Final Revision Sheet

Immune tolerance

Ability to prevent immune response against host’s cells/tissues.

Central tolerance

Eliminates self-reactive lymphocytes in primary lymphoid organs

Peripheral tolerance

Eliminates self-reactive lymphocytes in secondary lymphoid organs/peripheral tissues

Commponents of peripheral tolerance

Tregs, clonal anergy, peripheral detection

Anergy

Functional inactivation of lymphocyte due to lack of co-stimulation (needed for complete activation and differentiation)

True or false: Self antigens induce the expression of co-stimulatory proteins

False

Molecular mimicry

Foreign substance with similar proteins to host causes cross-reaction

Neoantigens

Novel antigens found on cancer cells from genetic mutations, unique to tumour

Sequestered antigens

Hidden from immune system to prevent response against them, often in immunologically privileged areas

Epitope spreading

Immune response expands to include other epitopes released from tissue damage

Autoimmunity

Failure of immune mechanisms to eliminate self-reactive cells

Acute inflammation is an immediate __ response aiming to __, and mainly consists of _. It’s usually resolved when the _ is removed, leading to tissue _ and ___.

Chronic inflammation is a __ response that can be due to persistence of _ or failure of ___, and mainly consists of _, lymphocytes, and __. Chronic inflammation often results in tissue _ and ___.

short-term, clear stimulus, neutrophils, stimulus, repair, restoration of function, long-term, stimulus, acute inflammatory response, macrophages, plasma cells, damage, loss of function

Components of acute inflammation

Vasodilation, increased vascular permeability, migration of leukocytes/recruitment of cells

Cardinal signs of acute inflammation

Rubor, calor, tumor, dolor, functio laesa

What causes rubor and calor?

Vasodilation

What causes tumor?

Increased vessel permeability and fluid leakage

What causes dolor?

Chemical mediators stimulating nerve endings e.g. prostaglandins

Functio laesa is a consequence of…

Tumor and dolor

Immune response

Body’s defence mechanism to harmful substances/those not recognised as host’s own constituent

The primary antibody response is the initial reaction to a new antigen and is _ and _, with a longer _ period and lower peak _ levels and _. It is initated by _ and mainly produces _ antibodies.

The secondary antibody response is a _, _, more _ reaction upon _ to the antigen. It has a __ period and higher peak __ and _. It is _ initiated by ___ and mainly produces _ antibodies due to the presence of these.

slow, weaker, latent, antibody, affinity, naive B cells, IgM, faster, stronger, prolonged, re-exposure, shorter latent, antibody levels, affinity, memory B cells, IgG

Serology

In-vitro study of blood serum and its components to detect antigens or antibodies

Uses of serological tests

Diagnosis of disease, treatment evaluation, prognosis, quantitation of antigen and antibody

Polyclonal antibody production for lab use

Generated in animals by different B cell clones, produced as defence mechanism by plasma B cells when exposed to antigen

Monoclonal antibody production for lab use

Produced by a single clone of plasma B cells using hybridoma cell technology and mouse myeloma cell lines

Monoclonal antibody production steps

B cell and myeloma cell fusion, HAT medium selection, screening, antibody production

Hybridoma

Antibody-producing tumour cells; produce many copies of same antibody and grow easily in the lab

Hybridoma role

Large-scale monoclonal antibody production

Ouchterlony technique

Qualitative technique demonstrating double diffusion in two dimensions

Total identity

Partial identity

Non-identity

Single diffusion, one dimension

Oudin Procedure

Single diffusion, two dimensions

Radial immunodiffusion (Mancini technique)

Double diffusion, one dimension

Oakley-Fulthrope procedure

Direct Coomb’s test

Detects antibodies/complements already bound to RBCs

Direct Coomb’s test steps

RBCs washed to remove unbound antibodies, tested with anti-human globulin reagent, agglutination observed

Direct Coomb’s test use

Diagnosing autoimmune conditions, haemolytic disease, transfusion reactions

Indirect Coomb’s test

Detects antibodies free in serum, unattached to RBCs

Indirect Coomb’s test steps

Serum mixed with RBCs of known antigen type, agglutination observed

Indirect Coomb’s test use

Checks for antibodies that could cause reaction with RBCs; cross-matching for transfusions, prenatal screening

The complement fixation test detects the presence of ___ by observing whether a _  amount of complement is _ to an antigen-antibody _

specific antigens or antibodies, known, bound, complexes

Complement fixation test: Fixation phase

Serum + specific antigen, complement added

In the complement fixation test, the complement will only be fixed if…

both antigen and antibody are present

Complement fixation test: Detection phase

Sensitised sRBCs added, if complements unbound haemolysis occurs

Monoclonal antibodies are a _ population of antibodies, produced by ___ of plasma B cells.

homogeneous, a single clone

Monoclonal antibody specificity

Highly specific for a single epitope on an antigen

Monoclonal antibody production is…

consistent across batches(high reproducibility), expensive

Monoclonal vs polyclonal antibody sensitivity

Monoclonal: moderate

Polyclonal: high

Polyclonal antibodies are a __ of antibodies, produced by ___.

heterogeneous mixture, different B cell clones

Polyclonal antibody specificity

Lower specificity, bind to same antigen but possibly different eptitopes

Polyclonal antibody production is…

cheap, varies across batches (low reproducibility)

Control

Benchmark with known value or property, processed alongside patient samples

Controls ensure _ and _ of results and provide a ___. They can detect issues that could lead to __.

accuracy, reliability, point of comparison, false results

Precipitation is…

• 2 soluble reactants join to make 1 insoluble product

• Formation of lattices

• Soluble antigen type (proteins, toxins)

Agglutination is…

• Development of ag-ab complexes in the form of particle clumps

• Lattices of polyvalent antigens

• Particulate antigen type (cells, beads)

Which immunoglobulin is a particularly good agglutanin? Why?

IgM, pentamer shape = high avidity

Factors affecting rate of diffusion in a precipitation reaction

Temperature, molecular size, gradient, viscosity of medium, distance and surface area

Factors affecting rate of diffusion in a precipitation reaction:

Temperature

Higher = faster

Lower = slower

Factors affecting rate of diffusion in a precipitation reaction:

Molecular size

Larger = slower

Slower = faster

Factors affecting rate of diffusion in a precipitation reaction:

Viscosity of medium

Higher = slower

Lower = faster

Zone of antibody excess

Prozone

Initial, insufficient antigen for visible lattice formation

Zone of equivalence

Optimal ag-ab itneraction, maximal precipitation

Zone of antigen excess

Postzone

Precipitation declines

Precipitin curve: To achieve the optimal ratio…

antigen is slowly added to antibody solution

Components of complement fixation test

Patient serum, known antigen, complement source, indicator system

ELISA principle

Binding target analyte to labelled particle, producing measurable visible signal

Direct ELISA

• Enzyme-labelled primary monoclonal antibody binds to antigen

• Enzyme reacts with substrate to produce signal

Indirect ELISA (compared to direct)

Labelled secondary antibody binds to unlabelled primary antibody

Sandwich ELISA

Antigen bonded between capture antibody on well floor and labelled detection antibody

Competitive ELISA

• Antigen competes with labelled antigen for antibody binding

• Labelled secondary antibodies produce inverse signal

Fluorescent Immunoassay (FIA)

Fluorescent-labelled antibodies used for detection

Radioimmunoassy (RIA)

Radioactive isotopes used for sensitive antigen detection

Neutralisation

Binding antibody to epitopes/soluble molecules to inhibit thier ability to bind to host cells

Cell flow cytometry

• Fluorescent-labelled cells pass through laser in single stream

• Physcial and chemical properties are measured

Nephelometry

• Light passes through soln. with suspended particles

• Light scatters upon contact

• Intensity of scattered light is measured

Turbidimetry (compared to nephelometry)

Intensity of light that passes through (not scattered) is measured

What types of antigens can be used in neutralisation tests?

Viruses (whole or parts), bacterial toxins, viral proteins

Cell flow cytometry: Forward-scatter (FSC)

Light scattered in forward direction (very slight angle from laser beam)

Cell flow cytometry: Side-scatter (SSC)

Light scattered at 90º angle to laser beam

SSC light is influenced by…

granularity, internal complexity, structure of particle/cell

ELISA: Biotin

Molecular tag, binds to antibodies or other target molecules

ELISA: Streptavidin

Protein with extremely high affinity for biotin

ELISA: Streptavidin and biotin role

Signal amplification

Turbidimetry: Light absorbance

Light absorbed by sample

Turbidimetry: Light transmittance

Light passed through sample

Non-heritable influences affecting immunity

Exposure to microbes, diet, stress, sleep, environment

Heritable influences affecting immunity

WBC counts, cytokine concentration/profiles, variants within cytokine genes and immune-mediated disease

Circadian rhythm

Natural cycle regulating sleep-wake cycle patterns, other physical/mental/behavioural changes

Primary immunodeficiency

Usually genetic and congenital and apparent by 6 months of age when maternally-derived antibodies disappear

Primary immunodeficiency example

Severe Combined Immunodeficiency (SCID)

Secondary immunodeficiency

Usually caused by environment and may occur at any time in life

Secondary immunodeficiency possible causes

Infection, therapeutic treatments, cancer, malnutrition

Secondary immunodeficiency example

Acquired Immunodeficiency Syndrome (AIDS)

Most common clinical characteristics of immunodeficiency

• Recurrent/chronic infections

• Inability to clear infectious agents after standard antibiotic therapy

• Unusual infectious agents