609 1st int slides

8/6/2019 609 1st Int Slides

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IMMUNOLOGY


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What does the immune system recognize? Most immunogens

contain multiple antigenic determinants called epitopes.Anepitope is the basic minimal structure on the Ag surface

recognized by the immune system.Most immunogens are

composed of a variety of epitopes, haptens comprise a single

epitope.Paratope is the corresponding specific binding site

on the Ab molecule

Antibodies with a complimentary shape will be formed that

can specifically combine with these epitopes on the antigen.

one Ag may stimulate the prod. of many Abs but only the bestfit will combine


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Antigen-Antibody binding


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ANTIBODY

REFERS TO A GRPOFSERUM GLYCOPROTEINS,FORMEDIN

RESPONSETO ANANTIGEN. ANTIBODIESFOUNDINSERUMCALLED HUMORAL ABS

BROADLY KNOWNASIMMUNOGLOBULINS ---BELONG

TO FIVECLASSES - IgG,IgM,IgD,IgA,IgE

MAYBEDESIGNATEDBYNAMES THATDESCRIBETHEIRREACTIONWITH CERTAINTYPES OFANTIGENS

ANTITOXINS (NEUTRALIZEMICROBIAL TOXINS),

AGGLUTININS(CAUSECLUMPING),PRECIPITINS(CAUSEPRECIPITATION),LYSINS(CAUSELYSES) COMPLEMENTFIXING

ANTIBODIES(CAUSES CFTREAC.),OPSONINS(MAKEMICROBESMORESENSITIVETO PHAGOCYTOSIS)


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Immunoglobulins & functions

There are 5 classes ofIg

class Mol.wt % in serum function

IgG 150,000 80 Involved in phagocytosis

CFT, found in lymph/serum,

crosses placental barrier

IgM 900,000 6 Found in serum & lymph, cidal

to Gm-ve bacteria

IgA 170,000 13 Found in tears,saliva otherbody secretions

IgD 185,000 1 Found in serum

&lymphocytes, contr B cell

stim

IgE 190.000 0.002% Causes allergies, Imm hyper


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Immunoglobulin structure Each Ig mol is made of 4 polypeptide chains :

2 identical light(short) & 2 identcal heavy(longer) heldtogether by interandintra-disulphide(S-S) bonds There are 2types of light chains-kappa & lamda

Basedon this the Ig mol may be kappa/lamda

In order to study the diff.portions of Ig mol, it was treatedwith

proteolytic enzymes-papain & pepsin.

Papain digestion results in3 pcs----2 identical pcsFab(fractionAg binding) each containing a light chain andhalfa heavychain. The 3rdsingle pcFc(fraction-crystallisable) consists of

the other 2 halves of the heavy chain still linkedby thedisulphide bonds

When Ig is digestedwith pepsin only one major fragment of 2Fab units is obtainedwhere the interdisulphide bondis stillexisting andan Fc region which loses its link& breaks up


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Structure of

Immunoglobulin


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Papain digestion results in 3 pieces-2 identical pcs (Fab) each containing a light chain

and half a heavy chain,the 3rd pc (Fc) consists of other 2 halves of the heavy chains

still linked at the hinge by the disulphide bonds


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When IgG is digested with pepsin, only one major fragment consisting of two Fab

Units is got, it is joined at the hinge with interdisulphide bonds. The Fc region loses

Its link with the hinge region and breaks into small fragments


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Based On cellular basis immunity may be of 2 types

Humoral Immunity Cell mediated immunity

Involves syn.& release the antigen stims

of free Ab into blood / the produc. Of sensi-

other body fluids tized lymphocytes which

Depend on B cells confer Host protection

depend on T cells

Cells involved in immunity are present throughout the body but

mainly found in Lymphoreticular organs

a) Primary lymphoid organs---thymus & bursa of fabricus

b) Secondary lymphoid organs---lymph nodes, spleen, tonsils


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Active immunityActive immunity is the resistance developed by an individual in

response to an antigenic stimulus. The antigen may gainentrance either by natural infection or through vaccination(artificial). Active immunity involves the active functioning of apersons immune system, resulting in the synthesis of specificantibodies (humoral immunity) or production of

immunologically active cells (cell mediated immunity)Humoral immunity-here specific antibodies are produced in in

plasma or lymph,in response to specific antigenic stimulus. Theantigen may then be clumped or their toxicity neutralized.Thisimmunity sets in after a latent/lag period needed for Ab prodn

Cell mediated immunity-here instead of specific antibodiessensitized lymphocytes are produced which react with thespecific antigens bringing about cytotoxic effects and lysis.If aperson is exposed to a parti. Ag for 2nd time, immune responseis quicker & abundant than 1st ---known as Secondary response


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Immune system The cells involved in the immune responses have their origin in

the stem cells of bone marrow.

There are 2 types of cells inthe bone marrow

Myeloid lineage---neutrophil, monocytes,basophil, esonophil

Lymphoid---B lymphocytes & T lymphocytes

M

ajority of lymphoid stem cells migrate to the thymus gland &get differentiated into T cells ofHelper,Suppresor& Cytotoxic

type. This starts shortly before birth, they are small, found in

blood, lymph, lymphoid tissue. T cells do not produce humoral

antibodies but indirectly control their production. T cells are

involved in cell mediated immunityOther lymphoid stem cells migrate to the Bursa(lymphoid area in

birds) equivalent eg payers patch & differentiate into B cells

when stimulated by antigen produce antibodies to be circulated

through out lymph & blood to protect the individual, lives for Ito 2 weeks.Involved in humoral immunit


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Primary lymphoid organs are the major site for lymphopoiesis. Here the

lymphoid Stem cells proliferate, differentiate and mature into immuno-

competent cells in the absence of antigenic stimulation.

The mature but nonfunctional lymphocytes migrate from the primary lymphoid

organs through the blood or lymph and accumulate in the peripheral or

secondary lymphoid organs.

Here the cells when exposed to antigenic stimulus become functional by

producing either 1) specific antibodies or 2) sensitized cells.

The spleen, lymph nodes, tonsils, appendix constitute the secondary lymphoid

organs. These second.Lymph.Orgs are poorly developed at birth and grow

progressively while the primary ones are large at birth and atrophies on age.


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IMMUNE RESPONSE

THE BODY CELLS IMPORTANT TO IMMUNE RESPONSE

B CELLSLYMPHOCYTES WHICH PRODUCE ABS. IN BLOOD

MACROPHAGES---PHAGOCYTIC CELLS WHICH ALERT HELPER T CELLS OF

PRESENCEOF PATHOGEN

HELPER T CELLS--- MASTER SWITCHES WHICH RELEASE LYMPHOKINES---STIMULATES RAPIDDIVISION OF T & B CELLS

NATURAL KILLER(NK) CELLS---LYMPHOCYTES THATRELEASELYMPHOTOXINS

DIRECTLYDESTROYBODYCELLS ALREADY INFECTED WITH PATHOGEN

SUPRESSOR T CELLS---LYMPHOCYTES WHICH SLOWS DOWN IMMUNE

RESPONSE BYINHIBITING THEPRODUCTION OF CYTOTOXIC T CELLSCHECKING THEM FROM CAUSING MORE DAMAGE THAN NECESSARY

MEMORY CELLS---A GROUP OF T&BCELLS PRODUCEDDURING PRIMARY

ENCOUNTERBUT ARERESERVED TORESPOND TOLATER ATTACKS BY SAME

ORGANISM (role in secondary immunity)


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Function of B cells & T cells

B cells have Ig receptors on their surface membrane which aresuitable for the specific Ab it will produce.If the B cells in the

lymph nodes encounter an antigen,which has been transported

to the lymph node via the lymphatics,the cells get

activated,divide and differentiate to become plasma cells

B cells must receive a signalfrom T cell/macrophage to

proliferate andonlyafteralag perioddifferentiate into plasma

cells in the bloodwhich produce the antibodies to fight.

The function of the T cells is to recognise an antigen andthen

release *lymphokines (helper T)---stim B & T cells to grow &

differentiate *enhance suppresor function ( supp T)---which

inhibit the production of killer T cells that actually secrete

lymphotoxins and bring about cell lysis


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Innate vs adaptive immunity

Innate system(non-specific, natural )Adaptive system( specific, acquired )

Does not take time to respond, as

the system is in place prior to

exposure to antigen

Takes some time to react to an

invading organism, since the system is

induced after exposure to antigen

It is not antigen specific andreacts

equally well to a variety of

organisms

It is antigen specific and reacts onlywith that particular organism

Does not demonstrate

immunologicalmemory

This system demonstrates

immunological memory. It remembers

that it had encountered an invading

Organism and reacts more rapidly on

subsequent exposure.Plays an

important role in secondary immunity


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Primary/secondary immune response

P

rimary immune responseimmune response produced byentry of the Ag for the first time---the 1st injection of the Ag is

called primary dose.

Secondary immune response if the same animal is exposed to

the same Ag for the second time the animal produces the

immune response for the 2nd time called secondary immuneresponse and the 2nd Ag is known as booster/secondary dose.

The humoral immunity results in the production of antibodies in

the blood. The amount of antibody produced is called the

antibody titre.If this is plotted against time a sigmoid curve isobtained having 4 phases lag, log, plateau and decline phases.

Graphical representation of primary/secondary immune

response


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comparisonPrimary immune response Secondary immune response

Lag phase- time after the Ag exposure

Varies from hours to days

Very short , the body responds quickly

Log phase rise in Ab content and this is

sharp

The Ab titre here exceeds that of primary

response ----10 folds more

Plateauthis is a time when the Ab titre

is constant, short duration

The time is much extended compared to

primary

Declinehere the catabolism of Ab

exceeds the production, so there is a fallin Ab titre

The fall in Ab level takes place very slowly


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Clonal selection theoryLong standing principle governing Ab production

Each B-lymphocyte during its development is committed to

respond to one antigenic determinant. (epitope)

Accordingly each lymphocyte has a single type ofAg-specific

receptoron its surface. (paratope)

Receptor occupation is required for B cell activation.

Following contact with Ag, the B cell expands and divides

producing aB cell clone ( clone is a genetically identical

progeny produced naturally/artificially by mitosis from a

single specific cell type). This will differentiate into plasmacells which will form antibodies having receptors of identical

specificityas the parent B cell


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IMMUNOTOLERANCE

ITIS A CONDITIONWHEREadministration OFANTIGENICMATERIAL DOESNOTEVOKEANIMMUNOLOGICAL RESPONSE

BOTHB& T CELLS AREMADEUNRESPONSIVE

immunotolerance

natural or self tolerance

Where body does not evoke

immune response to self

antigens

Induced tolerance

Where body does

Not evoke immune

Response to external

antigens

During pregnancy mothers tolerance to foetus/placenta ---eg of natural

In clinical practice during organ transplantation chances of rejection is major

Problem faced. To prevent rejection, drugs are used to induce tolerance


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Tolerance can occur in several ways -----

1) GENETIC UNRESPONSIVENESS-------ANIM AL LACKS THE GENETIC

ABILITY TO RECOGNIZE ANTIGENIC MATERIAL

2) TSUPRESSION------Supressor T CELLS MAY BE MORE EFFECTIVELY

ACTIVATED THANTHE helper T CELLS THUS SUPPRESSING THE

IMMUNESYSTEM

3) HELPLESSNESS----WHENT cells CANNOT ACTIVATETHEB CELLS THEY

AREREGARDED AS HELPLESS

4) CLONAL DELETION----CONTACT WITH ANTIGENINTHE NEONATE (

immature immune system) RESULTSINDEATH/PERMANENTINACTIVATION

OFdeveloping LYMPHOCYTES


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Autoimmunity

Antigens present in ones own cells are autoantigens or self

antigens. These generally do not elicit an immune response.

Only the foreign or nonself Ag evokes immune response.

However when they are altered by the exposure to some

bacteria/ toxic chemicals/drug, they are recognised as non-selfAgs & stim prod. of Abs against them called autoantibodies.

Hence autoimmunity is definedas humoral/cell mediated

immune response directedagainst the bodys own tissues or self

antigens

Autoimmune diseases are a group of disorders in which host

tissue damage is caused by autoimmune responses to self Ags


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Types of autoimmune(AI) disease

1. Haemolytic Auto Imm diseaseresults in destruction of

RBC/platelets/ as Ags on RBC etc become nonself Ag.

Haemolytic anaemia, leucopenia,thrombocytopenia (low

platelet count )2.Localised Auto Imm disease results in the destruction of a

particular organ eg Thyrotoxicosis, Addisons disease

Myasthenia gravis (weakening of skeletal muscles-autoAb

against Ach receptor cells thus Ach cannot be producedno

impulse passed)

3.Systemic Auto Imm disease affects the whole body or many

organs

eg Lupus erythematosus, rheumatoid arthritis


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Hypersensitivity Immune response is mostly directed towards the protection

of host, as the bacteria/virus is killed in the process. Howevernot all Ag-Ab reactions are beneficial.

In hypersensitivity the immune responses become

injurious/destructive to the host, killing or damaging body

cells Thus it may be definedas the violentadverse reaction

(malfunction) of the immune system leading to severe

symptoms & even death in a sensitizedanimal/human when it

is re-exposedto the same Ag for the 2nd time

Hypersensitivity occurs only in 10% of cases, in clinical terms

The agents causing hypty are called allergens

The effect is called allergy/hyperty


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Factors causing hypersensitivityAllergens may be extrinsic (enters into body) or intrinsic

(present inside body)Drugs penicillin, sulphacetamide, aspirin

Airborne particles pollen grains, dust, scales/feathers ofanimals eg dog, cat, bird

Food stuff shell fish, brinjal, pineappleInfectious organisms bacteria, virus, fungi, parasite

Blood transfusion mismatched blood

Common hypersensitive reactions---

Anaphylaxis Serum sickness

Transfusion reaction Mantoux reaction

Erythroblastosis foetalis Contact dermatitis

Arthus reaction Graves disease


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Classification

Based on time taken-*immediatesymptoms appear eg Hay fever within mins i.e

rapid onset (type 1, 2, 3), eg allergic reacts-urticaria (skin),

rhinitis, extrinsic asthma,involves B cells, can be transferred

through serum, controlled by antihistamines

*delayed symptoms within 24 to 48 hrs (type 4,5) eg Mantoux

test, involves T cells, transferred through T cells/corticosteroids

Based on different mech. of action

*type 1 :Anaphylactic hypersensitivity

*type ii:Antibody-dependent cytotoxic hyperty

*type iii:Immune-complex mediated hyperty

*type iv:Cell mediated hyperty

*type v: Stimulatory hyperty


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Typescontd.Anaphylaxisis defined as exaggerated reactions shown by the

body to a foreign substance to which it has previously become

sensitized.

Abs are fixed on mast cells in host causing release ofhistamine, serotonin on 2nd exposure eg penicillin allergy

Cytotoxic when antibodies attach to the antigens located on the

surface of cells eg agglutination & lysis occurs due to

mismatched blood groups, eg erythroblastosis ( RBC rupture)

Immune complexwhen huge amt of antigen enters body a large

conc. of Ab is produced which combine to form insol complex

which get attached around minute blood vessels leading to

hypersensitivity, Arthus reaction, serum sickness

Cell mediatedcaused by interaction between Ag & T cells, abs arenot involved Contact dermatitis, tuberculin reaction

Stimulatorycaused by interaction between Ab with cell surface

Ag where over stimulation of cells of the thyroid occur eg

Graves disease (thyrotoxicosis)


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ANTIGEN-ANTIBODY REACTIONSSEROLOGYIS THESTUDY OFAG-ABREACTIONS

USED------ TO TYPEBLOOD,DIAGNOSEDISEASE

IDENTIFYMICROBES,INTISSUETRANSPLANTATION, TO DETECT

ALLERGY/HYPERSENSITIVITY

THECOMMON Ag-Ab reactions (INVITRO TESTS)ARE---

AGGLUTINATION

PRECIPITATION

COMPLEMENTFIXATION

FLUORESCENT ANTIBODY TECHNIQUE

RADIOIMMUNOASSAY (RIA)ENZYMELINKEDIMMUNOSORBENT ASSAY(ELISA)

PURPOSE:THEPRESENCEOFSPECIFIC ANTIBODIESINTHEPATIENTSSERUM

WILL INDICATEWHETHER HEHAS THEDISEASE/HEISIMMUNETO

DISEASE/HEIS ALLERGIC TO ANTIGEN


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Antigen antibody reactionWhen an Ab and the Ag responsible for its production are mixed, the two

bind to form a Ag-Ab complex and such a reaction is called Ag-Ab react,represented by Ag + Ab ----------------- Ag-Ab complex

Characteristics of such a reaction:

1. Role -This immune complex forms the basis of humoral immunity

2. Specificityrefers to the indiscriminate ability of a particular Ab tocombine with only one Ag determinant responsible for its production.

Compared to lock and key system.

3. Binding sites of Ag and Ab. An Ag may contain 10-50 determinants

while most Abs are bivalent (2 paratopes) only IgM has 5-10

4. Binding forcesof Ag and Ab. The intermol forces are of non covalenttype eg hydrogen bonding/vanderwaal. Affinity refers to strength of bond

between a Ab mol and a single antigenic determinant

5. Avidity- It is the total strength of the bonds after formation of the

individual complexes on an Ag nAb + mAg--------------AbnAgm

n=no of Ab m=no of Ag determinants


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AGGLUTINATION --- is an antigen antibody reaction where the specific antibody

causes the insoluble cellular (particulate) antigens to adhere to one another to form

visible clumps,in presence of electrolyte and suitable temp, pH.

The particulate Ag include RBC, platelets, lymphocytes, bacteria, viruses etc.

Agglutination is used for ABO and Rh blood typing --- sample( A ,B, AB, O)

agglutination anti B

here the Abs are called agglutinins & Ags called agglutinogens

Applications----

Widal Test ---used for diagnosis of typhoid fever. The test uses antigen H & O, so if

the antiserum of patient carries specific Abs to these Ags, clumping seen.Well Felix reaction ---- used to test typhus fever

Coombs test---- devised for detection of anti Rh antibodies

sample+ +

Anti Aserum

AntiBserum


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Agglutination

Qualitativedone only to detect the presence of

Specific Ag or Ab in sample.

Eg patients blood can be mixed with serum having

anti A, B, AB, to see with which it agglutinates.

Grp A if agglutination with anti A serum(Ab in GrpB)

Quantitativedone to measure the level of AbsPresent in the serum with respect to Ag. Serial

dilutions of a serum sample is made and placed in

same vol in a 96 well plate.It is

Mixed with a fixed no. of the Ag(bacteria/RBC)

The max dilution that gives visible agglutination is

noted and is referred to as Titre.

A 4 fold rise intitre is generally taken as disease established.

Passive agglutination since agglutination takes place only with insoluble Ags

one modification can be done incase of soluble Ags (viral Ag, polysaca) the RBC is coated

with the sol Ag and the test is done as above. Hence by observing the clumping of RBC the

titre of the sol, Ab may be measured indirectly hence passive.


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Complement fixation test Complement refers to a group oflarge, thermolabile,complex

proteins(beta globulins),found in the serum and body fluids,

which completes the Ag-Ab complex, by fixing with it.

CFT is an immunological technique used for the detection of

unknown Ag or Ab by finding if the binding/fixing of the

complement to the Ag-Ab complex has occurred or not. When a complement is added to a serum containing Ag and

its Ab, the complement present is activated and immediately

binds to the Ag-Ab complex and is said to be fixed

Ag + Ab + * ======== Ag--*-Ab

compl fixed


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CFTThis test involves the operation of 2 immune systems

1st system ---is the unknown system under detection called testsystem

2nd system---is the known system containing sensitized RBC andacts as the indicator system as it indicates complementactivity

Steps in CFT

1.Serum to be tested for Ab + known Ag + compl = Ag-*--Ab

2. RBC (coated with its Ab ) is added to above = no haemolysisin the tube due to unavailability of free complement as it has

been used up in the 1st

complexResult : Absence of haemolysis is a +ve test indicating presence

of specific Ab in sample.Presence of haemolysis is ave test,indicating absence of specific Ab in sample

609 1st int slides

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