Immunology and Serology: Definitions and History

Immunology and Serology Definitions

• Immunology studies host reactions to foreign substances, not just the immune system.
• Immunology includes the unwanted effects of inflammation.
• Serology is the laboratory expression of immunology.
• Serology deals with the measurement and characterization of antibodies, antigens, and other immunological substances in blood and body fluids.

Historical Milestones in Immunology

• Chinese practices involved inhaling smallpox crusts for artificial adaptive immunity.
• Edward Jenner is dubbed as the father of immunology and initiated smallpox vaccination.
• Louis Pasteur introduced the practice of attenuated vaccines.
• Attenuated vaccines weaken modified organisms to produce a less drastic body response.
• lli Metchnikoff introduced the concept of phagocytosis.
• Robert Koch contributed to the understanding of cellular immunity in PB.
• Jonas Salk developed the polio vaccine.
• Berson and Yalow developed the radioimmunoassay technique.
• Tonegawa discovered the principle of antibody diversity.
• Kary Mullis discovered the polymerase chain reaction (PCR).

Divisions of Immunity

• Immunity is divided into natural and adaptive immunity.
• Natural immunity includes the first and second lines of defense.
• Adaptive immunity is the third line of defense.
• Natural immunity is present at birth, while adaptive immunity is underdeveloped at birth.
• Natural immunity is also known as innate or non-specific immunity.
• Natural immunity has a generic response to any antigen.
• Natural immunity has no immunologic memory.
• Adaptive immunity is referred to as acquired or specific immunity.

First Line of Defense

• The first line of defense includes physical and biochemical barriers and human reflexes.
• Examples of reflexes: Sneezing, coughing, urine flushing.
• The first line of defense is external and considered the lowest form of defense.
• The skin has a pH of 5.6, maintained by lactic acid and fatty acid excretions.
• Lysozyme in body fluids protects against gram-positive organisms by attacking the peptidoglycan layer.
• Normal flora provides competitive exclusion, preventing harmful organisms from inhabiting the same area.

Second Line of Defense

• The second line of defense is the internal component of natural immunity.
• Divided into cellular and humoral components.
• The cellular component's main component includes polymorphonuclear cells (PMNs), monocytes, macrophages, and natural killer (NK) cells.
• The humoral component refers to soluble factors.
• Complement is the main humoral component.
• The classical complement pathway is part of the third line of defense.
• Mannose-binding lectin and alternative pathways are part of the second line of defense.
• Interferons protect against viral replication.

Third Line of Defense

• The third line of defense also has cellular and humoral components.
• The cellular component includes specialized lymphocytes (T cells and B cells).
• The humoral component includes cytokines and antibodies.
• T cells mediate cellular immunity, while B cells mediate humoral immunity.

Types of Acquired Immunity

• Acquired immunity can be active or passive.
• In active immunity, the host produces antibodies.
• Passive immunity involves receiving antibodies from an external source.
• Active immunity can be acquired naturally through exposure to infections or artificially through vaccinations.
• Natural passive immunity occurs through transplacental transfer of antibodies (IgG) or breast milk (IgA).
• Artificial passive immunity involves the infusion of plasma/serum or artificial antibodies, such as anti-venom.

Duration of Response in Acquired Immunity

• Active immunity produces longer-lasting responses.
• Passive immunity provides short-term, immediate responses.

Inflammation

• Inflammation comes from the latin word "inflammat" means "to set on fire".
• Inflammation is a general reaction to tissue injury or infection.
• Goal of inflammation is to destroy and remove the pathogen.
• Inflammation prevents the spread of infection.
• Inflammation repairs tissue damage.
• Five cardinal signs of inflammation: rubor (redness), calor (heat), dolor (pain), tumor (swelling), and functio laesa (loss of function).

Stages of Inflammation: Vascular Response

• Injured cells cause mast cells to release histamine which results in dilation of small venules and constriction of large vessels.
• Increased blood flow brings heat to the injured area.
• Increased capillary permeability results in plasma leakage into tissue, causing swelling.
• Compression of nerves results in pain.
• Blood becomes viscous, leading to blood stasis and impaired delivery of blood which results in loss of function.
• First cardinal sign of inflammation is redness (rubor).

Stages of Inflammation: Cellular Response

• White blood cells, mainly neutrophils, migrate from capillaries to surrounding tissue within 30-60 minutes.
• Macrophage migration occurs hours after injury, peaking at 16-48 hours.
• Pain is caused by increased capillary permeability and the release of prostaglandins by cellular mediators.

Stages of Inflammation: Healing or Repair

• Initiated by fibroblasts, which produce acid mucopolysaccharides to neutralize toxic substances
• The ideal outcome is total repair of the affected area.
• If organisms cannot be killed, abscess formation with loss of function, granuloma formation.
• Granuloma formation is a hallmark of chronic inflammation: continuous tissue damage.
• A granuloma is a body's way to wall off offending agents.
• Diseases with granuloma formation: Tuberculosis and fungal infections.
• Edema is the hallmark of acute inflammation.

Acute Phase Reactants

• Acute phase reactants are normal serum constituents that increase rapidly during infection, injury, or trauma.
• Negative acute phase reactants decrease during inflammation.
• Produced primarily by liver cells (hepatocytes).
• Production is driven by interleukin-6
• Usually produced within 12-24 hours.
• They prevent further damage and promote inflammatory responses like phagocytosis, functioning as opsonins.
• Increase in C-reactive protein is an indicator of inflammation
• C-reactive protein was once thought to be an antibody due to its reaction with the C-substance of pneumococci.
• C-reactive protein levels increase more in bacterial infections than viral infections.
• C-reactive protein is a sensitive but non-specific indicator of acute inflammation.
• It can increase by 1000 times in cases of acute inflammation.

Serum Amyloid A

• Serum amyloid A is an apolipoprotein with high affinity to HDL.
• It activates monocytes and macrophages.
• Found in atherosclerotic lesions, contributing to localized inflammation in coronary artery diseases.

Alpha-1 Antitrypsin

• Alpha-1 antitrypsin inhibits proteases released from leukocytes, such as elastase.
• Elastase, if not controlled, can damage normal cells.
• Alpha-1 antitrypsin sweeps off excessive inflammation effects.
• Deficiency in alpha-1 antitrypsin can lead to pulmonary emphysema and juvenile cirrhosis.

Fibrinogen

• Fibrinogen helps to prevent the spread of microorganisms.
• Clots create a barrier.

Haptoglobin

• Haptoglobin binds free hemoglobin to prevent oxidative damage.
• Haptoglobin-hemoglobin complex is cleared by liver macrophages.
• Haptoglobin levels decrease early in the inflammatory response.

Ceruloplasmin

• Ceruloplasmin is a protein to carry with oxidase activity acts as the transport protein of copper
• One molecule can carry six copper molecules.
• Converts iron into the non-toxic form (ferric).

Complement Components

• Complement components function in opsonization, chemotaxis, and lysis of cells.

Phagocytosis

• Phagocytosis is a cellular response of inflammation.
• The most active phagocytes: Neutrophils, monocytes, macrophages, and dendritic cells.
• Dendritic cell is the most potent phagocyte and antigen-presenting cell (APC).

Factors Enhancing Phagocytosis

• Integrins: Surface receptors that promote cell-to-cell interaction.
• Integrins are important in diapedesis (crossing vessel walls).
• Opsonins: Substances that coat particles/organisms to make them susceptible to phagocytosis.
• Examples of opsonins: IgG and C3b.

Pathogen Recognition Receptors (PRRs)

• These are molecules on host cells that recognize substances only found on pathogens
• PRRs are found on macrophages and dendritic cells.
• Macrophages and dendritic cells: Approximately 10-15% of tissue cells.
• Macrophages and dendritic cells are the most important cells in pathogen recognition.
• PRRs are associated with pathogen-associated molecular patterns (PAMPs).
• Examples of PAMPs: Peptidoglycan from gram positive organism, lipoproteins, flagellin.

Toll-Like Receptors (TLRs)

• TLRs are proteins in fruit flies (Drosophila) responsible for immunity.
• TLRs were first discovered pathogen recognition receptors in humans, discovered by Charles Janeway.
• Divided into cell surface and cytoplasmic TLRs.
• The January board exam included questions about exact substance that is recognized by different TLRs

Other Pathogen Recognition Receptors (PRRs)

• C-type lectin receptors bind mannan and beta-glucan found in fungal cell walls.
• Retinoic acid-inducible gene 1 (RIG-1)-like receptors (RLRs) recognize RNA from RNA viruses such as influenza
• RLRs induce inflammatory response.
• RLRs promote production of interferon and viral replication.
• NOD-like receptors recognize intracellular peptidoglyans released from bacteria
• NOD-like receptors protect against intracellular protozoan parasites.
• Mutations in NOD-like receptors may result in Crohn's disease.

Steps in Phagocytosis

• Adherence, engulfment, phagosome formation, granule contact, phagolysosome information, digestion, and excretion.

Oxygen-Dependent Killing

• Production of reactive oxygen species for killing organisms.
• Requires enzymes: NADPH oxidase and superoxide dismutase.

Oxygen-Independent Killing

• Entry of hydrogen and potassium ions into the cell.
• Altered pH activates proteases.
• Proteases: Defensins and cathepsin G.
• The oxygen-dependent pathway involves respiratory or oxidative burst.
• Oxygen-independent involves activation of intracellular proteases.

Macrophages and Their Locations

• Macrophages differentiate from monocytes.
• They migrate to different tissues and differentiate with different names, depending on the location.
• Microglial cells are the macrophages in the brain.
  • Histiocytes are in connective tissues.
    • Red pulp macrophages (marginal zone macrophages) are in the spleen that remove red blood cells through calling and pitting.
    • Sinus histiocytes are in lymph nodes.
    • Hofbauer cells are in the placenta.
    • Messangel cells are in the kidneys. -Langerhan cells are in the skin.

Chronic Granulomatous Disease (CGD)

• CGD are a group of disorders involving inheritance of either an x-linked or autosomal recessive gene that affects this affects neutrophil macrobicidal function.
• Neutrophils phagocytize but cannot kill organisms.
• Caused by defective NADPH oxidase system.
• Characterized by severe, recurrent infections.

Tests for Chronic Granulomatous Disease

Screens

• Nitro blue tetrazolium (NBT) test.
  • Principle:Detect presence of O2 radicals produced by phagocytes. -Reagent : NBT (yellow) reduced by NADPH to blue precipitate, formazan -Negative Test: if neutrophils are unable to produce O2 radicals and the NBT remains blue. -Principle for CGD -Nitro blue tetrazolium is originally yellow or colorless. -The positive result for blue formazan dyre indicates the ability to convert yellow to blue indicates the prescience of NADPH oxidase, which is needed to produce oxygen species (hydrogen peroxide)

Flow Cyometric Assay- Dihydrorhodamine (DHR)

-Label neutrophils with with a certain type that whenever there is successful oxidative outbursts (fluorescent) -Activated with the use of four ball myristate acetate. activated neutrophils - DHR- is a nonfluorescents dye that is converts to fluorescent Rhodamine in presence of H2O2 or HOCL. -Measured fluorescent by flow cytometer -Positive Result : Fluorescence- ability to be reduced and is nonfluorescent.

Lymphoid Organs

• Divided into primary and secondary lymphoid organs. - Primary lymphoid organs are bone marrow and thymus -Site of lymphocyte maturation - T cell and B cells - Site for lymphocyte pro liver Asian - Antigen Independent - Secondary Liver Organs - Activation of Lymphocytes - antigen dependent process
• Spleen - Largest, Red vs White pulp regions and filtration of blood -Found in peri arteriole lymphoid sheath, B cells on primary follicles
• Lymph Nodes - Filter lymph and lymph fluid - Cortex = T cells, B calls on outer cortex

Lymphocytes

• Major populations: T cells, B cells, and natural killer (NK) cells. - Most common - T cells -10-20% - B cells -10-15% - Natural killer cells - Adaptive Immunity : T and B cells - B cells - mediators - cell mediated Immunity - T cells Surface Markers - flow of immunophenoting - Malignancy Diagnoses

• T cells (CD2, CD3, 4, 8)

  • T red blood cell Rosette receptor -CD3 - marker on all mature T cells.

• B cells - Bigger numbers compared to the cell Markers

• Natural Killer Cells

  • CD16 - igg - receptor and Antibody dependent
    • CD56, CD94 -Mitogens
    • Needed for cell proliferation and activating cellular response
    • Staphlococcus protein B - can prevent production and activation### Lymphocyte Populations

• T lymphocytes comprise approximately 60-80% of circulating lymphocytes.

• B cells are another type of lymphocyte found in circulation.

• Natural killer cells (NK cells) represent 10-15% of circulating lymphocytes.

• NK cells can be activated by interleukin-2.

• Interleukin-2-activated NK cells are also known as lymphokine-activated killer (LAK) cells.

B Cell Maturation Stages

• B cell precursors migrate to the bone marrow to mature before release into secondary lymphoid tissue.
• There are 6 stages of B cell maturation, each with important events.

Pro-B Cell

• Characterized by rearrangement of genes coding for heavy and light chains.
• Heavy chain genes are found on chromosome 14.
• Light chain genes originate from chromosomes 2 and 22
• Distinctive surface markers include CD19, CD45R, CD43, CD24, and c-kit.

Pre-B Cell

• Pre-B cell involves the synthesis of the heavy chain part of the antibody molecule
• The first heavy chain synthesized is the mu chain (µ chain).
• The mu chain is the heavy chain of the first complete antibody on the B cell surface.

Immature B Cell

• Marked by surface markers CD21, CD40, and MHC class II molecules.
• The first antibody found on a developing B cells surface is IgM
• Following IgM is IgD

Mature B Cell

• Mature B cells express both IgM and IgD on their surface.
• Mature B cells also express MHC class II molecules
• Also known as immunocompetent B cells
• Immuno competence is necessary for antigen presentation and recognition.

Activated B Cell

• Contact with foreign antigens leads to the appearance of CD25.
• CD25 serves as a receptor for interleukin-2.
• CD25 indicates is already an activated lymphocyte
• B cells activated by interleukin-2 transform into blasts.
• These blasts give rise to plasma cells and memory B cells.

Plasma Cells

• Plasma cells produce antibodies.
• Spherical or ellipsoidal cells, 10-20 micrometers in size.
• Characterized by abundant cytoplasmic immunoglobulins.
• Unlike other B cells they have cytoplasmic immunoglobulins and not suface immunoglobulins
• Also known as effector B cells.

T Cell Maturation

• T cell precursors migrate to the thymus to mature before release into secondary lymphoid tissue
• Early stages lack CD4 and CD8 markers ("double negative").
• Later stages express both CD4 and CD8 ("double positive").
• Surface markers include CD3, CD4, and CD8.
• CD3 is a component of the T cell receptor (TCR).
• Complete T cell receptor: CD3 alpha/beta receptor complex.
• Double negative stage involves rearrangement of genes coding for the beta chain of the TCR.
• Double positive stage involves rearrangement of genes coding for the alpha chain of the TCR.
• Developing T cells undergo selection to recognize self vs. non-self.
• T cells that survive selection become either CD4+ or CD8+ cells.
• CD4+ T cells recognize antigen with MHC class II, becoming T helper cells.
• CD8+ T cells interact with antigen via MHC class I, becoming cytotoxic T cells.
• Mature T cells spend ~12 days in the medulla for additional proliferation.
• Released to peripheral lymphoid organs to contact antigens.

T Cell Activation

• T cells in peripheral tissues recirculate every 12-24 hours.
• Antigen recognition activates and differentiates them into small lymphocytes.
• Activated T cells express CD25 as a receptor for interleukin-2.
• Mature T cells are either CD8+ or CD4+, having survived lymphocyte selection.

Lymphocyte Selection Process

Positive Selection

• Occurs in the thymic cortex.
• Double positive thymocytes must recognize foreign antigen with class I or II MHC molecules.
• Cells with functional TCRs survive by identifying the receptor.

Negative Selection

• Occurs in the thymic medulla.
• T cells interact with self antigens.
• T cells that bind self antigen are eliminated.
• Ensures that only T cells that do not recognize self-antigens are released into peripheral tissues (self vs non-self test).

T Helper Cell Subsets

• All T helper cells are CD4 positive.

Th1

• Produces interferon gamma, interleukin 2, and tumor necrosis factor beta.
• Protects cells against intracellular pathogens.
• Activates cytotoxic lymphocytes and macrophages.

Th2

• Produces interleukins 4, 5, 6, 9, 10, and 13.

T Helper Cell 2 Role

• Assists B cells in antibody production.
• Generally regulates B cell activity.

T Regulatory Cell (T-reg) Characteristics

• Possesses the CD4 antigen and CD25.
• Comprises about 5% of all CD4 positive T cells.
• Suppresses immune response to self-antigens, acting as a switch for the immune system.
• CD4 positive cell.
• Does not produce Interleukin-9.

TH17 Cell Function and Effects

• Produces interleukin-17 and interleukin-22.
• Pro-inflammatory effect increases inflammation and joint destruction.
• Associated with autoimmune diseases like rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (IBD).

Laboratory Detection of Lymphocytes

• Rosette Technique: Obsolete method using sheep red blood cells to detect T cells (CD2 antigen). Rosette formation is considered if at least three sheep red blood cells are attached to a single lymphocyte.
• Uses sheep red blood cells attached to the CD2 antigen found on T cells.

Flow Cytometry Technique

• Automated system analyzing single cells in fluid suspension.
• Evaluates cells based on light scattering characteristics and surface or cytoplasmic proteins.
• Often coupled with immunophenotyping to evaluate cell populations using surface markers.
• Uses fluorescent-labeled antibodies as probes to identify specific markers.
• Useful for leukemia and lymphoma evaluation and prognosis based on marker presence or absence.

Major Components of Flow Cytometry

• Fluidics: Cells pass through the laser one at a time for evaluation.
• Laser Light Source: Measures cell size with forward scatter and cell granularity/internal complexity with side scatter (90-degree angle).
• Uses fluorescently built antibodies to evaluate antigenic properties via CD markers.
• Optics and Photodetectors: Detect signals from cell interaction with the laser and convert data into quantitative measurements.

Sample Preparation for Lymphocyte Detection

• Commonly uses whole blood, bone marrow, and fluid aspirates.
• Preferred anticoagulant for whole blood is EDTA, processed within 30 hours of collection with use of heparin if cannot.
• Requires removal of red blood cells prior to testing using density gradient centrifugation or a red blood cell lysing agent.

Density Gradient Centrifugation

• Separates cell populations into layers using Ficoll-Hypaque agent.
• Three layers form: plasma, mononuclear cells (lymphocytes), and red blood cells/granulocytes (bottom).
• Disadvantage: selective loss of some cell populations, time-consuming.

Erythrocyte Lysing Agent Method

• Uses ammonium chloride.
• Preferred method for sample preparation compared to density gradient centrifugation.

Lymphocyte Defects: B Cell Related

• Developing B cells are arrested in the pre-beta cell stage.

A gamma globulinemia

• Leads to complete absence of B cells, plasma cells, and immunoglobulins of all classes.
• Genetic mechanism: lack of Bruton tyrosine kinase (BTK) enzyme.
• X-linked inheritance, affecting males more.

Selective IgA Immunodeficiency

• Most common congenital immunodeficiency.
• Usually asymptomatic, but symptomatic patients are highly susceptible to respiratory or gastrointestinal infections and autoimmune diseases.

Common Variable Immunodeficiency

• Deficiency of both IgA and IgG antibodies.
• Characterized by sinusitis, pneumonia, shingles, lymphoma, proliferative disorders, gastric carcinomas, and autoimmune disorders.

Isolated IgG Subclass Deficiency

• Deficiency of a single IgG subclass, most commonly affecting IgG4.

Lymphocyte Defects: T Cell Related

DiGeorge Anomaly

• Affects the thymus due to developmental abnormality of the third and fourth pharyngeal pouches.
• Results in a quantitative T cell deficiency, causing tetany and cardiac defects in severely affected neonates.

Lymphocyte Defects: Affecting Both T and B Cells

Wiskott-Aldrich Syndrome

• Rare X-linked recessive syndrome.
• Triad of immunodeficiency, eczema, and thrombocytopenia.
• Affects cellular and humoral immunity.
• Most consistent lab finding: absence of IgM antibodies.
• Primary molecular defect: abnormality in the integral membrane protein CD43, due to a mutation in the WASP gene.

Ataxia-Telangiectasia

• Rare autosomal recessive syndrome.
• Characterized by involuntary muscle movements (cerebral ataxia) and capillary swelling (telangiectasias).
• Defective ATM gene on chromosome 11, causing abnormalities in T cell receptor (TCR) and immunoglobulin gene rearrangement.

Severe Combined Immunodeficiency (SCID)

• Most serious congenital immunodeficiency, a group of related diseases affecting all T and B cell functions.
• Common form caused by a mutation in the interleukin-2 receptor gamma (IL-2RG) gene on the X chromosome, mainly affecting males.

Immunogens vs. Antigens

• Immunogen: Macromolecule triggering an adaptive immune response by inducing antibody production and T cell sensitization.
• Antigen: Reacts with an antibody or sensitized T cell but may not evoke an immune response initially.
• Not all antigens are immunogens, but all immunogens are antigens.

Factors Influencing Immunogenicity

• Macromolecular Size: Larger molecular weight makes a molecule more potent as an immunogen (at least 10,000 Daltons).
• Foreign Nature: Substances recognized as non-self are immunogenic.

Self Tolerance

• Achieved through a selection process to not evoke immune response under normal circumstances.

Chemical Composition and Molecular Complexity

• Proteins and polysaccharides are the most effective immunogens.
• Carbohydrates or polysaccharides are carbohydrates and are less immunogenic than proteins.
• Pure nucleic acids and lipids are not immunogenic.
• Nucleic acids exist in complex with histones (proteins).
• Synthetic polymers (e.g., Teflon, nylon) are non-immunogenic, used for artificial heart valves and elbow replacements.
• Ability to Be Processed and Presented: Immunogens must be degraded and presented to lymphocytes.

Relationship of Antigens to the Host

Alloantigen

• Antigens from other members of the host species.
• Capable of eliciting an immune response.

Autoantigen

• Antigens that belong to the host.
• Do not evoke an immune response under normal circumstances.

Heteroantigen

• Antigens from other species.

Heterophile Antigen

• Special type of heteroantigen in unrelated plants or animals that are identical or closely related in structure.
• Antibody to one will cross-react with antigen of the other.

Differential Diagnosis

• Differential diagnosis is considered to discern if infectious mother necrosis, serum sickness, or arthritis is the reason.

Epitopes

• Part of the immunogen that is actually recognized in the immune response.
• Also known as the antigenic determinant.

Linear Epitope

• Sequential epitopes, amino acids following one another on a single chain.

Conformational Epitope

• Results from the folding of one chain or multiple chains.
• B cells may react with linear and conformational epitopes.
• T cells recognize processed epitopes complexed with MHC proteins.

Happens

• Antigenic but non-immunogenic because they are not complex enough.
• Become immunogenic when attached to a carrier protein.
• Catechol is a hapten and is a group of compound, that are non-immunogenic.
• Urushiol (found in poison ivy): conjugates with tissue proteins to form an immunogen.

Adjuvants

• Substance administered with an immunogen.
• Increases the immune response to provide immunity to a particular disease.
• Target antigen-presenting cells and protect the immunogen from degradation, allowing a longer response time.
• FDA-licensed adjuvants: alum salt and oil-based substance MF59.

Major Histocompatibility Complex (MHC) also termed as human leukocyte antigens (HLA)

• MHC, also termed human leukocyte antigens (HLA), are coded in the short arm of chromosome six.
• Genes coding for MHC molecules are in the short arm of chromosome 6.

Functions and Applications of MHC

• Mounting the immune responses.
• Determination of histocompatibility for paternity testing.
• Disease association.
• Three classes of MHC: class 3 doesn't participate in antigen presentation but it has a role in immune response for the body.
• Class 1 and class 2 participate in antigen presentation where as Class 3 do not express on cell surfaces because it has a protein immune function, the complement component.
• Class 3: complement components (C4A, C4B, C2), tumor necrosis factor.
• Class 1 genetic locus: HLA-A, B, C. Class 2 genetic locus: HLA-DP, DQ, DR. Class 3 not found between Class 1 and Class 2.

Classical vs Non-Classical Antigens

Classical Antigens

• Class 1 classical antigens: A, B, C.
• Class 2 classical antigens: DP, DQ, DR.

Non-Classical Antigens:

• Class 1 non-classical antigens: E, F, G.
• Class 2 non-classical antigens: DM.

Structure of Class 1

• alpha chain and beta2 microglobulin.

Structure of Class 2

• alpha chain and beta chain.

Class 1 Cell Distribution

• Found in all nucleated cells.

Class 2 Cell Distribution

• Found in B cells, monocytes, macrophages.

Sizes of Peptides

• Class 1: attach to 8-11 amino acid sequences.
• Class 2: attach to 13-18 amino acid sequences.
• Nature of peptide binding cleft: Class 1 is closed at both ends, Class 2 is open at both ends.
• Class 1 presents endogenous antigens to CD8 positive T cells.
• Class 2 presents exogenous antigens to CD4 positive T cells.

Disease Association from Major Histocompatibility Complex (MHC)

• HLA B8: Celiac disease, myasthenia gravis, Addison's disease, chronic active hepatitis, dermatitis.
• HLA B27: Ankylosing spondylitis.
• HLA B51: Behcet’s disease.
• HLA DR2: SLE, epidermolysis bullosa acquisita, Goodpasture syndrome.
• HLA DR3: Systemic lupus erythematosus, autoimmune thyroid conditions (Hashimoto's thyroiditis and Graves' disease)
• HLA DR4: Rheumatoid arthritis.
• HLA DR5: Alopecia areata.
• HLA DR/DQ: Type 1 diabetes mellitus, multiple sclerosis (also associated with HLA B7).

Laboratory Detection Methods:

• Molecular method is the most widely used.
• Serological Cellular, Molecular
• Examples of Molecular: rapid results with high cost.
• Restriction fragment.
• Lymphocyte reaction: complement dependent.
• Serological CDC: the Serological method is capable of detecting both MHC class one and class two.
• CDCS: can detect both MCH 1 and 2 and is usually reserved for the detection of class two.
• Anticoagulant used: acid citrate dextrose or ACD.

Microscopy

• Microscopes a cdc, cdc is complement dependent cell cytotoxicity.
• Use inverted phase contrast microscope.
• Only dead cells are stained, appearing flat, large, dark, and non-refractile.
• Living/viable cells remain unstained: appearing small, bright, and refractile.

Interpretation

• 0-10% dead cells = negative result. 11-20% = doubtful negative. 21-50% = weak positive. 51-80% = positive. 81-100% = strong positive.

Cellular Method

• Mixed lymphocyte reaction (MLR): only detects HLA DR related antigens (MHC class II).
• Anticoagulant: ACD or heparin.

Immunoglobulins (Ig)

• Formerly called gamma globulins, they are now termed antibodies.
• Glycoproteins in the serum that constitute approximately 20% of plasma proteins.
• Composition: 86-98% polypeptide, 2-14% carbohydrate.
• Humoral component of adaptive immune response.

Functions of Antibodies

• Neutralize toxic substances.
• Facilitate phagocytosis (opsonization).
• Activate complement.
• Combine with antigens on cellular surfaces and cause their destruction.

Structure of Immunoglobulin

• Tetrapeptide structure made of four polypeptide chains.
• Two identical heavy chains (alpha, gamma, epsilon, or delta) determine the class (IgA, IgG, IgE, or IgD respectively)
• Two identical light chains (kappa or lambda).

Gene Coding

• Heavy chains coded in chromosome 14.
• Light chains coded in chromosomes 2 and 22.

Antibody Structure and Function

• Each chain has a single variable region and one or more constant regions.
• Heavy and light chains are held together by non-covalent forces and disulfide inter-chain bridges.
• Interactions only occur between a heavy and a light chain.
• Abnormal interactions include heavy-heavy or light-light chain interactions.
• The variable region is the first 110 amino acids of the light and heavy chains.
• Constant regions have the same amino acid sequences, while variable regions have varying amino acid sequences.

Hinge Region and Receptors

• The hinge region is located between CH1 and CH2.
• It is rich in proline and hydrophobic residues, providing antibody flexibility.
• CH2 contains the receptor for the complement.
• CH3 contains the receptor for phagocytes (Fc receptor).
• Fab stands for antigen-binding fragment.
• Fc stands for complement-binding fragment and crystallizes at 4 degrees Celsius.
• Only antibodies with gamma, delta, and alpha chains have hinge regions.
• These chains are paired to confer flexibility.
• IgMs do not have a hinge region; their heavy chains are paired to confer flexibility.

Immunoglobulin Variation

• Isotype variations are based on the type of heavy chain.
• Idiotype variations are based on the variable region.
• Allotype variations are based on minor amino acid sequence differences in the constant region.
• These variations give rise to different subclasses.

Fragmentation of Antibodies

• IGM is a pentamer; its J chain must be broken using 2-mercaptoethanol or dithiothreitol for fragmentation.
• Pepsin digestion cleaves the antibody below the hinge region, resulting in one F(ab)2 fragment and one Fc fragment.
• Papain digestion cleaves the antibody above the hinge region, resulting in three fragments.
• Pepsin digestion was introduced by Alfred Nisonov; papain digestion by Rodney Porter.

Immunoglobulin Properties: IgG

• IgG has the longest half-life, thus conferring long-term immunity.
• It has four subclasses: IgG1, IgG2, IgG3, and IgG4, in descending order of serum concentration.
• IgG is able to cross the placenta and enter fetal circulation.
• IgG coats antigens, acting as an opsonin, particularly IgG1 and IgG3.
• IgG activates the classical complement pathway, requiring antibody presence.
• IgG3 is the most efficient subclass for activating the classical complement pathway.
• IgG1 and IgG3 tend to bind to protein antigens, while IgG2 and IgG4 bind to polysaccharide antigens.

Immunoglobulin Properties: IgA, IgM, IgD, and IgE

• IgA is predominant in body secretions and is present in mother's milk.
• IgA has two forms: IgA1 (secreted) and IgA2 (secretory).
• IgA2 is a dimer, while IgA1 is a monomer.
• IgM can exist as a monomer on the surface of developing B cells or as a pentamer after antigen exposure.
• IgM is the first antibody formed by the fetus and appears during an immune response.
• IgM is most effective against gram-negative bacteria and in complement fixation and agglutination reactions.
• IgD has no inter-chain disulfide bonds between its heavy chains and plays a key role in antigen recognition by B cells.
• IgD functions as a receptor, signaling B cells for antibody production.
• IgE is the least abundant immunoglobulin in serum.
• IgE is found on the surface of basophils and mast cells and is involved in allergic and parasitic reactions.

Kinetics of Immune Response

• Lag phase: increase in antibody production.
• Plateau phase: antibody production equals antibody degradation.
• Decline phase: increased antibody degradation.
• Primary immune response: first encounter with a specific antigen, resulting in a longer lag phase and lower antibody titer (IgM).
• Secondary immune response: subsequent encounters with the same antigen, with a shorter lag phase, higher antibody titer (IgG), a longer plateau phase, and a more gradual decline.

Hybridoma Technique

• A hybridoma is an immortal cell capable of producing indefinite sequences of nucleotides.
• The hybridoma technique, introduced by Kohler and Milstein, involves fusing an activated B cell (plasma cell) with a myeloma cell.
• Polyethylene glycol is used as a surfactant to fuse the plasma cell with the myeloma cell.
• The selective medium should contain hypoxanthine.
• Hybridomas produce monoclonal antibodies, which exhibit exceptional purity and specificity.
• Mice are commonly used for the production of monoclonal antibodies.
• Monoclonal antibodies can identify and quantify hormones; are useful for tissue and blood typing, infectious agents, CD markers, tumor antigens, and delivering immunotherapy.

Monoclonal Antibodies for Cancer Therapy

• Rituximab targets the CD20 antigen on B cells and is used for B cell neoplasms.
• Alemtuzumab targets the CD52 antigen on mature lymphocytes and is used for chronic lymphocytic leukemia and T cell lymphomas.
• Panitumumab targets the epidermal growth factor receptor (EGFR) and is used for colorectal cancer.
• Trastuzumab targets the HER2 receptor and is used for breast and gastroesophageal tumors.
• Bevacizumab targets vascular endothelial growth factor (VEGF) and is used for glioblastoma, colon, lung, and renal cancers.
• Ipilimumab targets cytotoxic T lymphocyte antigen 4 (CTLA-4).
• Nivolumab targets programmed cell death protein 1 (PD-1) and is used for melanoma and colon cancer (molecules that block T cell activation and proliferation).
• Brentuximab and Vedotin and Trastuzumab emtansine (DM1) are antibody-drug conjugates directed against tumor-associated antigens (TSAs), such as CD30 and HER2.