Chapter 1: Collect immunological test sample

Immunological testing is a cornerstone of applied biology, supporting disease diagnosis, monitoring of immune status, and research in both clinical and environmental settings. In Kenya, professionals in hospitals, research laboratories, and quality control units rely on a deep understanding of immunological concepts to ensure accurate sample collection and analysis. The following chapter explores foundational immunology, covering essential terminology, cell types, and processes that underpin immunological testing. Mastery of these principles enables applied biology professionals to contribute effectively to public health, agriculture, and biomedical research across the country.

1.1 Introduction to Immunology

Immunology is the study of how an organism recognises and defends itself against infectious agents and other foreign substances. A technician in a biological research or teaching laboratory meets immunology daily: mice and rabbits are immunised to produce antibodies, sera are tested for antibodies by ELISA, vaccines are evaluated in animal houses, and blood cells are counted and sorted. Knowing how the immune system works is what allows a technician to collect the right specimen, choose the right test and read its result correctly. Example: a serum sample for antibody testing can give a wrong result if haemolysis interferes with the assay or repeated freezing and thawing has damaged the antibodies, and only a technician who understands antibodies will see why.

This topic builds the vocabulary and the basic concepts of immunology, from the terms and the kinds of immunity to the molecules, cells and organs involved and the ways in which the system can go wrong.

1.1.1 Terms Used in Immunology

Immunology has its own vocabulary, and the terms must be used exactly because results and procedures are recorded and reported with them.

Key immunological terms

• Immunity: the ability of an organism to resist infection and harmful substances by means of the immune system.
• Immune response: the coordinated reaction of cells and molecules to a foreign substance.
• Pathogen: a microorganism, such as a bacterium, virus, fungus or parasite, that can cause disease.
• Antigen: a substance that can be recognised by antibodies or by T cell receptors. It is usually a protein or a polysaccharide.
• Epitope: the small part of an antigen that an antibody or a receptor actually binds. One antigen carries many epitopes.
• Antibody: a protein made by B cells and plasma cells that binds a specific antigen.
• Lymphocyte: a white blood cell responsible for specific immunity, either a B cell or a T cell.
• Self and non-self: the immune system normally tolerates the body's own molecules (self) and responds to foreign ones (non-self).
• Immunological memory: the ability of the adaptive system to respond faster and more strongly to a second exposure to the same antigen.

1.1.1.1 Innate vs Adaptive

Innate immunity is present from birth, responds within minutes to hours and acts in the same way against many kinds of pathogen. Adaptive immunity develops after contact with a specific antigen, takes days to become effective and remembers the antigen. The two work together: the innate system holds the infection back and alerts the adaptive system, which then produces a specific and lasting response.

1.1.1.2 Active vs Passive

Active immunity is produced by the animal's own immune system after it meets an antigen, and it lasts for months or years. Passive immunity is obtained by receiving ready-made antibodies from another source, and it protects at once but lasts only for weeks, because the animal does not produce the antibodies itself.

• Natural active: immunity that follows infection.
• Artificial active: immunity produced by vaccination or by deliberate immunisation of a laboratory animal with an antigen.
• Natural passive: antibodies passed from mother to young across the placenta or in colostrum and milk. A newborn calf, for example, gets antibodies from colostrum because antibodies do not cross the placenta in cattle.
• Artificial passive: antibodies injected as antiserum or antitoxin, such as antiserum raised in rabbits or horses, or monoclonal antibodies.

1.1.1.3 Humoral vs Cell-mediated

Humoral immunity is the part of adaptive immunity carried out by antibodies in the body fluids, produced by B cells and plasma cells. It acts mainly against extracellular microbes and toxins. Cell-mediated immunity is carried out by T cells themselves: helper T cells coordinate the response and cytotoxic T cells kill infected cells. It acts mainly against viruses and bacteria that live inside cells.

• Humoral tests: measure antibodies, for example by ELISA, agglutination or precipitation.
• Cell-mediated tests: measure T cell activity, for example by skin tests, lymphocyte proliferation tests or flow cytometry.
• Cooperation: helper T cells are needed for most antibody responses, so the two arms cannot be separated completely.

1.1.2 Antigens, Haptens, and Immunogens

These three terms describe how a molecule relates to the immune response, and the difference between them matters when antibodies are produced in animals or detected in tests.

Antigens

An antigen is any molecule that is recognised by an antibody or a lymphocyte receptor. Proteins make the best antigens, followed by polysaccharides, and lipids and nucleic acids are weaker. Antigens on bacteria, viruses, parasites, pollen and transplanted cells are all recognised by the immune system.

Immunogens

An immunogen is an antigen that can actually start an immune response. All immunogens are antigens, but not all antigens are immunogens. Several properties make a molecule immunogenic:

• Foreignness: the molecule is clearly different from the host's own molecules.
• Size: molecules with a molecular mass above about 10,000 daltons are usually good immunogens, and very small molecules are not.
• Chemical complexity: proteins with many different amino acids work better than simple repeating polymers.
• Degradability: the molecule must be processed and presented by antigen-presenting cells.
• Route and dose: the route, amount and use of an adjuvant influence how strong the response is.

Haptens

A hapten is a small molecule, usually under about 1,000 daltons, that can be bound by an antibody but cannot start an immune response by itself. When the hapten is chemically linked to a large carrier protein, the combined molecule becomes an immunogen. The antibodies produced are able to bind the free hapten as well.

• Use in the laboratory: haptens such as dinitrophenol are linked to carrier proteins like bovine serum albumin or keyhole limpet haemocyanin to raise antibodies in rabbits or mice. These antibodies are then used to detect small molecules such as drugs, hormones and toxins.
• Hypersensitivity: some drugs act as haptens when they bind to body proteins, and this can lead to allergy.

1.1.3 Antibody or Immunoglobulins

Antibodies, also called immunoglobulins, are Y-shaped glycoproteins found in blood and tissue fluids. They are the products of B cells and plasma cells, and they are the main reagents and targets in serological testing.

Structure of antibodies

An antibody is made of four polypeptide chains: two identical heavy chains and two identical light chains, held together by disulphide bonds.

• Variable regions: at the tips of the arms, these regions form the antigen binding sites, and their shape decides which antigen the antibody binds.
• Constant regions: these regions determine the class of antibody and its effector functions, such as complement activation.
• Fab fragments: the two arms that bind antigen.
• Fc fragment: the stem, which binds to receptors on phagocytes and other cells.
• Hinge region: a flexible region that lets the arms move so that both can bind antigens at different distances.

Classes of immunoglobulins

• IgG: the main antibody of blood and tissue fluid, and the main antibody of secondary responses. In humans it crosses the placenta. It is also the antibody type that is usually raised in laboratory animals and purified for use in tests.
• IgM: the first antibody made in a primary response. It is a large pentamer, so it is very efficient at agglutination and at activating complement.
• IgA: found as a dimer in tears, saliva, milk and the gut, where it protects mucous surfaces.
• IgE: present at low concentration. It binds to mast cells and takes part in allergy and in defence against parasitic worms.
• IgD: found mainly on the surface of B cells, where it acts as an antigen receptor.

Functions of antibodies

• Neutralisation: antibodies bind toxins and viruses and block their attachment to cells.
• Opsonisation: antibody-coated microbes are taken up more efficiently by phagocytes.
• Agglutination and precipitation: antibodies cross-link particles or soluble antigens into clumps, the basis of many laboratory tests.
• Complement activation: antibody bound to a microbe starts the classical pathway of complement.
• Antibody-dependent cellular cytotoxicity: natural killer cells recognise antibody on a target cell and kill it.

1.1.4 Vaccines and Adjuvants

A vaccine is a preparation of an antigen, or of instructions to make an antigen, that is given to produce active immunity without causing the disease. Adjuvants are substances added to some vaccines to make the response stronger. Both are used in public health, in livestock and poultry production, and in research.

Meaning of vaccines

A vaccine works because it trains the adaptive system to recognise a pathogen, so that the memory cells made by vaccination respond quickly if the real pathogen is met later. Most vaccines must be stored at 2 to 8 °C, and keeping this cold chain unbroken from manufacture to use is essential to keep them effective.

Types of vaccines

• Live attenuated vaccines: contain living but weakened pathogens that multiply a little in the body. An example is the BCG vaccine. They give strong and lasting immunity, but are not suitable for animals with weak immune systems.
• Inactivated vaccines: contain killed pathogens. They are safe but often need boosters and an adjuvant.
• Subunit and recombinant vaccines: contain only selected antigens, such as the hepatitis B surface antigen made in yeast, so side effects are fewer.
• Toxoid vaccines: contain a toxin made harmless by chemical treatment, such as tetanus toxoid.
• Conjugate vaccines: link a polysaccharide to a protein carrier so that the immune system responds to the polysaccharide properly, the same principle as a hapten linked to a carrier.
• Nucleic acid and vector vaccines: mRNA vaccines and viral vector vaccines carry the instructions for the antigen, and the body's own cells make it.

Importance of vaccination

• Disease prevention: reduces illness and death from infectious disease in people and animals.
• Herd immunity: when enough individuals are immune, the spread of the pathogen falls and the unprotected are protected indirectly.
• Control of zoonoses: vaccinating animals reduces diseases that can pass to people.
• Research: vaccines are tested in animals to measure the antibody and T cell response before use elsewhere.

Role of adjuvants

Adjuvants are added to vaccines or to antigens used for immunisation to raise the strength and the length of the response, especially for weak antigens such as purified proteins.

• Aluminium salts (alum): widely used in vaccines. They hold the antigen at the injection site and stimulate innate cells.
• Oil emulsions: Freund's complete and incomplete adjuvants give strong antibody responses in rabbits and mice, but they cause marked tissue reactions, so complete adjuvant is restricted and used only where an ethics committee has approved it.
• Saponin-based and other newer adjuvants: are included in some modern vaccines to stimulate T cell responses.
• Functions: they create a depot that releases antigen slowly, activate innate immune cells, improve antigen uptake and allow a smaller dose of antigen.

1.1.5 Cytokines, Chemokines

Cytokines are small signalling proteins released by cells that change the behaviour of other cells. Chemokines are a subgroup of cytokines that attract cells to a site. Together they coordinate immune responses, since immune cells have no other means to communicate across a distance.

Cytokines: types and functions

• Interleukins: signal between white blood cells. For example, interleukin-2 stimulates the growth of T cells.
• Interferons: interferon-alpha and interferon-beta make nearby cells resistant to viruses, and interferon-gamma activates macrophages.
• Tumour necrosis factor: promotes inflammation and can kill some cells, and large amounts cause fever and shock.
• Colony-stimulating factors: direct the bone marrow to make more blood cells.
• Transforming growth factor-beta: damps down immune responses and helps tissue repair.

Chemokines: roles in immunity

Chemokines form a gradient in tissue so that cells move towards their highest concentration, a process called chemotaxis. Interleukin-8 (CXCL8) attracts neutrophils to a site of infection, and monocyte chemoattractant protein-1 (CCL2) attracts monocytes. They also guide lymphocytes into lymph nodes and organise lymphoid tissue.

Relevance of cytokines and chemokines in practice

• Measurement: cytokines are measured in serum or culture fluid by ELISA, by multiplex bead assays and by flow cytometry, to follow an immune response in a study.
• Disease markers: high levels of some cytokines mark infection, inflammation and autoimmune disease.
• Cytokine storm: a sudden excessive release of cytokines can cause tissue damage and shock.
• Research tools: cytokines are used to culture and activate immune cells, and drugs that block a cytokine are studied in animal models of inflammation.

1.1.6 Cells of the Innate and Adaptive Immune System

All immune cells originate from stem cells in the bone marrow. They are divided into those of the innate system and those of the adaptive system, but they communicate and depend on each other.

Cells of the innate immune system

• Neutrophils: the most numerous white blood cells in humans, which engulf and destroy bacteria and are the first to arrive at an infection.
• Monocytes and macrophages: monocytes circulate in blood and become macrophages in tissues, where they engulf microbes, clear dead cells and present antigens to T cells.
• Dendritic cells: capture antigens in tissue, move to lymph nodes and activate naive T cells, which makes them the main link between the two systems.
• Natural killer cells: kill virus-infected and abnormal cells without prior exposure.
• Eosinophils: attack parasitic worms and contribute to allergic reactions.
• Basophils and mast cells: release histamine and other mediators in allergy and inflammation.

Cells of the adaptive immune system

• B lymphocytes: recognise antigen with their surface antibody and, with help, develop into plasma cells.
• Plasma cells: produce and release large amounts of antibody.
• Helper T cells (CD4): coordinate the response by releasing cytokines and helping B cells and cytotoxic T cells.
• Cytotoxic T cells (CD8): kill cells infected by viruses and some abnormal cells.
• Regulatory T cells: restrain immune responses and maintain tolerance to self.
• Memory cells: long-lived B and T cells that respond rapidly when the same antigen returns.

How the cells work together

An infection begins with phagocytes and natural killer cells. Dendritic cells and macrophages carry antigen to the lymph nodes and present it to helper T cells, which then activate B cells and cytotoxic T cells. B cells become plasma cells that make antibodies, and the antibodies and cytokines direct the innate cells back to the infection. After the infection, memory cells remain.

1.1.7 Lymphoid Tissues and Organs

Lymphoid organs are the places where immune cells are made, matured and activated. Together with the lymphatic vessels, they form the lymphatic system, which drains fluid from tissues and returns it to the blood.

Primary lymphoid organs

Primary lymphoid organs are where lymphocytes are produced and become mature.

• Bone marrow: produces all blood cells, and B cells mature here in mammals.
• Thymus: T cells mature here and learn to tolerate self molecules. It is largest in young animals and shrinks with age.
• Bursa of Fabricius: in birds, B cells mature in this organ near the cloaca, which is how B cells got their name.

Secondary lymphoid organs

Secondary lymphoid organs are where mature lymphocytes meet antigen and are activated.

• Lymph nodes: filter lymph, and are found along lymphatic vessels. They swell during infection as lymphocytes multiply.
• Spleen: filters blood, removes old red blood cells, and responds to antigens in the blood.
• Mucosa-associated lymphoid tissue: includes the tonsils, Peyer's patches in the intestine and lymphoid tissue in the airways, guarding the mucous surfaces where most pathogens enter.

Functions of lymphoid tissues

• Development: the primary organs supply mature, tested lymphocytes.
• Antigen trapping: secondary organs collect antigen from tissue, lymph and blood.
• Activation: lymphocytes multiply and differentiate into effector and memory cells here.
• Storage: memory cells are kept in these tissues.
• Laboratory use: the spleen and lymph nodes of immunised animals are the source of cells for antibody-producing hybridoma work and for cell-mediated immunity tests.

1.1.8 Inflammation

Inflammation is the response of living tissue to injury or infection. It brings blood, fluid, proteins and white blood cells to the site to remove the cause and start repair. It protects the body, but if it is excessive or continues too long it damages tissue.

Signs of inflammation

• Redness (rubor): more blood flows to the area.
• Heat (calor): warm blood and raised metabolism warm the tissue.
• Swelling (tumor): fluid leaves the leaky vessels.
• Pain (dolor): mediators such as prostaglandins and bradykinin act on nerve endings.
• Loss of function: swelling and pain make it difficult to use the part.

Stages of inflammation

1. Injury or microbes release chemical signals and alert tissue macrophages and mast cells.
2. Histamine and other mediators widen the blood vessels, so more blood flows to the site.
3. The vessel walls become permeable and fluid and proteins leave the blood.
4. Neutrophils and later monocytes leave the blood and move to the site along chemokine gradients.
5. The phagocytes remove microbes and dead tissue.
6. Anti-inflammatory signals end the response and the tissue is repaired.

Outcomes of inflammation

• Resolution: normal structure and function return.
• Abscess: pus collects when infection persists.
• Chronic inflammation: the response continues and damages tissue.
• Fibrosis: scar tissue replaces normal tissue and can impair the organ.
• Systemic effects: fever, raised acute-phase proteins such as C-reactive protein and, when severe, shock.

Inflammation in the laboratory

Inflammation is studied in animal models. For example, an injection of carrageenan into a rat's hind paw produces a measurable swelling, which is used to test anti-inflammatory drugs. Paw volume is measured with a plethysmograph at set intervals.

1.1.9 Complement System

Complement is a group of more than 30 plasma and cell surface proteins that work in sequence to destroy microbes and clear immune complexes. It is part of the innate system and it is activated and directed by antibodies.

Components of the complement system

• C1 to C9: the main proteins, which are activated one after another in a cascade.
• C3: the central protein. Its fragment C3b coats microbes and C3a attracts cells.
• Anaphylatoxins: the small fragments C3a, C4a and C5a that cause inflammation and mast cell release.
• Membrane attack complex: the assembly of C5b to C9 that forms a pore in the membrane of a microbe.
• Regulatory proteins: these protect the body's own cells from complement damage.

Activation pathways

• Classical pathway: begins when C1 binds antibody that is attached to an antigen.
• Alternative pathway: is triggered directly on microbial surfaces without antibody.
• Lectin pathway: begins when mannose-binding lectin binds sugars on microbes.

All three pathways lead to the cleavage of C3.

Functions of the complement system

• Opsonisation: C3b coats microbes so that phagocytes take them up.
• Lysis: the membrane attack complex punches holes in the membranes of some bacteria and other cells.
• Inflammation: C3a and C5a attract and activate cells.
• Clearance of immune complexes: complement keeps antigen-antibody complexes from collecting in tissues.

Handling complement in the laboratory

Complement is fragile. To prevent complement from interfering with serological tests, serum is often heated at 56 °C for 30 minutes, which destroys it. Serum that is to be used as a source of complement is stored frozen at a very low temperature and thawed only once.

1.1.10 Hypersensitivity Reactions

Hypersensitivity reactions are immune responses that are excessive or inappropriate and damage the body's own tissue. They are divided into four main types, following the classification of Gell and Coombs.

Types of hypersensitivity reactions

• Type I (immediate): IgE bound to mast cells reacts with an allergen, and histamine is released within minutes. Allergic rhinitis, asthma and anaphylaxis are examples.
• Type II (antibody-mediated): antibodies attach to cells or tissues and destroy them through complement or phagocytes. Transfusion reactions and haemolytic anaemia are examples.
• Type III (immune complex): antigen and antibody form complexes that are deposited in tissue and cause inflammation. The Arthus reaction, first described in rabbits, is an example.
• Type IV (delayed): T cells, not antibodies, cause the reaction after one to three days. Contact dermatitis and the tuberculin skin test are examples.
• Type V: some classifications add a fifth type, in which antibodies stimulate a receptor, as in Graves' disease.

Laboratory diagnosis

• Skin prick and intradermal tests: detect type I sensitivity to a particular allergen, and tuberculin-type tests detect type IV sensitivity in animals.
• Serum IgE measurement: measures total and allergen-specific IgE by immunoassay.
• Direct antiglobulin (Coombs) test: detects antibody on red blood cells in type II reactions.
• Complement and immune complex assays: assess type III reactions.
• Lymphocyte proliferation tests: measure type IV responses to an antigen in culture.

1.1.11 Autoimmune Disorders

Autoimmune disorders occur when the immune system loses tolerance and attacks the body's own tissues. They may affect a single organ or many organs.

Mechanisms of autoimmunity

• Loss of tolerance: self-reactive T and B cells that should have been removed or controlled survive and become active.
• Molecular mimicry: a pathogen antigen resembles a self molecule, so the response to the pathogen also attacks the body.
• Genetic predisposition: certain inherited genes make some individuals more susceptible.
• Hormonal influence: many autoimmune diseases are more frequent in females.
• Environmental triggers: infections, drugs and other exposures can start the disease in a susceptible individual.

Common autoimmune disorders

• Rheumatoid arthritis: chronic inflammation of the joints.
• Type 1 diabetes mellitus: immune destruction of the insulin-producing beta cells of the pancreas.
• Systemic lupus erythematosus: attack on many tissues by antibodies against nuclear components.
• Multiple sclerosis: immune attack on the myelin sheath of nerves.
• Hashimoto thyroiditis: destruction of thyroid tissue.

Animal models help research on these disorders, for example the non-obese diabetic mouse for type 1 diabetes, the MRL/lpr mouse for lupus and experimental autoimmune encephalomyelitis in mice for multiple sclerosis.

Laboratory diagnosis of autoimmunity

• Autoantibody detection: ELISA or indirect immunofluorescence on cultured cells detects antibodies against self antigens, such as antinuclear antibodies.
• Inflammatory markers: C-reactive protein and the erythrocyte sedimentation rate are raised.
• Organ function tests: for example, blood glucose measurement in diabetes.
• Tissue examination: histology shows immune cells in the affected organ.
• Flow cytometry: identifies and counts the immune cell populations.

Chapter Summary

This chapter begins by introducing the foundational concepts of immunology, including the main terms that distinguish innate from adaptive immunity, active from passive responses, and humoral from cell-mediated mechanisms. It explores the roles of antigens, haptens, and immunogens in triggering immune reactions, as well as the structure and function of antibodies or immunoglobulins. The discussion extends to vaccines, adjuvants, cytokines, and chemokines, highlighting their importance in modulating immune responses. Key cellular players of both the innate and adaptive immune systems are described, along with the lymphoid tissues and organs that coordinate immune activity. The chapter also addresses the processes of inflammation, the complement system, types of hypersensitivity reactions, and autoimmune disorders. It then shifts focus to immunological specimens, detailing the various types such as serum, virology swabs, biopsy and necropsy tissues, cerebrospinal fluid, whole blood, urine, and sputum, before concluding with an overview of specimen processing techniques necessary for accurate immunological testing.