Immunity -dr Shahat

*- Immune respons

: its characterized by the produuction of

proteins ( Igs) and specificially reactive lymphocytes (T-cells ) when an animal encounters a foreign macromolecules or cells. The inducing substances are called antigens i.e. antibody generators or immunogens. *- Immunogenicity

*- Antigenicity

: it the inherent ability of the immunogen (complete antigen ) to induce a specific immune response and to react with the products of this response (i.e. antibodies or the immune reactive lymphocytes) . : It is the ability of the foreign substance to react with the products of that response .

Therefore, Antigens are the ligands that react with the products of an immune response

Also, the immunogenicity & antigenicity

are two interchangeable terms which will be used during discussion of the immune reponse during the period of this course.

In addition, HAPTEN HAS AN ANTIGENICITY but HAPTEN PLUS PROTIEN CARRIER IS IMMUNOGEN

Overview of the Immune System Immune System

Innate (Nonspecific) 1o line of defense

Cellular Components

Humoral Components

Adaptive (Specific) o 2 line of defense Protects/re-exposure

Cellular Components

Interactions between the two systems

Humoral Components

Comparison of Innate and Adaptive Immunity Innate Immunity *

No time lag

* Not

*

antigen specific

No memory developed

Adaptive Immunity *

A lag period

*

Antigen specific

Memory developed

Functions of the Immune System (Self/Non-self Discrimination) • To protect from pathogens • •

Intracellular (e.g. viruses and some bacteria and parasites) Extracellular (e.g. most bacteria, fungi and parasites)

• To eliminate modified or altered self

Infection and Immunity Balance infection

Disease =

immunity

Bolus of infection x virulence immunity

Effects of the Immune System • Beneficial:

•

•

Protection from Invaders

•

Elimination of Altered Self

Detrimental: •

Discomfort and collateral damage (inflammation)

•

Damage to self (hypersensitivity or autoimmunity)

Innate (Nonspecific) Immunity

Innate Host Defenses Against Infection • Anatomical barriers – Mechanical factors – Chemical factors – Biological factors

• Humoral components – Complement – Coagulation system – Cytokines

• Cellular components – – – –

Neutrophils Monocytes and macrophages NK cells Eosinophils

Anatomical Barriers - Mechanical Factors System or Organ

Cell type

Mechanism

Skin

Squamous epithelium

Physical barrier Desquamation

Mucous Membranes Non-ciliated epithelium (e.g. GI tract)

Peristalsis

Ciliated epithelium (e.g. respiratory tract)

Mucociliary elevator

Epithelium (e.g. nasopharynx)

Flushing action of tears, saliva, mucus, urine

Anatomical Barriers - Chemical Factors System or Organ

Component

Mechanism

Skin

Sweat

Anti-microbial fatty acids

Mucous Membranes HCl (parietal cells) Tears and saliva

Low pH Lysozymes and Phospholipase A

Defensins (respiratory & GI Antimicrobial tract) Sufactants (lung)

Opsonin

Anatomical Barriers - Biological Factors System or Organ Skin and mucous membranes

Component Normal flora

Mechanism ☻Antimicrobial substances ☻Competition for nutrients and colonization

Natural immune response (i-Humoral Components) Component Complement

Mechanism ☻- Lysis of bacteria and some viruses ☻- Opsonin ☻- Increase in vascular permeability ☻- Recruitment and activation of phagocytic cells ☻- With the help of antibodies, they can destroy the pathogens

Coagulation system

☻- Increase vascular permeability ☻- Recruitment of phagocytic cells ☻- Β-lysine from platelets (a cationic detergent)

Lactoferrin and transferrin Lysozymes

☻- Compete with bacteria for iron, therefore, cause bacterial death.

Cytokines (Interleukins

☻- They had various immunological effects

, Interferon's)

☻- Breaks down bacterial cell walls causing their lysis.

Interferons (IFNs ) They are natural proteins produced by the cells of the immune system of most vertebrates in response to challenges by foreign agents such as viruses, bacteria, parasites and tumor cells. Interferons belong to the large class of glycoproteins known as cytokines.

;The discovery of interferon While aiming to develop an improved vaccine for smallpox, two Japanese virologists, working at the then Institute for Infection Disease at the University of Tokyo, noticed that rabbit-skin or testis previously inoculated with UV-inactivated virus exhibited inhibited viral growth inhibitory factor, and began to characterise it by fractionation of the UV.irradiated viral homogenates using an ultracentrifuge

:Functions of inteferons

.Interferons in general have several effects in common They are antiviral and possess antioncogenic properties ► Macrophage and natural killer lymphocyte activation, and enhancement of major ► histocompatibility complex glycoprotein classes I and II, and thus presentation of .foreign (microbial) peptides to T cells In a majority of cases, the production of interferons is induced in response to ► microbes such as viruses and bacteria and their products (viral glycoproteins, viral RNA, bacterial endotoxin, bacterial flagella, CpG DNA), as well as mitogens and , tumor12interleukin- ,2interleukin ,1other cytokines, for example interleukin necrosis factor and colony-stimulating factor, that are synthesised in the response .to the appearance of various antigens in the body

Their metabolism and excretion take place mainly in the liver and kidneys. They rarely pass the placenta and the blood-brain barrier

Type I interferons IFN-α and IFN-β are secreted by many cell types including lymphocytes )NK cells, Bcells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others. They stimulate both macrophages and NK cells to elicit and anti-viral response, and are also active against tumors. IFN-ω is released by leukocytes at the site of viral .infection or tumors

Type II interferons IFN-γ is involved in the regulation of the immune and inflammatory responses; in humans, there is only one type of interferon-gamma. It is produced in activated T-cells and natural killer cells. IFN-γ has some anti-viral and anti-tumor effects, but these are .generally weak 1 However, this cytokine potentiates the effects of the type I IFNs. IFN-γ released by Th cells recruits leukocytes to a site of infection, resulting in increased inflammation. It also stimulates macrophages to kill bacteria that have been engulfed. IFN-γ released cells is also important in regulating the1 by Th response. As IFN-γ is vitally implicated in the regulation of immune response, its 2 Th production can lead to autoimmune disorders

Autoimmune diseases i-What are autoimmune diseases? Our bodies have an immune system that protects us from disease and infection. But if you have an autoimmune disease, your immune system attacks itself by mistake, and you can get sick. Autoimmune diseases can affect connective tissue in your body (the tissue which binds together body tissues and organs). Autoimmune disease can affect many parts of your body, like your nerves, muscles, endocrine system (system that directs your body’s hormones and other chemicals), and digestive system. Autoimmunity is the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as "self", which results in an immune

response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmune diseases, therefore are a large group of diseases characterized by abnormal functioning of the immune system that causes your immune system to produce antibodies against your

own tissues - the prominent examples being Crohn's disease, Diabetes Type 1, Coeliac disease, Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and Rheumatoid arthritis (RA).

Prognosis of Autoimmune diseases Although autoimmune diseases are chronic, the course they take is unpredictable. A doctor cannot foresee what will happen to the patient based on how the disease starts. Patients should be monitored closely by their doctors so environmental factors

or triggers that may worsen the disease can be discussed and avoided and new medical

therapy can be started as soon as possible. Frequent visits to a doctor are important in order for the physician to manage complex treatment regimens and watch for medication side effects.

Who is at risk for getting autoimmune diseases? Most autoimmune diseases occur in women, and most often during their childbearing years. Some of these diseases also affect African American, American Indian, and Latina women more than white women. These diseases tend to run in families, so your genes, along with the way your immune system responds to certain triggers or things in the environment, affect your chances of getting one of these diseases. If you think you may have an autoimmune disease, ask your family members if they have had symptoms like yours. The good news is that if you have an autoimmune disease, there ARE things you can do to feel better!

What are the most common symptoms of autoimmune diseases? There are more than 80 types of autoimmune diseases. Learning the symptoms of some of the more common autoimmune diseases can help you recognize the signs if you get one. But some autoimmune diseases share similar symptoms. This makes it hard for doctors to find out if you really have one of these diseases, and which one it might be. This can make your trip to doctors long and stressful. The most common symptoms of the autoimmune diseases include tiredness, depression , sensitivity to cold, weight gain, muscle weakness and cramps, dry hair tough skin, constipation and sometimes there are no symptoms

Natural immune response ii-Cellular Components Cell

Functions

Neutrophils

☻-Phagocytosis and intracellular killing ☻- Inflammation and tissue damage

Macrophages

☻- Phagocytosis and intracellular killing ☻- Extracellular killing of infected or altered self targets ☻- Tissue repair ☻- Antigen presentation for specific immune response

NK and LAK cells

☻- Killing of virus-infected cells and altered self targets

Eosinophils

☻- Killing of certain parasites

Phagocytosis and Intracellular Killing

Phagocyte Response to Infection • The Signals –N-formyl methionine-containing peptides –Clotting system peptides –Complement products –Cytokines released by tissue macrophages

• Phagocyte response –Vascular adherence –Diapedesis –Chemotaxis –Activation –Phagocytosis and killing

Phagocytosis

Steps of Phagocytosis •Attachment

•Pseudopod extension •Phagosome formation •Granule fusion

•Phagolysosome formation

Initiation of Phagocytosis Attachment via Receptors: IgG FcR Complement R ScavengerR Toll-like R

Phagocytes - Neutrophils (PNMs)

☻- Characteristic

nucleus and cytoplasm

☻-

specific granules

☻-

CD 66 membrane marker

Phagocytes - Macrophages

•

Characteristic nucleus

•

Lysosomes

•

CD14 membrane marker

Natural Killer (NK) cells  Also known as large granular lymphocytes (LGL)

 Kill virus-infected or malignant cells  Identified by the presence of CD56 & CD16 and absence of CD3  Activated by IL2 and IFN-γ to become LAK cells

What are Natural Killer Cells? Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no immunological memory and are independent of the adaptive immune system. Natural killer cells constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and Most of us have enough natural killer cells (cell .).cancerous cells (AAA Reference Laboratories, Inc counts) in our body, however many of us don't have enough natural killer cells that are active. These inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue, but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly affect all of us .

What You Should Know about Natural Killer Cells Activity? It is known that: Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20 Many patients with chronic diseases including Fibromyalgia and Chronic Fatigue Syndrome have low levels in the range of 10 to 30. A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and others have low levels in the 10 to 30 range Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis, Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80 years of age which may partially explain why the risk of cancer increases with each decade of life Low natural killer cell activity is a significant independent risk factor for the future development of cancer ,as well as other chronic diseases and illnesses. Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients. Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for survival

Non-specific Killer Cells

NK and LAK cells ADCC (K) cell

Activated macrophages Eosinophils

They all kill foreign and altered self targets

Intracellular Killing Pathways a-Respiratory Burst a-1- Oxygen-Dependent Myeloperoxidase-Independent Reactions

+

Glucose +NADP

NADPH + O2 -

2O2 + 2H+

G-6-P-dehydrogenase

Pentose-P + NADPH

NADPH oxidase Cytochrome b558

NADP++ O2

Superoxide dismutase

H2O2 + 1O2

-

-

-

OH* + OH + 1O2

2O2 + H2O2

Toxic compounds are : Superoxide anion (O2 -), Hydrogen peroxide

(H2O2), Singlet oxygen (1O2) and Hydroxyl radical (OH*)

Respiratory Burst (continued) a-2- Oxygen-Dependent

H2O2 + Cl -

-

2OCl + H2O

Myeloperoxidase-Dependent Reactions

myeloperoxidase

-

OCl + H2O 1O 2

-

+ Cl + H2O

Toxic compounds: Hypochlorous acid (OCl-), and Singlet oxygen (1O2)

Respiratory Burst (continued) Detoxification Reactions Superoxide dismutase -

H2O2 + O2

2O2 + 2H+ Catalase 2 H2O2

H2O + O2

b-Oxygen-Independent Killing in the Phagolysosome Effector Molecule

Function

Cationic proteins (cathepsin)

Damage to microbial membranes

Lysozyme

Hydrolyses mucopeptides in the cell wall

Lactoferrin

Deprives pathogens of iron

Hydrolytic enzymes (proteases)

Digests killed organisms

Summary of Intracellular Killing Pathways Intracellular Killing

Oxygen Dependent

Myleoperoxidase Dependent

Oxygen Independent

Myleoperoxidase Independent

Nitric Oxide Dependent Killing

TNF TNF

Nitric Oxide Nitric Oxide

Lymphokine Activated Killer (LAK) cell

kills kills transformed malignant and malignant cells cells

Regulation of NK Cell Function

•MHC I •KIR

•No Killing

•KAR •KAL

•Killing

Their metabolism and excretion take place mainly in the liver and kidneys. They rarely pass the placenta and the blood-brain barrier

Type I interferons IFN-α and IFN-β are secreted by many cell types including lymphocytes )NK cells, Bcells and T-cells), macrophages, fibroblasts, endothelial cells, osteoblasts and others. They stimulate both macrophages and NK cells to elicit and anti-viral response, and are also active against tumors. IFN-ω is released by leukocytes at the site of viral .infection or tumors

Type II interferons IFN-γ is involved in the regulation of the immune and inflammatory responses; in humans, there is only one type of interferon-gamma. It is produced in activated T-cells and natural killer cells. IFN-γ has some anti-viral and anti-tumor effects, but these are .generally weak 1 However, this cytokine potentiates the effects of the type I IFNs. IFN-γ released by Th cells recruits leukocytes to a site of infection, resulting in increased inflammation. It also stimulates macrophages to kill bacteria that have been engulfed. IFN-γ released cells is also important in regulating the1 by Th response. As IFN-γ is vitally implicated in the regulation of immune response, its 2 Th production can lead to autoimmune disorders

Autoimmune diseases Autoimmunity is the failure of an organism to recognize its own constituent parts (down to the sub-molecular levels) as "self", which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmune diseases, therefore are a large group of diseases characterized by abnormal functioning of the immune system that causes your immune system to produce antibodies against your own tissues - the prominent examples being Crohn's disease, Diabetes Type 1, Coeliac disease, Systemic Lupus Erythematosus (SLE), Sjögren's syndrome and Rheumatoid arthritis (RA).

Prognosis of Autoimmune diseases Although autoimmune diseases are chronic, the course they take is unpredictable. A doctor cannot foresee what will happen to the patient based on how the disease starts. Patients should be monitored closely by their doctors so environmental factors or triggers that may worsen the disease can be discussed and avoided and new medical therapy can be started as soon as possible. Frequent visits to a doctor are important in order for the physician to manage complex treatment regimens and watch for medication side effects.

What are autoimmune diseases? Our bodies have an immune system that protects us from disease and infection. But if you have an autoimmune disease, your immune system attacks itself by mistake, and you can get sick. Autoimmune diseases can affect connective tissue in your body (the tissue which binds together body tissues and organs). Autoimmune disease can affect many parts of your body, like your nerves, muscles, endocrine system (system that directs your body’s hormones and other chemicals), and digestive system.

Who is at risk for getting autoimmune diseases? Most autoimmune diseases occur in women, and most often during their childbearing years. Some of these diseases also affect African American, American Indian, and Latina women more than white women. These diseases tend to run in families, so your genes, along with the way your immune system responds to certain triggers or things in the environment, affect your chances of getting one of these diseases. If you think you may have an autoimmune disease, ask your family members if they have had symptoms like yours. The good news is that if you have an autoimmune disease, there ARE things you can do to feel better!

What are the most common symptoms of autoimmune diseases? There are more than 80 types of autoimmune diseases. Learning the symptoms of some of the more common autoimmune diseases can help you recognize the signs if you get one. But some autoimmune diseases share similar symptoms. This makes it hard for doctors to find out if you really have one of these diseases, and which one it might be. This can make your trip to doctors long and stressful. The most common common symptoms of the autoimmune diseases include tiredness depression sensitivity to cold weight gain muscle weakness and cramps dry hair tough skin constipation sometimes there are no symptoms

What are Natural Killer Cells? Natural killer (NK) cells are an important first line of defense against newly arising malignant cells and cells infected with viruses, bacteria, and protozoa. They form a distinct group of lymphocytes with no immunological memory and are independent of the adaptive immune system. Natural killer cells constitute 5 to 16 percent of the total lymphocyte population. Their specific function is to kill infected and Most of us have enough natural killer cells (cell .).cancerous cells (AAA Reference Laboratories, Inc counts) in our body, however many of us don't have enough natural killer cells that are active. These inactive natural killer cells are present in great numbers in our blood, lymph nodes, organs, and tissue, but they are not killing foreign invaders such as infectious organisms and malignant cells that constantly affect all of us .

What You Should Know about Natural Killer Cells Activity? It is known that: Almost all cancer patients have very low levels of natural killer cell activity: usually 0 to 20 Many patients with chronic diseases including Fibromyalgia and Chronic Fatigue Syndrome have low levels in the range of 10 to 30. A wide variety of Auto Immune Disorders including Rheumatoid Arthritis, Lupus, Multiple Sclerosis and others have low levels in the 10 to 30 range Most patients with chronic and/or recurrent infections (such as Staph, Sinusitis, Bronchitis, Tonsillitis, Pneumonia, and ear infections, etc.) have low levels in the 10 to 50 range Many patients with symptomatic EBV, CMV, HPV and other chronic viral infections are in the 0-20 range It is also known that, there is a direct age related decrease in natural killer cell activity from 20 to 80 years of age which may partially explain why the risk of cancer increases with each decade of life Low natural killer cell activity is a significant independent risk factor for the future development of cancer ,as well as other chronic diseases and illnesses. Also, low natural killer cell activity is a strong predictor of poor prognosis of survival for cancer patients. Therefore, the higher the natural killer cell activity in patients with cancer the better their prognosis is for survival

Complement: History Discovered in 1894 by Bordet

It represents lytic activity of fresh serum Its lytic activity destroyed when heated at 56C for 30 min

Complement functions • Host benefits: – – – – – –

Opsonization to enhance phagocytosis Phagocyte attraction and activation Lysis of bacteria and infected cells Regulation of antibody responses Clearance of immune complexes Clearance of apoptotic cells

• Host detriments: – Inflammation, anaphylaxis

Proteins of the complement system (nomenclature) • C1(qrs), C2, C3, C4, C5, C6, C7, C8, C9 • factors B, D, H and I, properdin (P)

• mannose binding lectin (MBL), MBL associated serine proteases (MASP-1 MASP-2) • C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF), Complement receptor 1 (CR1), proteinS (vitronectin)

Definitions • C-activation: alteration of C proteins such that they interact with the next component

• C-fixation: utilization of C by Ag-Ab complexes • Hemolytic units (CH50): dilution of serum which lyses 50% of a standardized suspension of Ab-coated r.b.c

• C-inactivation: denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity

• Convertase/esterase: altered C-protein which acts as a proteolytic enzyme for another C-component

Activation product of complement proteins (nomenclature) Activated component are usually over-lined: e.g. C1qrs When enzymatically cleaved, the larger moiety, binds to the activation complex or membrane and the smaller peptide is released in the microenvironment Letter “b” is usually added to the larger, membrane-binding, peptide and “a” to the smaller peptide (e.g., C3b/C3a, C4b/C4a, C5b/C5a), EXCEPT C2 (the larger, membranebinding moiety is C2a; the smaller one is C2b)

Pathways of complement activation CLASSICAL PATHWAY antibody dependent

LECTIN PATHWAY

ALTERNATIVE PATHWAY

antibody independent

Activation of C3 and generation of C5 convertase activation of C5

LYTIC ATTACK PATHWAY

Components of the Classical Pathway

C3 C1 complex

C4

Classical Pathway Generation of C3-convertase

Classical Pathway Generation of C3-convertase

_____ C4b2a is C3 convertase

C4b

Classical Pathway Generation of C5-convertase

________ C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway

C4b

C3b

Biological Activities of Classical Pathway Components Component

Biological Activity

C2b

Prokinin; cleaved by plasmin to yield kinin, which results in edema

C3a

Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead to anaphylaxis

C3b

Opsonin Activation of phagocytic cells

C4a

Anaphylaotoxin

C4b

Opsonin 54

C1-inhibitor deficiency: hereditary angioedema

Components of mannose-binding lectin pathway

MBL

MASP1

Mannose-binding lectin pathway _____ C4b2a is C3 convertase; it will lead to the generation of C5 convertase MASP1

MBL

Components of the alternative pathway

C3

Spontaneous C3 activation

Generation of C3 convertase

C3 i

b

C3b

C3iBb complex has a very short half life

C3-activation the amplification loop If spontaneously-generated C3b is not degraded

C3b

b

C3 b

C3-activation the amplification loop

C3 b

C3b

b

C3b

General Introduction The immune system is a set of mechanisms that protect an organism from◙ .infection by identifying and killing pathogens This task is extremely difficult, since pathogens range from viruses to parasitic worms ◙ .

and these diverse threats must be detected with absolute specificity amongst normal cells and tissues

Pathogens are also constantly evolving new ways to avoid detection by the immune ◙ .system and successfully infect their hosts .To meet this challenge, multiple mechanisms have evolved to recognize and neutralize pathogens

These mechanisms include antimicrobial peptides called defensins, pattern ◙ .recognition receptors, and the complement system More sophisticated mechanisms, however, developed relatively recently, with the ◙ evolution of vertebrates. The immune systems of vertebrates such as humans consist of many types of proteins, cells, organs, and tissues, which interact in an .elaborate and dynamic network As part of this more complex immune response, the vertebrate system adapts over ◙ .time to recognize particular pathogens more efficiently The adaptation process creates immunological memories and allows even ◙ more effective protection during future encounters with these pathogens.This

.process of acquired immunity is the basis of vaccination

◙

General Introduction (continued) ◙Disorders in the immune system can cause diseases. ◙Immunodeficiency diseases occur when the immune system is less active than normal, resulting in recurring and life-threatening infections. ◙Immunodeficiency can either be the result of a genetic disease, such as severe combined immunodeficiency, or be produced by pharmaceuticals or an infection, such as the acquired immune deficiency syndrome (AIDS) that is caused by the retrovirus HIV . ◙In contrast, autoimmune diseases result from a hyperactive immune system attacking normal tissues as if they were foreign organisms. Common autoimmune diseases include rheumatoid arthritis, diabetes mellitus type 1 and lupus erythematosus. ◙ Therefore, immunity or the resistance is the sum of all naturally occurring and acquired defense mechanisms that protect the organism from infectious diseases.and the study of this mechanisms that a host has evolved to get rid itself of pathogens and other foreign substances. ◙The immune system so, has at least three major functional properties that distinguish it from all the body's other defenses:

Immunity (resistance): It the sum of all naturally occurring defense mechanisms that protect the organism (or host) from infectious diseases. In addition, it include the study of the mechanisms that a host has evolved to get rid itself from the invading pathogens and other foreign substances.

. The immune system so, has at least three major functional properties that distinguish it from all the body's other defenses:

The first: Is its extreme specificity, the ability to recognize and distinguish among a large or vast number of different target molecules, and to respond (or not respond) to each of these individually

Second: The immune system discriminates between self (body ingredients ) and non self ( foreign bodies), so that it normally coexists peacefully with all of the immunerable proteins and other organic materials that make up the host but responds vigorously against foreign substances, including cells or tissues from other people .

Third:

The immune system has memory, that is, the ability to be molded by its experiences so that subsequent encounters with a particular foreign pathogen provoke more rapid and more vigorous responses than occurred at the initial encounter.

A- Non a specific or innate immune response: This consists of the pre-existing defenses of an animal, such as barrier layers and secretions. It has the following properties: i- It does not exhibit high specificity. ii- It does not depend on a complete (specific) recognition of the antigen. iii- A single mechanism protect against many pathogen.

B- Specific or adaptive immune response: This response involves the cells of the immune system and frequently leads to a state of immune memory, and finally destroying the invading organisms.

Comparison between the non-specific and the specific immunity Non-specific Immunity Response is antigenindependent (Not antigen-

Specific Immunity Response is antigendependent (antigen-specific)

specific)

There is immediate maximal response Exposure to the Pathogen did not produce immunological memory

There is a lag time between exposure and maximal response Exposure to the Pathogen produce immunological memory

2- The Non-Specific (Innate Immune) Response 1- First defense line: a-Anatomical barriers: - Skin which physically preventing the interaction between the host and the pathogen. - Intestinal movement and mucus coating their walls. - Oscillation of broncho-pulmonary cilia.

b-secretory molecules: -These secretions include organic acids in skin secretions, thiocyanate in saliva, low molecular weight fatty acids, bile acids in lower gastric intestinal tract, transferring, lactoferrin, lyzozyme, interferons, fibronectin, complement, etc. in serum, interferons and tumor necrosis factor at the site of inflammation.

2. Second defense line: They represent the Cellular components, and they include phagocytic cells either polynuclear phagocytes or mononuclear phagocytes and NK cells.

Polynuclear phagocytes: Neutrophils (Polymorph nuclear cells PMNs) are the most important cellular components in bacterial destruction. They are relatively large and most abundant white blood cells (65% of leucocytes) with lobed nucleus and cytoplasmic granules (lysosomes All phagocytic cells have receptors for a variety of molecules. Most pertinent to non-specific immune response are receptors for IgG-Fc, complement, interferon, TNF and certain bacterial components. Receptor interactions with these ligands promote phagocytosis and activation for efficient killing of pathogens

The figure shows a Neutrophil in a blood film

Example of Phagocytosis: A macrophage attacking E.coli

Chemotactic response to inflammatory stimulus And the steps of this type of response 1- Adherence 2- Diapedesis 3- going to the inflammatory site 4- Re-activation of adherence via histamine and thrombin secretions.

Histopathology of bladder shows eggs of Schistosoma haematobium surrounded by intense infiltrates of eosinophils

3-Front defense line:

The major physiologic roles of natural killer cell (NK cells) appear to be in the early host defense against microbial agents. Nk cells, therefore, help to protect against a range of infections before the T-cell and B-cell response have developed. NK cells may thus function as a bridge between the innate and the acquired immune systems, acting as a front line of defense , while producing cytokines to promote the development of a specific immune response.

NK cells and their activation

1- Derived from bone marrow. 2- Lack most markers for T and B cells (no TCR or CD3). 3- Don’t undergo thymic maturation. 4- Express CD56, a specific NKs marker 5- Express a low affinity receptor for Fc portion of IgG called FcR (CD16), also expressed on granulocytes and macrophages. 6-Cytokines especially IL -2 promotes further differentiation in to lymphokine – activated killer cells (LAK).

Acute-phase Response Most soluble mediators of innate immune response are found in relatively small amounts, with the exception of C3, in the serum under normal conditions. The concentrations of several of these proteins, however, can increase as much as 1000-fold during serious infections, as part of accordinated protective reaction called the hepatic acute-phase-response. In this response, the liver temporarily increases its synthesis of more than adozen different serum proteins that participate in antimicrobial defense, including complement factors C3 and B, the mannose binding proteins, C-reactive protein, serum amyloid protein P, and others. The response occurs when hepatocytes are exposed to certain cytokines

3.1 Cells of the immune system

Immunity (resistance): It the sum of all naturally occurring defense mechanisms that protect human from infectious disease

Non – specific

Naturally acquired

( Innate )

Specific ( Acquired )

- Mucous membranes - Phagocytic cells

- Placental transfer of antibodies( Passive )

- Enzymes in secretion

- Recovery from disease ( Active )

-Interferons ( α,β,γ) -

- Administration of antitoxin ( Passive )

- NKCs-

- Vaccinations ( Active )

-Skin. Macrophages-

Artificially acquired

Natural ( Innate )

Specific ( Adaptive )

-Less specific . - Skin & mucous membrane .

or (Acquired)

-NK cells .

- Complement cascade . - Phagocytosis . - C- reactive protein .

Active

Passive

-Induced by contact with foreign antigens .- Consist of clinical infection , immunization with live or -

killed infectious agents or their toxins . -

- Long term.

-

- Induced by antibody performed in -

another host- Ab injected in the incubation period - Short term .

Artificially acquired

Naturally acquired

active

passive

active

passive

First: Non – specific Immunity ( Innate) :-

This is a physiologic mechanism which is inherent or innate with the following properties

It does not exhibit specificity

Do not depends on specific recognition of a foreign material

A single mechanisms Protect Against many paths

Definition :- the body forms his OWN IMMUNITY when stimulated (sensitized ) by introduction of immunogenic agent. Natural Types

Artificial

Infection *living attenuated vaccine * killed vaccine . bacterial products *Endotoxins. * Exotoxins.

Others . Characters :- * slowly developed . *longer duration (and leave a potential immunity , so there is A rapid response in the future to the Same antigen ) leads to ?? *-Homogenous antibodies *- Cellular defense mechanism play a role

Mechanism of Acquired immunity :Humeral Cellular

Ab T_Cells

classification of acquired Immunity:- passive Acquired Immunity :Definition: acquired Immunity by given already form antibodies or antitoxic serum or gamma globulins from normal or convalescent individuals or Trans placental or lactation . Trans placental . Natural Types Lactation (Colostrum). Antitoxin serum tetanus. (Anti_ cobra venom) Artificial Gamma globulins.

- characters :- * -Rapidly developed . * -Short duration . [ Rapidly eliminated in 2-4 WKS due to the formation of anti – antibodies (a disadvantage )]. *-Heterogeneous antibodies . * -Cellular mechanism not stimulated . (No memory ). * - Side effects:*- Hyper sensitivity reactions against the foreign serum *-Neurological affection in some cases ( Encephalitis ). *-Superadded in infections e.g. (AIDS & HEPAT) .

- Humoral immunity

- Cellular immunity .

Antibody mediated immunity.

- Cell mediated immunity .

( B- lymphocyte)

- (T- lymphocytes-Mediated)

help

help CD4

B- lymphocyte

Helper T- LYMPH

CD8

.

Cytotoxic T-lymphocyte

(Protection is mediated by the produced antibodies)

TH1

TH2

Haematopoietic stem cell

Lymphoid stem cell

B-lymphocyte

monocyte

NK cell

Myeloerythroid progenitor

T-lymphocyte

neutrophil eosinophil macrophage basophile

RBC

platelets

* BLymphocytes

This cell type consists ( 20 – 25%) of the total peripheral lymphocytes n mammals , they mature in bone marrow , then, migrate to secondary lymphoid organs ( e.g. spleen & Lymph nodes ) . * Upon exposure to antigen , B-Lymphocytes are stimulated to proliferate , (large lymphocytes) differentiate and mature into LARGE PLASMA CELLS * The large mature B-Lymphocytes have short life span ( days to weeks ) . Immunoglobulins or Humoral antibodies L-CHAIN

Antibody H-CHAIN

Secreted

* Some large mature B-Lymphocytes (B- cells ) can be converted into small B- cells which have long life span

Secondary Immune Response

This type of cells is involved in the Memory cells

And serve as

Activation & differentiation of B-Lymphocytes , in certain instances , needs a Helper T- Lymphocytes activity to enhance the above to

processes in that B-Lymphocytes.

T-Lymphocytes :*- THEY CONSTITUTE 65 -80 10 of total peripheral lymphocytes . *- They have long life span ( months to years ). *- They mature in thymus gland before migrating to lymphoid organs *- Upon exposure to antigen , T -cell proliferate .

How ever , their specific effectors molecules are not secreted and remains firmly Attached to their cellular membranes Giving what is called cell-mediated immune response

*- They are involved in a variety of cell-mediated immunological responses defense against malignant cells

graft rejection hyper sensitivity reactions

bacteria & protozoa Fungi

viruses

T-CELLS

T-HELPER (TH) T-SUPRESSOR(TS) T-CYTOTOXIC (TCs) T- Helper :Their Surface Antigen : is T4 (CD4) . *They Promote Maturation Of Antigen . *Stimulated B and T cells. And Enhance their response T – suppressor cells: * Their Surface antigen is T8(CD8). * they suppress the effect of T – helper cells . i.e. *Suppress T &B – response . T –cytotoxrc: * their Surface antigen T8(CD). * they specifically destroy target cells. virus infected cells

T-DELAYED HelperSensitivity Cells ( TdH) and T-CELL MEDIATED IMMUNITY (Tcmi )

unacceptable grafted cells

tumor cells T – delayed hypersensitivity & T cell mediated immunity.CD4 (T4) *they are responsible for delayed hypersensitivity reactions to different antigens , particularly those of intra cellular parasites & contact allergen . In general : * some of the stimulated T-cells release soluble substances lymphokines that modulate the behavior of other cells.

*- most antigens which have a small number of epitopes and require carrier need T – cell cooperation with B- cells for antibodies production . * Deficiency of B – cells (and\or) T-helper cells leads to defective synthesis of antibodies. * its over activity lead to

Autoimmune

disorders

the majority of B-lymphocytes express both surface IgM & IgD, very few express surface IgG & IgA or IgE in the circulation. *the majority of B-cells also carry class 2 major histocompatibility complex )class П MHC) products which are functionally important in

Regulation of immune response

2- T cell Activation When a T cell encounters an antigen presenting cell (APC), the specificity of its TCR determines the outcome. Only if the TCR recognizes its particular antigen MHC combination does activation occur. The recognition of appropriately presented antigen activates T cells to proliferate, differentiate and perform their effector functions. Activation of helper T-cells leads to the production of lymphokines that promote cellular and humoral immune responses, whereas activation of cytotoxic T cells results in killing of the antigen bearing cells.

Co -operation of innate & specific Immunity in Host defense against infection *Antibodies promote Phagocytosis or activate complement to kill microbes

*T-lymphocytes enhance phogocytic and microbial functions of macrophages

INNATE IMMUNITY

SPECIFIC IMMUNITY complement In direct lyses by C.

+ BACTERIA PHOGOCYTE

+

PHOGOCYTE INEFFECTIVE

Opsonization B-Lymph

BACTERIA

And Phagocytosis

Direct lyses

BACTERIA

+ SERM Bacterial lyses COMPLEMENT

+ BACTERIA

B-Lymph

+ BACTERIA

a Ab b s

LYSIS

Cell

Mediated PHOGOCYTE

bacteria

T-Lymph

response

Embryo Liver

stem cell

In

Bone marrow

central or primary lymphoid organs (tissues)

Secondary Lymphoid Organs Spleen or Bone marrow

+

+

A9 Effector Killer cells

A9 T_Cells

B_Cells

PLASMA memory cells B

T

CELLS HUMORAL ANTIBODIES

Specific memory and self-limitation of Immune response Ag A Serum

Secondary anti A

infection

AB

Primary Anti A RESPONSE

weeks

*- Antigen

enhance

response

12 weeks

THE production of specific Antibody A.

*- the secondary response to Ag A is more rapid and larger then the primary response ( memory cells ) . *- Antibodies Titer decline ( with time ) after each immunization .

Specific immune response : It is developed as a result of exposure to a variety of agents capable of inducing an immune response ( i.e. immunogens ) vaccines

microbes that colonize

A special case

in the body Antigen

Macro molecules in the diet

in the form of hapten

Hapten is a micomolecule may be conjugate with a carrier protein in the blood to be immunogen (antigen) Specific immune response

Humoral

cellular

B. Cells

T-CELLS

*- They are two interrelated & interdependent mechanisms .

Specific immune response can be further Classified according to its components into primary

secondary

Initial exposure to a particular

on farther or

Infectious agent or immunogen

repeated exposure

Induction phase of lymphocytes

to antigen ( same )

proliferation

T-CELLS B-CELLS

PLASMA CELLS

increased resistance

Sensitized T-CELLS Cellular Immune response

Antibodies humoral

Humoral response

develops through

Cellular response

Acquired immune response Has both good ( desirable ) and Bad ( undesirable ) consequence

undesirable

Desirable

Protection

Allergies (hypersensitivity)

From infections agents

Control of Pre-cancerous growths

Immune response

Autoimmune diseases

Graft rejection

Interactions & functions of the major components of the immune system

ANTIBODY – MEDIATED IMMUNE RESPONSE

CELL MEDIATED REPONSE Two major components

Main defense against * exteracellular, encapsulated pathogenic bacteria e.g. streptococci & staphylococci *Neutralizations of toxins e.g.( • tetanus)

* viruses ) Hepatitis C,A,B…….(•

T-HLPER & MACROPHAGES

Cytotoxic

Intracellular bacteria

T-CELLS

* ( mycobacterium &tuberculosis) Viruses * Fungi Acts by Destroying Virus- infected cells

T-CELLS

B-CELLS LYMPHOKINES IL-2,IL-4.IL-5

HELPER

CYTOTOXIC

CD4

CD8

PLASMA CELL IL_2

IL_2

ANTIBODIES +

Neutralize Toxins

ACTIVATED HELPER

Activated

AND MACRO PHAGES

Cytotoxic cells

INHIBIT

Kill

INTRACELLULAR

Virus – infected

Bacteria

cells

COMPLEMENT + NEUTROPHILS

KILLING OF BACTERIA

& fungi

VIRUS infection cell MHC Class I T-Cell receptor virus MHC Killing IgM

CD8 CYTOTOXIC

Class II T-Cell

RECEPTOR B-CELLS CD4

INTERLEUKIN-4

( T_HELPER )

INTERLEUKIN-5 VIRUS ANTIGEN VIRUS

Defense mechanism against viral infection

*- Recognition of phases :- antigen recognition ( binding of Ag to specific receptor on mature lymphocyte ( exist prior to ag exposure ) *- activation proliferation & differentiation of lymphocytes is the sequence of events induced in lymphocytes as result of Ag recognition . *- Effectors phases

elimination of antigen [ is the stage of the response

At which the sensitized cells perform the function that

(eliminate of Ag)

Some antigen – stimulated lymphocytes die by process called programmed cell death ( apaptosis ). Elimination T B

OF Ag OR

+ Ag

Phagocytosis

NATIVE

complement

LYMPHOCYTES Recognition

ACTIVATION

phase

PHASE

Effector programmed cell Death

phase

Immunogenicety

ability to induce immune response

Antigenicity immune system

ability of the substance to react specifically with

must be Immunogenic

Antigenic are not necessary to be

Happen is incomplete antigen ( di nitro phenol or penicillin)

It cannot stimulate humoral or cellular reactions but can react with these products specifically so it is Antigenic not immunogenic If they reacted with larger carrier protein (e.g., albumin , globulin or synthetic poly peptide ) . It will be Immunogenic Animals injected with this hapten – protein Complex will make

antibodies to this hapten ,

Only if it is ( hapten ) covalently linked to the carrier (chemically bonded)

PRTOCAL

CARRIER PROTEIN

HAPTEN

ANTIBODY

i

NO

YES

NO

II

YES YES ( not chemically linked) YES (CHEMICALLY LINKED )

NO YES

Anti carrier only Anti carrier only

YES

Anti carrier & Anti hapten

III IV

Immune response :- its characterized by the production of proteins

called immunoglobulins( Igs) and specificially reactive lymphocytes (Tcells ), which carry their own effector molecules on their surfaces, when an animal encounters a foreign macromolecules or cells . The inducing substances are called antigens i.e ( antibody generators ) or immunogens *- Immunogenicity & antigenicity : Interchangeable terms used during discussion of the immune reponse. *- Immunogenicity : it the inherent ability of asubstance ( Immunogen ( complete antigen ) to induce a specific immune response and to react with the product of this response . *- Antigenicity : the ability of the inducing substance (Antigen) to react with the products of the immune response (i.e. the antibodies and|or the effector molecules of the T-lymphocytes). . HAPTEN HAS AN ANTIGENIC Properties but the HAPTEN PLUS PROTIEN CARRIER IS IMMUNOGEN Antigens are the a ligands that react with the products of an immune response .

Hapten-carrier conjugates have native antigenic determinants of the carrier as well as new determinants of the hapten

Epitope ( - antigenic determinants ) :are the sites either (on or) within the antigen with which antibodies or T-cells receptor reacts paratope :- the sites on antibodies which react with the antigen . epitope

size ( small ) conformational site

linear conformational are on antigen surface or internal that expressed only when the antigen has been partially degraded in vivo

valency of antigen :- e.g multivalent i.e the antigen molecule carry a number of different epitopes ( some times 2 or>)

some of which specify antibody A others specify antibody B . valency = total no . of epitopes the antigen pocesses .

Antigenic determinants are usually limited to those portions of the antigen that are accessible to antibodies shown in black for this iron-containing protein

EPITOPE (ANTIGENIC DETEREMNANT):The portion of Ag that binds specifically with the binding site of Ab (paratope) or a receptor(s) on T_lymphocyte

SIZE CONFORMATIONAL STRUCTURE The size and the structure of epitope are complementary to that of paratope .i.e. they must have approximately the same dimensions WITH RESPECT TO THEIR STRUCTURE ,A g MAY HAVE THE FOLLOWING CHRACTES :Ag may have only a single epitope of a given specificity on its surface which is capable to bind with antibodies , such Ag is called UNIVALENT AND UNIDETRMINANT (one kind of specificity ) for example hapten Ag may have two or more epitopes (which determine the specificity ), the A g in this case is called MULTIVALENT (which determine the number). If the epitopes are of the same type, the Ag is called also UNIDETERMINANT (UNIDETERMINANT MULTIVALENT and if they are of different types called MULTIDETERMINANT (specificity, MULTIDETERMINANT- MULTIVALENT ).

UNIVALENT UNIDETREMNANT

MULTIVALENT UNIDETREMINANT

MULTIVALENT MULTIDETRMENANT

In an antigen, the same antigenic determinant repeated many times

T-dependent antigens are characterized by a few copies of many different antigenic determinants

.factors Affecting Immunogenicity

Foreigness

chemical complexity

molecular size

A – foreigness :the immunogenic substance must be forign to prduce immune response . The greater the foreignness, the more will be the reponse *- identical twins smaller or no response *- brothers with the higher immune response same tissues compatibility the same blood groups .etc …………. B.CHEMICAL COMPLEXITY :*- MOST of organic molecules are immunogenic expert lipids *- proteins are the strongest immunogenic substance . *-Polysaccharides most of them are haptens but they become complete Ag in cases of * peneumococcal polysaccharide . * Lip polysaccharides in cell membrane of gram (– ve ) bacteria.

*- Glycoprotein's :Are immunogenic ex blood group Ags ( A,B,AB,O,RH ) *- POLYPEPTIDES & nucleic acids :-

Are weak immunogens *- lipids :- are not antigenic or immunogenic C.molecular size :-

usually the larger the molecule

M.Wt

below 5000 DA

MACRO MOLECULES

ARE NOT IMMUNOGENIC

are the most potent immunogens .

( e.g. albumin

m.wt 40.000 Da

Globulin

m.wt 160 kDa

Macrocyanin

the stronger the Immunogenicety .

m.wt 1000 kDa

The valence of A.g increases proportionally with molecular size . Macro molecules are easily to induce phagocytic ( as example ) and easier to be phagocytosed Quaternary structure are the most Immunogenic The more complexity , the more Immunogenicety

Superantigens activate a large fraction of T cells in contrast to conventional Tdependent antigens

MULTIVALENT since it has only one kind of determinant but many of such determinant on each molecule Ex. Many poly saccharides & homo polymer (e-g peptide chain of the some .A. Acids .) *- some antigens are multi determinant & valent such molecules have many epitopes of different kinds (multi specificity ) but only one of each kind ( mono valent ) Ex. Most proteins . *- High M.WT , chemically complexed compounds or polymerized proteins (quaternary structure or heteropolymerized proteins are usually . *-Multi determinant Ag ( multi specific) , multivalent Ag (more than one epitope of each kind) )What kind &How many of such kind )

Antibody binding site ( Paratope ).

Binding of Ag & Ab Affinity :- the strength of attraction and binding between an epitope( valent ) of an Ag and the antigen combining site of Ab molecule ( ). Avidity :- The strength with which ( multivalcnt ) Ag bind to its antibodies ( Abs). ( chemical complexity )

This depends on the affinities of the individual combining sites of the determinants on the antigen

mono Paratope

ANTIBODIES and their STRUCTURES

Electrophoretic separation of serum proteins

ANTIBODY STRUCTUR

*Classes of antibodies . IgM , IgG , IgE , IgA & IgD .

CH1

CH3

Heavy chain

VARIBLE = ANTIGEN BINDING SITE .

Constant A

B = NEUTROPHILS & MACRO- PHAGE BINDING SITE Hinge bonds

Constant B

CH2

Constant A

SITE

Constant B

A = COMPLEMENT BINDING

The basic structure of immunoglobulins

Rotating antibody

ANTI BODIES

POLYCLONAL ANTI BODIES

MONOCLONAL ANTIBODIES

- INDUCED AGAINST WHOLE ANTIGEN .

INDUCED AGAINST ONE EPITOPE .

- LESS SPECIFIC

(I.E . SMALL PART OF ANTIGEN )

- PRESENT IN SERUM

- MORE SPECIFIC .

EPITOPE EPITOPE

- PRODUCCED BY HYBRIDOMA TECHNOLOGY .

INFECTION POLYCLONAL Ab.

MONOCLONAL Ab

MAb

MAb MAb

II_ FUSION

i-Immunization

antigen

+ MICE + MYELOMA CELLS fusion

B-CELLS TUMOR MICE

MICE

Ascetic fluid

selection of Desired Clone

supernatant Fluid

Tissue culture

HybrIdoma technique

HYPRID CELLS

Immunoglobulins "Humoral antibodies” They are formed of two identical units each of them is formed of :A) heavy chain

B) light chain

A) Light chain

C) hing region

2( lambda)

but never 1 and 1K K ( Kappa ) 2 B) Heavy chains : * M-Wt 53.000 - 75.000 Da *- heavy chains are hold together with (disulphide bonds) . *- Fixed region contain 2k or 2  . *- The variable region contain a mixture of K,.

*- both L& H chains contains the following region : Light chain contain variable (VL) and hyper variable (VH) regions Heavy chain contain variable and hyper variable regions.

* Amino terminal * The amino acids differ on  to another specificity.

* The VL & VH are adjacent to each other forming paratope . * They have sub-regions of the variable region (hypervariable) These regions have extreme variability in their A .As sequence in different antibodies and they are responsible for binding with Ag(s)

* carboxyl terminal * A. As are similar in different * it contain the effectors domain which is responsible for the initiation of the process by which the body gets-rid of Ag. .

It is responsible for Designation of Ab class & its distribution. -. -

CDRs}{ comptementary detemining regions

)hyper variable (

C) Hinge region :* CH 1, CH 2 , CH 3 : occupies ¾ that of Heavy chains the other ¼ is VH . * The Hinge region lies between CH 1 & CH 2 . * It is flexible & allows movement between the two antibody binding sites . * The hinge region is digested by protease (e.g. pa pain ) which splits it into :( i ) antigen binding fragments (fab) = They are 2 identical fragments containing the antigen binding site . )ii ) crystallization fragment (FC ):-It contains the effectors (

Structures and function Of Specific Immunoglabulins *- Ig(s) are glycoprotein's in the gamma globulin fraction of serum proteins (albumin , fibrinogen , globulins ( ,  and  ) . *- they are produced by B- lymphocytes or plasma cells in response on immunogen (or Ag ). General Ig structure :*- 4 poly peptide chains. *- they are linked covalently by disulphide bonds *- the 4 chains , monomeric Ig structure ,are composed of 2 identical heavy poly peptide chains (H) 2 identical light poly peptide chains (L)

*- Heavy and light chains : *- H- chain : *- Have a M.wt of 50-75 KD (Twice that of L chain ) *- H chains contain  400 A.As (Twice that of L chain ) *-A. As differences in the .COOH terminal portion of the heavy chain (CH) identify 5 distinct H-chains isotypes .

* Each H chain has 4 or 5 domains :

1 domain in the variable and 3 or 4 in the constant 3 IgG (  ), IgA( )&IgD( )

Total

= 1 Variable +

3 constant

Or

or

4 Igm ( )&IgE(  )

=

4constant +1 Variable

Notes (1) -Each L- chain has 2 domains

1 VL 1 CL (2)- Folding of the polypeptides chains brings the hyper variable regions of the VH and VL domains into close proximity . (3)- this folding creates a 3-dimensional structure that is complementary to the epitope (last figure )

The Hinge region *- It is the portion of the H-chain between CH 1& CH 2. *- there is no homology between it and the other H- chain domains, thus .its sequence is unique (sole) for each Ig type and subclass

IgM & IgE do not possess a hinge region but have one more CH domain. These structure explain why both IgM & IgE have 4 domains on the CH chains but not like the other types (which have only 3 domains on CH) *-In this region (hinge), inter chain disulphide bonds forms between the arms of the Fab fragments preventing them from folding and therefore , rendering this portion of the molecule highly susceptible to fragmentation by enzymatic attach . * -The hinge region is highly flexible and allows for movement of the Fab arms in relation to each other .This motility explain why native antibody molecule do not activate complement , whereas those in an immune complex do .This is because , the native Ab is not in the appropriate configuration t1/2 or half life of( Abs) . *- These heavy chain isotypes form the basis of the 5 Class of Immunoglabulins molecule IgG () ,IgA ( ) ,IgM ( ),IgD ( )and IgE ( ). *- H chains Classes  and  are subdivided into subclasses of molecules  1 , 2 , 3 , and 4 And   1 and  2

The subdivision is based on the greater similarity of A.As sequence shown by subclasses of the same class i.e. 1 , 2 , 3 etc,. Than is shown by different classes (i.e.  ,  , or ) *- The heavy chain subclasses determine immunoglobulin subclasses e.g. 1 = IgG1 2 = IgG2 , 3 = IgG3 etc,. *-L-chains :* Are composed of  200 A. As . * They are of 2 types ( K= Kappa or  = lambda ) . { based on their structural (antigenic) differences } * All Igs classes have 2K or 2 chains but not k or  k . ex. * The proportion of K/ = 3/2 (human Ig) .

- chain Isotypes :-

*- There is no isotypic variations in K chains *- There are 4 distinct  chains 4 different isotypes . *- All the 4 subclasses are present in each of the Ig classes i.e. in IgM , IgE , IgD etc. *- Disulphide bonds Hold together the 4 polypeptide chains in Ig molecules . -*There are 2 types of disulphide bonds-:

1- Inter- chain disulphide bonds : occur between

H – H chains

Hinge region

But also in COOH-terminal of the H chain

They can be 1:15 depending On the class & subclass types

H – L chain

occurs in all Ig(s)except Ig A2M (1) which Lacks an inter chain disulphide bond

L – L chains

Single L-L bond only in Ig A2M (1) such bond can occur under pathogenic conditions. (e.g. Bence Jones protein ) seen in urine of some patients with multiple myeloma

INTRA CHAIN DISULPHIDE BONDS : *- occurs within an individual chain . *- they are stronger than inter chain bonds . *- they no. of intra-chain disulphide bonds varies depending only on the number of domains in the molecular . Light chain have 2 intra-chain bonds . *- human IgG, IgA, IgD heavy chains have 4 intra_chain bonds *- human IgM , IgE heavy chains have 5 intra-chain bonds . *- Each H& L-Chain has a variable (v) and constant (c) region *- V region lies in the – NH2 terminal portion of the molecule . *- The V region has a wide variation in it’s A.A composition . *- The C region lies in the - COOH terminal end of the molecule . *- The C region has a much more constant A.A Sequence except for minor inherited changes

*- The variable regions associate with appropiate constant regions . so that a variable H – Chain regions (VH) does not occur in an variable L – Chain (VL) and Vise versa . *- However , a particular VH chain sequence may occur in more than one H – Chain class ( i.e IgG, IgM , IgD ,IgA and IgE ) . *- Thus during class switching in an immune response e.g when B – cells change their production from IgM to IgG heavy chain only the constant regions of the H (CH) changes and the antibody specificity remains the same . HYPER variable regions

*- they are particular areas within the variable regions That are highly variable in A. As sequence . *- THESE hyper variable regions often called complentary determining regions

*- THESE regions occurs at simillar A.A positions in an relatively invariant molecules .

CDRs :- they are short polypeptide segments lining near A. As positions 30,50 CDR1 CDR2 CDR3 AND 90 in the variable regions of both L and H chains . FR1

FR2

FR3

FR4

variability

FRs

CDRS variable region

24-34 50-56 NO OF AgS

Note :- the variability range ( index ) used is an arbitrary scales of the no. of different A.AS found in each position if 100 different Light chain were analyzed . *- the hyper variable regions are important in the structure of the Ag binding site ( paratope ) . *- L – chain have 3 hyper variable regions ( the last figure ) *- H – chain have 4 hyper variable regions although, ONLY 3 OF THE 4 have been associated with epitope recognition *- each Ig chain consists of a series of globular regions or domains enclosed by disulphide bonds ( intra or inter ) ?? Chain disulphide bond . *- The A.AS sequence of the domains show a high degree of homology ( i.e the sequences are very similar ) .

89-97

Structure of the variable region framework regions

Properties of Ig :IgG H – CHAIN  H – CHAIN SUBCLASS 1, 2 M.Wt 150 Carbohydrate (%) 3 Serum conc(mg %) 1200 Seru t ½ ) days( 21 Functions :Complement activation ++ Opsonization ++++ Antiviral activation ++ Mast cell sensitization -

IgA

IgM

IgD

IgE

 1 2 160-400 7 200 6

 900 12 120 10

 180 13 3 3

 190 11 0.05 2

+ +++ -

++++ + -

-

? +

Immunoglobulin are glycoproteins :- (3-13 % of their M.WT ) OLIGOSACCHARIGES + PROTEIN *- THESE oligosaccharides are present in CH2

or CH3 .

*- N -glycosidic bonds usually link N- acetylglucosamine in the carbohydrate moiety to asparagine residue in the peptide c-chain of Ab [ linkage with the enzyme N -acetylglucosamine . - Asparagine transglycosylase ] transferase *- t ½ of Abs in the circulation depends on the status of oligosaccharide side chain *- the oligosaccharide side chain of Ab terminate with galactose to which sialic acid is bind . *- when Abs have the sialic acid removed by the enzyme neuraminidase , they become susceptible to degradation in the liver . *- in this case the terminal galactase bind to a receptor on hepatocytes and the entire molecule is , then , interenalized to the cell for degradation via Proteolytic enzymes in lysosomes of the cells .

Immunoglobulin fragments: Structure/function relationships

Digestion of Abs with Restriction enzymes (Immunoglobulin fragmentation) as well as Structure/function relationships: S-S

S-S

F(ab)2 Ab

FC

Restriction enzymes digestion of Abs : 1) Papain : digest above hinge region so it leaves 2 Fab fragments each is monovalent S-S

And crystalline fragment (FC) papain

Fab Monovalent

FC

2)Pepsin: digest away most of FC Fragments below the Interchain disulphide bond (below the hinge region) it give one large fragmentsF(AB)2 which is consist of two Fab fragments joined by the disulphide bond Thus , it is bivalent ,possessing the ability to bind and form agglutination

S-S

F(ab)2 Ab

FC

Figure 4 Immunoglobulin fragments: Structure/function relationships

Classes of antibodies They are 5 isotypes The class of Ab depends on the A.A: sequence of the constant regions of the heavy chain .

IgM

*- Immunoglobulin M (IgM) :* it is a pentamer ( 5 molecules ) . * they are linked together by disulphide bridges at the COOH terminal end of the heavy chains as well as an additional poly peptide chain ( joining chain) * this type of Ab account for 8-10% of the total PLASMA ANTIBODIES . * it is the most abundant Ab produced by the faetus . * it binds with viruses and bacteria *- Immunoglobulin g ( IgG ) :* it is a monomer * it accounts of ~ 75 % of the total antibodies . * it is important for elicit ting the immune response to Ags * it is only antibody which pass through the placenta to protect the faetus.

*-

immunoglobulin D ( IgD):-

* It is a monomer ACCOUNTING FOR < 1% & TOTAL ANTIBODIS . * Its function is controversial . *- immunoglobulin E ( IgE):* It is a monomer ( below 0.004 % & the total Abs) * It is present in spleen , tonsils , mucus membrane of lungs GI * On binding with ag it releases histamine from mast cells leading to hypersensitivity .

* It provides immunity to intestinal parasites . *- immunoglobulin A ( IgA):* MONOMER , DIMER or TRIMER( mostly dimer )

* Like IgM the units are linked by disulphide and j chain * it is found in tears , saliva , intestinal treat secretions * it binds with Ag preventing them from tissue adherence , colonization ,and making them more phagocytosed .

Laboratory Methods Serology In vitro Ag & Ab reactions called serology It provide methods for i) Identification (Diagnosing)

( ii ) quantization of titre of Ab (and \ or) Ag

Titre : or the level of Ab (s) in the serum can be measured by using known Ag The titre may have

or

diagnostic prognostic

Ex. A rise in Ab titre between acute &convalescent serum can be used as a diagnostic tool for a specific disease The titre is defined as the greatest dilution of serum (which contain the Ab under consideration ) that reacts which the antigen ( i.e. gives +ve result ) .

- the forces involved in Ag-Ab reactions are greatly affected by various environmental factors :*- The Ag- Ab complex is not bound firmly together . *_This complex may even dissociate spontaneously . * physiologic ph & salt concentration promote optimal union of them . *- the force of attraction tend to be weaker in a) very acidic .e.g. 0.01M b) very alkaline medium i.e

pH 4 and alkaline ( i.e. above pH 10 )

- temperature :- it plays an important role : * the higher the temp ( up to 50 – 55 0 c ) , the more rapid is the rate of reaction between Ag & Ab .

* the reason is the increase in kinetic motions of the reactants ( Ag & Ab )

various forces act to hold the Ag-Ab complex together :* The maximum attractive forces stabilizing Ag-Ab complexes Are van der weal forces Ionic bonds

1- van der weal forces :* occurs because of spatial fit ( the below fig ) * these forces of attraction hold Ag to Ab only When the two molecules have complementary shapes (a) puratope 2 puratope 2

Epitope

(a) (i) significant changes In the shape of epitope 2 Into 2a

2

Epitope

2a

(b)

these change precludes its ( 2a ) interactions with the matching binding site of the original Ab . * When the molecules have less similar shapes ( b) , these forces are less effective (b) 2-Ionic bonds :* They are patterns of complementary electric charges on the molecule . * The electrostatic interactions tend to hold the molecules together . COO

NH3 COO

NH3+

COO +NH3 Affinity :- the strength of attraction between a single epitope and its matching paratope is the referred to as the affinity of the reaction between the two reactants . Ag-Ab complex of low affinity dissociate readily

Avidity :* It is a related term to affinity * It refers to the strength of the interactions between multivalent antigens and the population of Abs that they have included .

*- Avidity is influenced by the affinity of individual Abs for their (A) epitope (B) the valency of Ag and (C) the valency of Ab tertiary structure of protein : *- the ability of Ab to bind with Ag can be affected by altering the tertiary structure of any of them ex. insulin which is composed of A&B chains Ab to either one of these chains can be produced by (a) splitting the chains (b) purifying tem (b) injecting ) .e.g. a pig) them into foreign host the pig will produces Ab to the particular chain that was injected *- if the host (pig) Abs are injected back into the animal species that supplied the original insulin (man) , the abs will not react with intact insulin molecules . *- This is because the tertiary structure of native insulin is such that the on the A & B chains are not accessible .. epitopes Now , it is generally accepted that in a given poly peptide the A .As that are spatially accessible because of Tertiary structure of this protein are only immune reactive

*- The physical state of the antigen is responsible for the identification of Ag –Ab reactions and the naming of Abs . *- The same Ab molecule could , in fact , be described by each of the following terms : (1) Agglutinins are Abs that aggregate cellular Ags. (2) Lysins are abs that cause dissolution of cell membrane . (3) Precipitins are abs that form precipitate with soluble Ags . (4) Antitoxins are abs that neutralize toxins . procedures must be involving direct demonstration and observation of reaction .. The relative sensitivities of the tests for Ags and Abs are Presented in table 8.1 page 156 [ immunology , 3rd edn ]. A- Agglutination Reactions :b a Serve to detect and quantities Agglutinins and identify cellular Ags Bacterial cell white blood cells red blood cells . **-- when the cells intact with the appropriate Ab , they clump together and eventually

form

masses

Large enough to be visible with naked eye

*- When Ab agglutinates bacteria in the body opsonization occur . *- Agglutination occurs because Abs and at least bivalent . *- Two sites on the Ab and multiple sites on the Ag

Ag – Ab

lattice formation that can build up into increasingly larges coupled

lattice structure

Example widal test :- (diagnostic test of typhoid ) *-Ab of patient serum is measured by adding a constant mount Ag (e.g. salmonella typhi ) to serially diluted serum . *- After incubation , the test tubes are examined for visible agglutination . *- the last tube (i.e the highest dilation of serum ) showing agglutination is referred as the titre.

B- lyses Reactions :In the presence of a complement an Ag – Ab reaction , on a cell membrane , may result in membrane damage that leads to cell lyses This phenomenon is important in the host's defense against condition such as microbial infection or cancer ( graft cell , virus infected cells , etc…………….( *i)- Haemolysis :In which the Hemoglobin is released from R.B.C, is a requisite phenomenon for the complement fixation test .

*ii)-- bacteriolysis :cells of gram (– ve) bacteria are undergoes immune lyses under certain condition . *iii)-- cytolysis :involves the destruction of other cells types (e.g. lymphocytes ).

C- precipitation:* occurs when the Ag is soluble instead of cellular *therefore a large number of molecules are required for lattice formation and a large no .of lattice must be formed for an aggregate to be formed and visibly seen.

*when soluble Ag (s) come intact with specific Ab. They aggregate (i.e precipitate ) Three conditions are present A- where the (Ag) is very low with excess Ab (zone of Ab excess ), Formation of complex occurs But Residual Ab remains in the supernatant B- As more Ag is added , large aggregate is formed In the (zone of equivalence) , maximal Ag-Ab complex are formed and precipitated C- Instead of reaching a plateau , this curve comes back down to zero with increasing the mount of Ag (zone of Ag excess ) * this is because the lattice size becomes too small to precipitate . * In extreme Ag excess . the complex will be trimmer i.e one Ab +2Ag Note:- the soluble Immune complex are not processed efficiently by the reticuloendothelial system ,and ,this cause damage (how??)

Amount of precipitate

Zone Of equivalence

Effects of increasing amounts of Ag on the total immune precipitate obtained from a mixture of soluble Ag and its homologous Ab

INDIRECT HCG : Examples 1 :- determination( and\ or) detection of HCG by using indirect methods . (i) an Ag will be added

( HCG ) .(from the kit)

(ii) Urine will be added ( excess Ag ) from a female may be pregnant . (iii) Ab to HCG will be added

In case of positive

In case of negative pregnancy

A state of Ag excess

a state of equivalence will be reached

Therefore, no precipitation

therefore, precipitation occur

Direct HCG assay : * (i) Ab to HCG will be added (from the kit) * (ii) Ag ( HCG of the test sample will be added) . If precipitation occur ( positive) if, no precipitation occur ( negative )

Hyaluronic acid (HA) assay using excess HA binding protein (HABP) :* HABP will be added in excess ( known excess ) (ACT AS Ab) * Sample will be added ( containing HA) (ACT AS Ag) *

[ A state of Ab excess

no ppt ]

* An radiolabelled HA will be added ( Ag ) Thus, precipitation occur ( IF +ve sample) and immune complex will be separated and quantities by radio- immune assay technique ,in case of no precipitation, the sample is negative

Immune diffusion * It used for quantization of Ag (s) * Thus, precipitate will also be demonstrated . * If an Ag – Ab reaction takes place in semisolid medium (e.g. agar ) , band of precipitate will be formed . * The reason of precipitation , is the diffusion of the components (Ag & Ab ) towards each other .

* A useful example is a double immune diffusion technique :-

Procedure :* Ag

& Ab preparations are placed in separate wells that are cut into a thin layer of agar in a Petri dish . * The reactants diffuse towards each other through the agar until they meet an optimal proportions [ zone of equivalence ] and forms ( ppt ) bands Solid Chevron PPT

Zone of

Fig (a) Ag Ag

Ab

equivalence

The advantages of the procedure is that antigenic relationship can be detected by the precipitation pattern (s)

3 basic patterns are given : (a)- in reaction of identity , the 2 Ags are similar , they will diffuse at the same rate and the two precipitations bonds merges into a solid chevron ( fig b) Aga Aga

Fig

b

Ab

2- in reactions of non-identity , the two Ags are completely different and the lines of the precipitate cross (fig c) Ayab Agac

Aga Agb Fig Aba Abb

c Aba

Abb

3- reaction of partial identity :* It is indicated by spur formation indicating that one of the Ag(s) is cross-reactive ( but not identical ) to the other one . * The spur occurs because one the Abs (b) does not react with the cross-reacting Ag (Ag ac) but migrate past that Ag (Ag ac ) until it reaches an Ag (Ag ab ) that

Has an epitope for which it has specificity .

B- quantitative radial immune diffusion * It is used routinely to quantities Ab in serum . * For this purpose , an agar coated slide is used . * The agar being impregnated by anti sera ( antibody to human IgG ) * SERUM samples are placed in wells in the sugar . * As it diffuse through the agar and encounters the Ab, the IgG in the sample form a concentric ring or halo precipitate . * The diameter of the halo of precipitate directly correlate with the [ IgG] in the sample .

Thus , the levels of IgG in the sample can be determined by referring a standard curve based on halo diameter (s) of known concentration (s) of IgG

C-immune electrophoresis :* It was developed because the double immune diffusion technique. (i) Could not resolve high complex mixtures of Ags . (ii) and, a more sophisticated technique was needed .

In this procedure : (a) Ag is placed in wells in agar on a glass slide and then , subjected to electrophoresis through application of an electric current . (b) Under these conditions , the individual Ags or antigenic components ( in the same sample ) migrate through the agar at variable rates . (c) If Ab is placed in a well that runs the length of the slide parallel to the path of migration , the reactants will diffuse towards one another and form separate arcs of precipitate for each antigenic determinant

D- counter-immune ectrophoresis (CIE) This technique Is the double diffusion method + an electric current

Which plays as the migratory force which: (i) – amplify the speed of reaction ( 24 hrs to

30 min )

(ii) Intensifies the precipitation bonds .

(iii) Increasing the sensitivity of the assay about 10 fold .

Procedure :(i) Ag & Ab are placed in wells and the current is applied . (ii) in suitable buffer ( eg ph 8.6 ) the negativity charged Ags migrate towards the anode , whereas the Ab [ which has no sufficient net charge ] migrate in the opposite or counter-direction , as a result of endosmosis . Precipitation occurs where the reactants melt .•

D- Antitoxin :* If a serum contain an antitoxin ( i.e. antibody to a toxin ) , the Ab . Will neutralize the toxin examples :-

- Suppose serum containing antitoxin is mixed with toxin ( in vitro ). - Then , after a few minutes , a small amount of the mixture is injected into an experimental animal ( in vivo ) . - The animal will be protected against the introduced toxin , and thus , its deleterious affects disappear because of antitoxin is present .

Clinical example :-

( the virus haemagglutinate R.BCs)

* To examine the serum of a patient suspected of having influenza , *1) The patient serum is mixed with known influenza 2)add red blood cells i- if Ab is present

haemagglutination will be prevented

.i.e the sample is positive this is due to the ability of Ab to bind with the virus and block its ability to haemagglutinate the R.B.Cs ii- if no Ab is present , haemagglutinate will occur . virus + R.B.Cs

haemagglutination occur

.i.e the sample is negative

E-Flacculation :it is another form of Ag –Ab reaction that occurs if the Ag is

neither cellular nor soluble

but it

is an insoluble particulate

VDRL TEST FOR Syphilis The venereal disease research laboratory (VDRL) test is a slide flocculation test used for the diagnosis of syphilis .

The VDRL make use of heterogenetic ( heterophillic ) antigen shared between the • Spirochete of syphilis & normal beef heart . * The Ag used is a water insoluble cardiolipin that had coated the surface of cholesterol particles that were added to the system .

* These form visible aggregate indicate to the presence of Ab ( reagin ) in the serum of patient for syphilis [ reagin is Ab type which flocculate (or ppt) an Ag that is neither cellular nor soluble but it is insoluble ] * The test can be performed on a glass slide .

Technique :cholesterol particles ( inert support )

+

normal beef heart extract ( antigen like substance )

Insoluble antigen serum ( A.Bsource ) visible aggregate Which can either seen by Using a microscope and green filter

The naked eye

COMPLEX SEROLOGICAL PROCEDURES Ag-Ab reactions in which the visible manifestation requires Participation of:

a) Accessory factors b) Indicators system c) Specialized equipment

A- fluorescent dyes :e.g fluorescein isothiocyanate ( FITC) * FITC can be conjugated to Ab. Molecules to visualize of the molecule under (uv) or (b)

a fluorescence microscope . * such labeled Ab. May then be used to identify Ag(s) (i) Direct immunofluorescence assay :* The method uses Ab. That is specific for

a particular Ag or

parasite * This Ab is labeled with a fluorescent dye (FITC) * This conjugate is allowed to react with unknown tissue or organism . * IF the Ab reacts ( i.e +VE the result ) , it will visualized as green stain on the specimen when it examined under the fluorescence microscope by

using uv light

Examples :Identification of Trepenoma palladium ( syphilis ) in an extracted from a patient suspected of having syphilis . - Procedure :*1) The slide is coated with the Ag .

*2) Ab tagged with FITC is added . * 3)Excess Ab is then washed . * 4)Then , the slide is examined with uv fluorescent microscope .

* Trepenoma palladium is fluoresce against the black back ground this methods can be extended for other pathogens .

(ii)-immune peroxides technique : If viral antigen in tissues will be detected, horse radish peroxides is conjugated with the Ab .

*-(1) After the enzyme – Ab complex has reacted with the tissue (Ag) . *- (2) Excess Ab is washed . *-(3) And , an appropriate enzyme substrate is added to the tissue section . *- the bound Ab. Is detected by the presence of a dark precipitate *- Advantage of immune peroxides technique over the immune fluorescent technique:*- The specimen can be stained with conventional Histochemical dyes So structural details can be seen ( noted ) *- the tissue con be examined by standard light microscope . (iii)- Indirect immune fluoresce technique:*- The procedure use Ab ( secondary ) ( against ) another Ab ( primary ) of patient .

*- the primary Ab is the patient' serum detection of Abs. *- the secondary Ab is covalently conjugated whit fluorescent compound (FITC) *-*- ex. Of secondary Ab is rabbit antihuman( INJECTED Ab in host is FROM HUMAN ) gamma globulin anti sera . .i.e Produced against Ab used to immunize rabbit and that Ab which will be examined latter on (unknown conc.)

Technique :- (this procedure allows for detection of Abs,) Example serodiagnosis of syphilis by the fluorescent trepenomal antibody absorption (FTA-Abs) test *-(i) -T. pallidum is fixed to a slide *-(ii)- the slide is flooded with the patient serum (staining Ab ) *-(iii)-If Ab to spirochete are present, the Ab will reacts (bind) with the organism on the slide * (iv)-Excess Ab (serum) must be removed with washing , to detect the bound Ab only . *-(v) the Ag-Ab complex formed is them treated with the fluorescein – tagged Ab to human gamma globulin, the excess Ab is washed carefully. *-If the patient's serum contains Ab (+ v e) against the T. pallidum, fluorescein organism will be seen when the slide is examined with fluorescence microscope

FITC

Slide coated with ag in (excess)

Patient’s

**-- Ab fluorescence conjugate is binded .

Serum is

Microscope (uv ) is used

added - Indirect Immune fluorescence assay is also used for Detection of Antinuclear Antibodies (Ana) :( e.g. DNA , RNA & His tone) ANA are present in systemic lupus erythroMatosis ( SLE ) , some Times in rheumatoid arthritis and other autoimmune collagen – Vascular diseases .

Example (SLE ) :* The procedure is similes to that of T . palladium . * The Ag is ( DNA ) histone in form such as Animal Buffy coat calls Human Buffy coat calls

Rat kidney section beef thymus Lymphoid Thymus Lymphoid organs

Haem agglutination Inhibition teat :It involves The agglutination of R.B.cs by

(haemagglutinin(s)) Ab(s)

Certain virus particles (influenza)

Or other substances

It demonstrates the presence of serum Ab to haemagglutinating viral substance .

Technique :-

R.B.C s

serum sample Which contain Ab Prevent haemagglutination

Agglutination occur The sample is negative

Ab from the kit that make haemagglutination

no agglutination the sample is positive

Similar test can be used to detect soluble Ag(s) which able to react with and neutralize a haemagglutinating Ab . R.B.Cs from kit

serum Ag which Prevent haemagglutination

Abs from kit which Capable of haemagglutination

haemagglutination takesplace Sample is negative

haemagglutination Inhibition Sample is positive

- passive agglutination:in the conversion of a reaction system from one that precipitate one that agglutinate Thus yields a more sensitive indication of the presence of antibodies .

Example :RHEUMATOID ARTHRITISThe use of latex particles in the diagnosis of rheumatoid arthritis (soluble Antibodies ) is an example of passive agglutination . Principle :In this disease ,the patient produces an Ab (Mainly IgM) to his own IgG Technique :*-(i) latex particles were coated with IgG. *-(ii) patient 's serum is added (which contains antibodies IgM) *-(iii) Agglutination indicates the presence of Antibodies (Ig?) (i.e The test is positive ) The detectable antibody is called rheumatoid factor

Bis-diazotized diphenyl :it is a coupling reagent that can be used to proteins to conjugate : or R.B.Cs Haptens

Passive haemagglutination Occur.

and thus

Thus : *- Addition of serum containing Antibodies to these substances (proteins or haptens ) allow the detection of these specific antibodies to these substance by a technique called Passive Haemagglutination rose –waaler Test : which detect rheumatoid factor in serum of the patients suspected to have an anti-IgG auto-antibody .i.e. .(IgM to an accumulated IgG Tannic acid –treated a sheep R.B.Cs (S R.B.Cs) are coated with rabbit IgG Antibodies specific for these S. R.B.Cs

from the kit

Tannic acid coated by IgG

R.B.Cs

serum from a patient suspected to have autoantibodies

haemagglutination

the sample is positive IgM

Coomb's (antiglobulin) Test :* In certain people .Abs directed against antigenic determinants (e.g. R.B.Cs antigens )are able to form visible aggregates when subjected to : 1- precipitation 2- Agglutination *-To demonstrate the presence of Abs in such cases the coomb's (antiglobulin)test may be used .

*- The test involves the addition of Ab direction against gamma globulin : which provides a bridge between two antibodies coated call or particle Thus ,The major use of the coomb's test is to detect the Non agglutinating (haemagglutinating )anti-red blood Cs Abs.

(1) – Direct coomb's test * It is used to detect call bound antibodies Technique :You must use EDTA BLOOD then centrifugation *-(1) The red blood calls (bound antibodies )are washed free from serum and the unbound antibodies (to be leaving the bound ones ). (2) Antiglobulin serum is added directly to this call suspension -*

*- The direct coomb's test is of value in the detection of antibodies to R.B.Cs associated with hemolytic disease of new born (e.g. erythroblastosis fetalis ) and auto immune anemia or disease . *- The Abs associated with these diseases have the ability to attach to but not agglutinate the target R.B.Cs . *- These absorbed Abs can be detected by the use of Ab (i.e. coomb's Kit serum ) to This human gamma globulin .

(2) indirect coomb's test :*- It is used to detect the presence of Circulating Antibodies. *- It is of value in detecting IgG - associated antibodies in the serum of woman who is though to be (a) sensitized to Rh antigen And (b) at risk for carrying an erythoblastbotic febus . Technique :*(1)- Serum sample (containing Ab ) is incubated with donor R.B.Cs( contain Rh antigen). Faetus like blood *- (2) Then ,the cells R.B.Cs are washed off ( to remove excess Ab ) . *- (3) The anti globulin (coomb reagent) reagent is added ( kit(

Serum

serum

Ab is absorbed

haemagglutination ( + ve ).

No Ab .(No absorption )

haemagglutination ( - ve ).

Anti globulin commb reagent

Viral Neutralization :It is very similar to haemagglutination Inhibition on ( i.e. it is a neutralization event ) . *- Principle :The assays is based on the ability of specific Abs to interfere with Some biological function of the virus under consideration ( usually The infective property is blacked ) .

( 1 )- Cyotpathic effect ( CPE ) :-certain virus + cells ( in tissue culture ) cell destruction . 2 )- the CPE is useful in the search for virus neutralizing Abs in serum sample.

*-Technique

:**_ serum suspected of containing Ab is added to a virus suspension . **_ Susceptible cell culture is inoculated with the mixture .

If the culture fail to develop CPE (no killing ) ( + ve )

CPE developed (killing ) no Neutralizations ( culture Cell death ) ( - ve )

Abs are interfere with the Ability of the virus to kill Tissue culture cells

– Radio immune assay (RIA ):* - It is and extremely sensitive method for quantization of any substance that is Immunogenic or heptenic and can be labeled with radioactive isotope e .g. I (25I) .

Liquid phase RIA :It depends on the competition between labeled (Known ) and Unlabeled ( unknown )antigen for the same antibody .

Labeled Ag (known amount)

Unlabled Ag[unknown amount]

Ab specific known amount

Immune reaction product (Immune complex) ( i ) Separation of this complex by immunological method by secondary Ab or by Precipitating agent . [ (NH4)(SO4)] ii) Separation of this complex by physiological methods (a) centrifugation (b) Decantation .

. The

supernatant

A Calibration curve based on using serial dilution of known unlabeled standard ( instead of the serum ) is used for calculating of un known samples

CPM

The radioactivity of either Is then determined

PPT (commonly used)

conc.

*- Solid phase RIA :Liquid phase is modified by :( i ) adsorption or covalently linkage of Ab to solid matrix ( solid phase RIA) . ( ii ) The unlabeled Ag (sample ) is added followed by the labeled one ( Antigen or Antibody ) . Then The bound versus free Ag can be determined by Using reference calibration curve ( as before)

[ washing steps]

Enzyme linked Immune Sorbet Assays { ELISA } :*- ELISA is both highly sensitive ( > 99% ) and specific ( > 99% in high – risk populations ) . *- It can be for the assay of either Ag(s) or Ab(s) . *- Ag or Ab can be attached to solid phase support ( plastic surfaces , paper disks ) and still retains its immunologic activity. *- Either Ag or Ab can linked with an enzyme e.g. ( horse reddish peroxidease alkaline phosphates ) . *- substrate is added and the color absorbed by the enzymatic procluct is then quantization and compared with a calibration curve .

Example :- detection of Ab(s) to the human HIV :*- The virus is grown in vitro in a human T-cell culture . *- purified whole virus is disrupted 8 viral proteins are immobilized onto plastic beads or multi well trays . *- Abs to any of these antigens will bind with them & immobilized . *- Excess proteins are removed by washing the beads ( or wells ) and an enzyme linked anti human gamma globulin antibody is added . *- The presence of this second Ab can be detected calorimetrically by adding a substrate for the enzyme that will yield a colored end product .

*- The rate of substrate degradation is determined by the amount of enzyme – labeled Ab that is bound which is proportional with the amount of Ag in the solution being tested. *- the color change can be measured quantitatively in a spectrophotometer .

* Double Antibody sandwich ELISA :It is used for the assay of Ag ( e.g. HBSAg ) uses tow Abs as below :( i ) first Ab ( specific e.g. HBs Ag ) is coated on a plastic surface ( poly styrene), the solution being tested for HBs Ag is then applied to the surface . ( ii ) Washing of any un reacted material . ( iii ) The second Ab ( ie enzyme linked anti HBs Ag specific Ab is then applied . ( iv ) Any excess conjugate is rewove by washing . (v) finally substrate is added to the detest the present of En2

ABO group & Transfusion Reactions *- ALL human erythrocytes contain all antigens ( i.e. Antigens that vary among individual members of a species ) of the ABO group . *- This is important system , which . is the basis for blood typing & transfusions . *- The A & B antigens are carbohydrates that differ by a single sugar .

*- Despite this small difference , A & B antigens do not cross – react *- R.B.Cs have 3 terminal sugars ( in common ) on there are surface..

N – acetyl glucose amine

Galactose

Fucose

H antigen

**-- Type A cells have an additional N –acetylgalatose . **-- Type B cells have an additional galactose. N.B . Type A & B genes code for transferaes that add the respective Sugar . *-* Type O have only the H antigen : To avoid Ag – Ab reactions that would result in transfusion, all blood for transfusion must be carefully cross matched . *-* So , Ag the corresponding Ab do not coexist in the some person's blood . *-* Transfusion reactions result when incompatible donor's R.B.Cs In to are transfused e . g . group A group B .

Ag – Ab – Reaction Involving R . B . Cs antigens

:-

ABO blood group . Structure of the terminal sugars that Determine ABO blood groups .

Group

Antigen on R.B.Cs

Antibody in plasma

A

A

Anti B

B

B

Anti A

AB

A&B

No Anti A NOR Anti B

O

No A nor B

Anti A & Anti B

4 POSSIBILITES OF CONBINATION