulose or polyvinylidene difluoride (PVDF) membrane matrix has good non-specific protein binding properties
• Electroblotting uses an electric current which is passed through the gel to facilitate the transfer of protein to the membrane
• The charged proteins move from within the gel onto the membrane while maintaining the organization they had within the gel that increases its antibody detection
• Block the membrane to prevent non-specific binding to ensure the membrane and antibody used for detection of target protein is not bonded
• Non-specific binding is blocked by placing the membrane in a dilute solution of protein – typically non-fat dry milk with small percentage (0.05%) of detergent such as Tween 20
• The protein in the dilute solution attaches to the membrane in all places where the target proteins have not attached so when the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein
• This reduces background in the final product of the Western blot and leads to clearer results that eliminates false positives
• Membrane is incubated with a primary antibody (monoclonal antibody) directed against the antigen -protein you are looking for
• In the second step, it is incubated with a secondary antibody (conjugated/linked to a reporter enzyme) that is specific for the primary antibody.
• The enzyme's substrate then reveals the antibody-bound antigen, then precipitates next to the enzyme and stains the membrane
• Development of the blot is stopped by washing away the soluble dye where the most common detection methods use secondary antibodies conjugated to alkaline phosphatase (AP) or horseradish peroxidase (HRP).

Lateral Flow Assays

• Can be used in sandwich or competitive format
• Typically large epitopes = sandwich, smaller epitopes = competitve
• Used for SNAP tests, rapid COVID tests, home pregnancy tests, recombinant growth hormone, and for drug detection
• Delivers qualitative test results as opposed to quantitative