Research Methods in Immunology

ResearchResearch methods in methods in

Immunology and Immunology and

geneticsgeneticsgeneticsgenetics

Medical studies in EnglishMedical studies in English

MEFSTMEFST, 201, 2012/132/13..

Techniques in ImmunologyTechniques in Immunology

Monoclonal and polyclonal antibodies productionMonoclonal and polyclonal antibodies production

Flow cytometryFlow cytometry

ELISA ELISA -- Enzime Enzime linkedlinked immunosorbentimmunosorbent assayassay

RIA RIA Radio immunosorbent assayRadio immunosorbent assay

IP IP immunoprecipitationimmunoprecipitation

Western blot, immunofluorescenceWestern blot, immunofluorescence Western blot, immunofluorescenceWestern blot, immunofluorescence

Blood group typingBlood group typing

Coombs test (direct and indirect)Coombs test (direct and indirect)

Affinity chromatographyAffinity chromatography

Other techniques: Haematopoietic cells concentration, Other techniques: Haematopoietic cells concentration, complement fixation test, contact precipitation, gel complement fixation test, contact precipitation, gel immunodiffusion, protein electrophoresis3immunodiffusion, protein electrophoresis3

Polyclonal antibodies

Monoclonal antibodiesMonoclonal antibodies

Antigenes have many epitopesAntigenes have many epitopes

Inoculated to experimental animal induce production Inoculated to experimental animal induce production of policlonal antibodiesof policlonal antibodies

By isolating plasma cells from spleen and fusing By isolating plasma cells from spleen and fusing them with B myeloma cells (which do not produce them with B myeloma cells (which do not produce antibodies), clones of plasma cells are immortalised antibodies), clones of plasma cells are immortalised antibodies), clones of plasma cells are immortalised antibodies), clones of plasma cells are immortalised hybridomas.hybridomas.

Uppon sellection in specific media, single cell clones Uppon sellection in specific media, single cell clones are propagated, and checked for antibody are propagated, and checked for antibody production and qualityproduction and quality

Monoclonal antibodies are used for:Monoclonal antibodies are used for: Radio labelled or fluorescente labelled applicationRadio labelled or fluorescente labelled application

Immunofluorescence (IF, IHC, IP, WB, experimental use)Immunofluorescence (IF, IHC, IP, WB, experimental use)

Immunotoxic tumor therapyImmunotoxic tumor therapy

Many other aplicationsMany other aplications

(PEG)

Flow cytometry

ELISAELISA

EnzymeEnzyme--Linked Linked ImmunoSorbentImmunoSorbent Assay Assay

Each sample is pored into a well with immobilised Each sample is pored into a well with immobilised antibodies specific for tested molecule (hormon, enzime)antibodies specific for tested molecule (hormon, enzime)

Different specific antibody is added to each well in Different specific antibody is added to each well in excess. This ab is bound to a colour producing enzime excess. This ab is bound to a colour producing enzime

Well os washed out from unbound componentsWell os washed out from unbound components Well os washed out from unbound componentsWell os washed out from unbound components

A substrate specifis for an enzime is that added, and A substrate specifis for an enzime is that added, and colour development is measured by spectrophotometrycolour development is measured by spectrophotometry

Sandwich ELISASandwich ELISA-- for very low concentration antigenes for very low concentration antigenes (egz. Cytokines)(egz. Cytokines)

Antibodies used are speciffic for opposite sides of Antibodies used are speciffic for opposite sides of moleculemolecule

ELISA

RIA RIA

RadioimmunoassayRadioimmunoassay

More sensitive than ELISA testMore sensitive than ELISA test

Used for measuring of Used for measuring of blood hormone blood hormone concentrationconcentration

-- Radioisotope bound (marked) antigene Radioisotope bound (marked) antigene ((hormon) hormon) -- Radioisotope bound (marked) antigene Radioisotope bound (marked) antigene ((hormon) hormon) binds to antibodybinds to antibody

-- After adding unmarked antigene in a known After adding unmarked antigene in a known concentration, some of the marked antigene is concentration, some of the marked antigene is displaced from antibody, due to competition.displaced from antibody, due to competition.

-- A binding curve of radioactivity is ploted.A binding curve of radioactivity is ploted.

-- Now, an unknown hormon concentration can be Now, an unknown hormon concentration can be measured by comparing to ploted curve.measured by comparing to ploted curve.

+ + +Centrifuging and

washing

ImmunoprecipitationImmunoprecipitation

Protein A or G coated

sepharose

Protein lysates

Protein electrophoresis

Western blottingWestern blotting

Blood group typingBlood group typing

Agglutination can happen because every Agglutination can happen because every

monomeric antibody has two antigen monomeric antibody has two antigen

binding placesbinding places

Hemagglutination uses erithrocite Hemagglutination uses erithrocite

aglutination for blood group typingaglutination for blood group typing

Blood group typingBlood group typing

Coombs testCoombs test

Uses antibodies against human antibodies Uses antibodies against human antibodies (Coombs reagent = anti human IgG)(Coombs reagent = anti human IgG)

direct Coombs testdirect Coombs test

For For antigene testing antigene testing (Rh (Rh blood group blood group

antigenes)antigenes)antigenes)antigenes)

indirect indirect Coombs Coombs ttestest

For antibody testing (antiFor antibody testing (anti--Rh IgG) Rh IgG) egz. For egz. For

prenatal testing of pregnant woman or blood prenatal testing of pregnant woman or blood

testing prior to transfusion.testing prior to transfusion.

Visualisation = hemagglutinationVisualisation = hemagglutination

(Immuno)affinity chromatography(Immuno)affinity chromatography

Monoclonal antibodies are bound to gel Monoclonal antibodies are bound to gel matrixmatrix

Mixture of proteins is passed throug the Mixture of proteins is passed throug the columncolumncolumncolumn

Column is washed and unbound proteins Column is washed and unbound proteins are discardedare discarded

By changing the pH of the elution buffer, By changing the pH of the elution buffer, ag/ab bound is broken, antigenes are ag/ab bound is broken, antigenes are washed away from the antibodies and washed away from the antibodies and collected in a tubecollected in a tube

Affinity chromatography

Chromatography Chromatography size based sepparationsize based sepparation

Stem cellsStem cells

Hematopoietic stem cell therapy Hematopoietic stem cell therapy for for patients with disfunctional hematopoietic patients with disfunctional hematopoietic

Injected cells find their way to bone Injected cells find their way to bone marrow through blood streemmarrow through blood streem

Approximately 10% of donor bone marrow Approximately 10% of donor bone marrow Approximately 10% of donor bone marrow Approximately 10% of donor bone marrow is transplantedis transplanted

Transplant types: Transplant types:

Autologous (frozen, or geneticaly Autologous (frozen, or geneticaly mannipulated)mannipulated)

Singenic (identical twins)Singenic (identical twins)

Alogenic (possibility of GVHD) Alogenic (possibility of GVHD)

Haematopoietic cells concentrationHaematopoietic cells concentration

Bone marrow is mixed Bone marrow is mixed with antibodies against with antibodies against mature red and white mature red and white blood cellsblood cells

Using flow cytometry cell Using flow cytometry cell Using flow cytometry cell Using flow cytometry cell sorter ab marked cells are sorter ab marked cells are selected and discardedselected and discarded

Finaly, unmarked Finaly, unmarked nondifferentiated nondifferentiated hematopoietic stem cells hematopoietic stem cells are concentratedare concentrated

Techniques in GeneticsTechniques in Genetics

Cloning (genetic engeneering)Cloning (genetic engeneering)

Production Production ofof KnockKnock--outout and and transgenic transgenic mice mice mice mice

Stem cell productionStem cell production

PlasmidsPlasmids

IsolatedIsolated segmentssegments ofof bacterialbacterial DNA, DNA, capablecapable

to to replicatereplicate independentlyindependently ofof chromosomalchromosomal

DNADNA

DoubleDouble strandedstranded andand circularcircular DoubleDouble strandedstranded andand circularcircular

One One bacterialbacterial cellcell cancan havehave upup to 1000 to 1000

copiescopies

UsedUsed as as vectorsvectors inin researchresearch for gene for gene

transferetransfere

PlasmidPlasmid as a as a vectorvector

(multiple cloning site)

RestrictionRestriction endonucleasesendonucleases

(PCR product)(multiple cloning site)

Restriction endonucleases

DNA ligase

Recombinate

plasmid vector

CuttingCutting

withwith

EcoR1EcoR1

CloningCloning intointo a a plasmidplasmid vectorvector

Insert DNA fragments

CloningCloning intointo a a plasmidplasmid vectorvector

KnockKnock--out out micemice

(2007(2007. . Nobel prize in Physiology)Nobel prize in Physiology)

Production of knockProduction of knock--out mice enabled us to out mice enabled us to study the influence of particular gene product study the influence of particular gene product on growth, developmen and function on growth, developmen and function

Procedure includes selective inactivation of a Procedure includes selective inactivation of a certain gene in a fase of blastocist certain gene in a fase of blastocist

Today all known genes of a mouse genome Today all known genes of a mouse genome Today all known genes of a mouse genome Today all known genes of a mouse genome is being tested through thisis being tested through this

Mouse genome will have a:Mouse genome will have a: Resistency towards one of the antibiotics Resistency towards one of the antibiotics

(necessery for the process of cell selection)(necessery for the process of cell selection)

Gene for making a fenotipe for changed genotype Gene for making a fenotipe for changed genotype easily detectable (crosseasily detectable (cross--breading of white and breading of white and black mouse)black mouse)

Making a knockout mouse: Stage 1, creating stem

cells with an interrupted gene 8. Sellect with G418 and gangcyclovir

TK- tymidin kinase

NEO- neomycine cassette

G418 - geneticine

Making a knockout mouse: Stage 2, placing

the errupted gene in the animal