Lect 08 Serology

8/2/2019 Lect 08 Serology

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Specific immune factors

Cellular

humoral

AG( antigene)--AB(antibody) complex

Serology


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SPECIFIC IMMUNE RESPONSEability to detect foreign substances

specificity different response to different antigensmemory permits a rapid and specific secondary response

IMMUNOCOMPETENT CELLSAntigen-presenting cells(APC): mononuclear macrophages,

dendritic cells, B lymphocytesAntigen-recognizing cells: T helpers (CD3,CD4)

Effector cells: B lymphocytes, plasma cells and memorycells (CD19, CD20, CD21)antibody productionCTL cytotoxic T lymphocytes (CD3,CD8), NK natural killer

cells (CD16),cytotoxicity


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ANTIGENS

Anti against // genos genus

organic substances of a colloid structure, which upon injection intothe body are capable to cause the production of antibodies and to react

specifically with them

substances of any nature, that are recognized by cells of macroorganismimmune system as genetically foreign and bring about immune response

AG MAJOR PROPERTIESImmunogenicity ability to cause an immune response

(to cause the production of AB)Antigenicityability to interact with AB

Specificityinteraction only with homologousAB


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complete AG

immunogen incomplete (partial)AGhapten

Substances most high-molecular(proteins, P, PS, NP, LP,

LPS, GP)low-molecular

Antigenicity + +Specificity + +Immunogenicity + (+ if coupled to a

carrier)


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antigenic determinant(epitope) certain chemical group in AGmolecule that determines reaction specificity and binds to

antigen binding site of AB monovalentAG (haptens),bivalent(contain 2 epitopes),

polyvalent(multivalent) AG

adjuvant chemicals that enhance the immunogenicity ofantigens and also stimulate phagocytosis, thus increasing the

immune response(aluminum compounds -(Al(OH)3), oil emulsion, Freund adjuvant mineral oil + killed Mycobacteriumtuberculosiscells)


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According the particular structure of bacterial cellthat AG are associated with:

H-AGflagellar, heat-labile, remain immunogenic after treatmentwith phenolO-AGsomatic(associated with LPSof cell wall), heat-stableK-AGcapsule(surface layer PS or protein)Vi-AG also capsuleAG, found in some enteropathogenic(high-virulent) bacteria

every microorganism contains many different AGs, that reflects

by it antigenic formula (e. g. Salmonella typhi- group D 9, 12, Vi.E. coli- O153:H1:K7 etc.)

AGs of MICROORGANISM


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Definitions of antigenes

protectiveAG high-antigenic and low-toxic, cause theproduction of blocking AB

cross-reacting AGare common for human andmicroorganism (phenomenon of microbial mimicry)

isoantigenes substances which have antigenic

properties and are contained in some individuals of agiven species (blood groups serum antibodies)autoantigenes substances capable of immunizing the

body from which they are obtained (eye lens,spermatozoids, emulsions of kidney, liver, lungs and

other)

According to the specificity AG could be:

Group-specificAG AG common for different microbial species

Species-specificAG

AG associated with particular speciesType-specificAG (serotype, serovar) found in different variantsof one species


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ANTIBODIES

are globular glycoproteins (-globulin fraction in serum proteinsof blood, tissue fluids and some secretions

are made in response to specific antigen (foreign macromolecule)by B cells (plasmatic cells)

are capable of specific binding to appropriate antigen (AG)could be produced to every antigen


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The five classes of antibodies


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PROPERTY IgG IgA IgM IgD IgE

Number of monomers 1 1, 2 5 1 1

Number of AG-binding sites 2 2, 4 10 2 2

Percentage of total immunoglobulin 80 9-10 5-10 0-1 0.005

Complement fixation (Class. orAltern.) + C,A A + C - -

Crosses the placenta + - - - -

Fixes to basophils and mast cells - - - - +

Binds to macrophages and neutrophils + - - -


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Types of AB reactivity:Toxin neutralizationantitoxinsPrecipitation of soluble AGprecipitinsAgglutination of corpuscular AGagglutininsEnhancement of phagocytic activityof leukocytesopsoninsComplement bindingcomplement-bindingAB

completeAB

form visual reactions, have 2 and more active(AG-binding) centersincomplete (blocked) AB have only 1 active center, other isblocked; require Kumbs method for their detection

monoclonal AB each specific AB is synthesized by particular

cell line, that could be developed trough hybridoma culturetechnique (lymphocytes are fused with myeloma cells);generally after immunizationin Ig pool are polyclonalAB


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T- independet T-dependentIs realised by B- cells which are

constantly carrying surface

receptors Ig M Ig D that

recognize epitopes of bacteria.

After binding with correspondent

AG

(Signal with IL-7 and tyrosine-

kynase) the polyclonal activation

is started and plasmatic cells

begins to produce Ig with low

affinity - Ig M Ig D.

Is realised by B- cells only if they

have the assistance of antigen

presenting cells and Th.

high affinity -Ig A, Ig G, Ig E

Antibody response on microbial antigenes may be:


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COOPERATIVE INTERACTION OF IMMUNOCOMPETENT CELLSMain features:Cell interaction only when they are genetically identical (MHC II class)Double recognition presented AG is recognized by immunocompetent cell

only in complex with I class (target cells) or II class (APCs) MHC

IMMUNE RESPONSEPhagocytosis of foreign AG( cell, particle) by macrophage Processing and presentation of this AG in association with II class MHC +secretion of IL-1 by APCs Recognition of presented AG by Th (CD3-CD4) complex + activation of Th byIL-1 secretion of humoral factors, including IL-2, IL-4, IL-5, -interferonRecognition of AG by B lymphocytes their activation, including synthesisof receptor for humoral factorsActivation of B cells by humoral factors and their differentiation into AB-producing cells (plasmatic cell)

Production of antibodyBinding of AB to AG and elimination of such immune complexes fromorganism by specific or non-specific mechanisms (phagocytosis, lysis bycomplement, destroying of foreign cells by CTL, NK and K cells etc.)


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DYNAMIC of AB PRODUCTION

depends from:

power of AG affection (AG dose),

frequency of exposure to AG,

general health of the host organism,particularly its immune system

dynamics differ in primary (I) andsecondary (II) response at all stages:

I II


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I II

1. Latent (lag) stage 35 days 12 days

2. Logariphmic 715 days differs, but generally has rapid rise in ABtiters, high AB titers, long stationary

period and slow decrease

3. Stationary 1530 days

4. Decrease 16 months

Ig isotype IgM, than later

IgGOnly IgG

secondary response goes due topreexistence of memory cells


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CHARACTERISTICS of SEROLOGICAL REACTION

specificity reaction of antigen with homologous antibodysensitivity minimum quantity of AG or AB detected byparticular serological reaction

STAGES of SEROLOGICAL REACTIONSpecific interaction of AG with ABNon-specific formation of visible result (usually in the presenceof electrolyte), depends from the characteristics of AG and couldbe developed in different forms (agglutinates, precipitates etc.)


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COMPONENTS of SEROLOGICAL REACTIONS

AG in form of corpuscular diagnosticums (suspension of live orfixated bacteria, biochemicals adsorbed on different particles

etc.) or solutions (extractions, isolated chemical fractions frombacteria, viruses, toxins etc.)

AB in form of immune serum (obtained by immunization ofexperimental animals) or AB-diagnosticums (AB adsorbed on

different particles)

ElectrolyteAdditional developing system (hemolytic system, complement,

markers etc.) if necessary


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SEROLOGICAL REACTIONSIMMUNE SERUM must have following determined characteristics:Serum titer(AB level) maximum serum dilution that gives positive

reactionSerum specificity reaction only with homologous bacterial cultures;

could be restricted to a genus specificity, species, variant etc.

DIAGNOSTIC IMMUNE SERA:

agglutinating sera(identification of bacterial genus, species, serovar),precipitating sera(AG detection in examines sample),hemolytic sera(are used in additional developing system, e. g. in RCB),

antitoxic sera(are used for toxin neutralization)

TYPES of IMMUNE SERUM

polyvalent(non-adsorbed) low specific; high AB titers; contain AB tomicrobes, that have common group, genus, family AG, i. e. can elicit

group cross-reactionsmono-receptor(adsorbed) high specific; low AB titers (1:40, 1:300);are generally used for agglutination on slide,for determination of serovar


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AGGLUTINATION REACTIONS

are used both for identification and examination of infectious agent(e. g.agglutination on slide, Gruber method etc.) and detection of specific ABin

patients serum (Vidal method for typhoid, Right, Heddlson method forbrucellosis etc.)

are based on the formation of AG-AB aggregates or latticeat optimal

concentrations of both componentsdirectand indirect(AG or AB are adsorbed on the cell or particle surface)hemagglutination AG are located on the surface of red blood cells, andthe addition of AB leads to their clumping (e.g. is basis for blood typing

etc.)DIAGNOSTIC TITER maximum immune serum dilution that elicit positive

resultto get reliable diagnose pair seraare taken (to follow the dynamic of AB

titers rise)


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KASTELLANI METHOD of IMMUNE SERUM ADSORBTION

relative groups of microorganism are used to bind and

exclude group-specific and species-specific AB out ofpolyvalent serum to get finally mono-receptor immuneserum (only type-specific AB left examination ofbacterial antigenic structure)


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PRECIPITATION REACTIONS

are generally used for detection and examination of AG using diagnosticserum

are also based on lattice formation and strongly require optimal AG and

AB proportions (zone of equivalence) and have very high sensitivity;are performed in solutions (ring-test) or in gels (Ouchterlony method,

Manchini, immunoelectrophoresis etc.)

NEUTRALIZATION REACTIONS

are based on the ability of antitoxic serum to bind and inactivate toxinsare used for detection of toxin production by microbial cells and detection ofpresence of antitoxin AB in patients blood (diagnostic of infectious disease)


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REACTION of COMPLEMENT BINDINGis based on the ability of AB to bind complement thus excluding it out of

solution

LYSIS REACTIONS

are based on the ability of opsonins to activate complement system

REACTIONS using LABELED AB (ELISA, RIA, RIF etc.)


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Lect 08 Serology

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