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Complement, Complement, Immunoglobulins, Antigen, Immunoglobulins, Antigen,
Hapten, Hapten, - Antigen Antibody Reaction- Antigen Antibody Reaction
Sakchai Dettrairat Sakchai DettrairatDivision of Division of Clinical Immunology Clinical Immunology
Department of Department of Medical TechnologyMedical Technology Faculty of Associated Medical Sciences Faculty of Associated Medical Sciences Chiang Mai University Chiang Mai University
03/06/2009
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macrophages, neutrophils
complement, interferon, TNF etc.
T cells; other effectors cells
antibodies
Components of the Immune SystemComponents of the Immune System
Immune System
Nonspecific Specific
Humoral Cellular Humoral Cellular
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Complement Complement SystemSystem• Serum globulins• Z ymogens or proenzyme• >20 components
– 1 1 1 4 2 3 5 6 7C q, C r, C s, C , C , C , C , C , C , C 8 , 9C
– DD DDDDDDDDDD ,, ,– etc.
• HH - eat labile (56 C, 30 min)- eat labile (56 C, 30 min)• RDDDD DDDD DD-DD DDDDDDDDD DDDDD DDD DDDD• D ausing lysis of some Ab sensitized cells• NDD-DDDDDDD DDDDDDDD• M ore active in fresh serum D Guinea pig)
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ComplementComplement Activation Activation PathwayPathway
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Function of Complement
• Opsonization of cellular (bacterial) antigen
• Provoke inflammation• Poke holes in membranes
leading to lysis of bacteria• Clear immune complexes• Activates antigen-specific B cell
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Function of Complement
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Regulation of Complement Activation
C1 inhibitor (C1INH)
Factor I, Factor H
Factor I 4, C bp
Protein S, SP40,40
DAF
Factor I, Factor H
Factor H
DAF
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Antigen (Ag)
Antibody (Ab)
specifically react
Immunoglobulins (Igs)
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Antibody Serum Protein
Protein Electrophoresis
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Glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies
Immune serum
Ag adsorbed serum or normal serum
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Protein Structure: Polypeptide chain
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Protein Structure
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Immunoglobulin Structure
• Ig Subunits• Ig Fragments• Relation of Ig Subunits and
Fragments
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Determining Ab Structure
• Work in 1950s and 60s using biochemical techniques
• Rodney Porter used partial proteolysis with papain - 2 identical antigen-binding fragments (Fab) and one “tail” fragment (Fc)
• Alfred Nisinoff used pepsin - one fragment with divalent antigen binding (F(ab2)’)
• Gary Edelman used b-ME to reduce Ig, resolving heavy (H) and light (L) chains
• Rodney Porter probed H and L chains with anti- Fab and anti-Fc antibodies: anti-Fab detected both H & L, but anti-Fc detected only H
• Combined work earned Porter and Edelman 1972 Nobel Prize
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Ig Reduction Subunits
Immunoglobulin Subunits
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Light chain (L)
Immunoglobulin Subunits
Heavy chain (H)
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Ig papain digestion fragments
Immunoglobulin fragments
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Immunoglobulin fragments (Fab and Fc)
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Relation of Fab and Fc to H and L chains
Anti-Fab Anti-Fc
H + +
L + -
•rabbit Fab goat goat anti-Fab
•Rabbit Fc goat goat anti-FC•anti-Fab, anti-Fc react with H and L chains
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Immunoglobulin StructureImmunoglobulin Structure
• Subunits:– 2 2H+ L
• Fragments:– 2 1Fab + Fc
• Rel at i on of Fab t o H and L chains
and Fc to H chain
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Fig. 3.3 The Y-shaped immunoglobulin molecule can be dissected by partial digestion with proteases.
Fab-Fragment antigen bindingFc-Fragment crystallizableFv-Fragment variable
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Structure of the Variable Region
• Hypervariable (HVR) or complimentarity determining regions (CDR)
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Human Immunoglobulin Classes
Heavy Chain Types• IgG - Gamma heavy chains• IgM - Mu heavy chains• IgA - Alpha heavy chains• IgD - Delta heavy chains• IgE - Epsilon heavy chains
Light Chain Types• Kappa • Lambda
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IgG• Structure
– Monomer (7S)
• Properties– Major serum Ig– Major Ig in extravascular spaces– Placental transfer – Does not require Ag
binding – Fixes complement – Binds to Fc receptors
• Phagocytes - opsonization• K cells - ADCC
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IgM• Structure
– Pentamer (19S)– Extra domain (CH4)– J chain
• Properties– 3rd highest serum Ig– First Ig made by fetus and B cells– Fixes complement– Agglutinating Ig– Binds to Fc receptors– B cell surface Ig
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IgA• Structure
– Serum - monomer– Secretions (sIgA)
• Dimer (11S)• J chain• Secretory component
• Properties– 2nd highest serum Ig– Major secretory Ig (Mucosal or Local Immunity)
• Tears, saliva, gastric and pulmonary secretions – Does not fix complement (unless aggregated)– Binds to Fc receptors on some cells
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Secretory IgA (SIgA)
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IgD
• Structure– Monomer– Tail piece
• Properties– 4th highest serum Ig– B cell surface Ig– Does not bind complement
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IgE• Structure
– Monomer– Extra domain (CH4)
• Properties– Least common serum Ig
• Binds to basophils and mast cells (Does not require Ag binding)
– Allergic reactions– Parasitic infections (Helminths)
• Binds to Fc receptor on eosinophils– Does not fix complement
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Kinetics of the Ab ResponseT-dependent Ag; 1o Response
• Lag phase
• Log phase
• Plateau phase
• Decline phaseAg
D a y s A f t e r I m m u n i z a t i o n
A b
T i
t e
r
LAG LOG DECLINEPLATEAU
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Kinetics of the Ab ResponseT-dependent Ag; 2o Response
1o Ag 2o Ag
D a y s A f t e r I m m u n i z a t i o n
A b
T i
t e
r
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Qualitative Ab Changes during 1o and 2o Responses
• Class variation
– 1o - IgM
– 2o - IgG, IgA or IgE
1o Ag
2o Ag
Total Ab
IgM Ab
IgG Ab
D a y s A f t e r I m m u n i z a t i o n
A b
T i t
e r
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Monoclonal Antibody
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Antigen & Hapten• What is an antigen?
– a substance that can induce a specific immune response
• An immunogen induces a humoral (B-cell) or cell-mediated (T-cell) response
• Haptens are too small to induce an IR unless coupled to a carrier (an antigen)
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Antigen• Foreigness• High Moleclular Weight
– >10,000 Da– <1000 Da are not usually immunogenic
• Chemical Complexity – Proteins are often good immunogens.– Homopolymers are not good
immunogens• Induces Immune Response
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Hapten• Low MW Chemicals• Does not induce IR by itself• Reacts specifically with Anti-hapten
Abs
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How to produce Abs to Hapten?
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- Antigen Antibody Reacti- Antigen Antibody Reactionon
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Antigen-Antibody ReactionAntigen-Antibody Reaction
Ag + Ab - Ag Ab complex
1. Primary Reaction
(Invisible)
2. Secondary Reaction (Visible)
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Forces binding Antigen to Forces binding Antigen to AntibodyAntibody
Non-covalent forces Origin
Electrostatic forces Attraction between opposite charges
Hydrogen bondsHydrogen shared between electronegative atoms (N, O)
van der Waal forces
Fluctuation in electron clouds around molecules oppositely polarize neighboring atoms
Hydrophobic forces
Hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der Waal forces
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Forces binding Antigen to AntibodyForces binding Antigen to Antibody
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- Factors Affecting Ag Ab React - Factors Affecting Ag Ab Reactionion
• Temperature Temperature :: - 4 40o C (RT,
37oC)• pHpH : - 72 74 • Ionic strength Ionic strength : 0 .1 5 M NaCl
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PrecipitationPrecipitation
• Soluble Ag + specific Ab Soluble Ag + specific Ab• Precipitation in Liquid Media Precipitation in Liquid Media• - Precipitation in Semi solid me - Precipitation in Semi solid me
diadia
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Precipitation in Liquid MediaPrecipitation in Liquid Media
• Constant amo Constant amo unt of Ab unt of Ab
• Varied amoun Varied amoun
t of Ag t of Ag
• Amount of Pre Amount of Precipitatecipitate
……....
? ? ? ? ? ? ……….. ?
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Quantitative Precipitation Curve Quantitative Precipitation Curve
• Constant amo unt of Ab
• Varied amoun t of Ag
• Amount of Precipitate
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• Ab excess zone (Prozone)
• Equivalence zone
• Antigen excess zone (Post zone)
(Lattice formation)
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Precipitation in Semi-solid media (Gel)Precipitation in Semi-solid media (Gel)
• Double diffusion in one dimensionDouble diffusion in one dimension
• Single diffusion in one dimensionSingle diffusion in one dimension
in Agar
in Agar
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Single diffusion in two dimensions Single diffusion in two dimensions((MaMa ncini’ s technique) ncini’ s technique)
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2
• Radial immunodiffusion Radial immunodiffusion • Precipitin ring Precipitin ring• Diameter ~> Ag concentration Diameter ~> Ag concentration• Quantitation of Ag concentration Quantitation of Ag concentration
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Antibody Antigen
Agar matrix
Double diffusion in two dimensio Double diffusion in two dimensionsns (( Ouchterlony’ s technique) Ouchterlony’ s technique)
Double Immunodiffusion
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Reaction of Identity / Non-Identity / Partial Identity
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Reaction of Identity, - Non Identityor Partial Identity
Ag A
Ag C
Ag BAg D Ab
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Immunoelectrophoresis (I Immunoelectrophoresis (IEP)EP) Protein electrophoresis in
Gel + Double immunodiffusion
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Counter immunoelectrophoresi Counter immunoelectrophoresi s (CIE) s (CIE)
Anode Cathode
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Electroimmunodiffusion (EID) Electroimmunodiffusion (EID)
• Ag Quantitation
Ag well
Cathode (-)
Anode (+)
- Ab containing gel
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AgglutinationAgglutination
• Particulate Ag + specific Ab
• -- RBC Ag + Ab > HemagglutinationHemagglutination
• Reaction in liquid media• Direct Agglutination
• Indirect (or Passive) Agglutination
• Reverse Passive Agglutination
• Agglutination Inhibition
• Antiglobulin Test (Coombs’ test)
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Direct AgglutinationDirect Agglutination
• Ag or Ab assay
• Bacterial Agglutination (Bacterial typing)
• RBC Agglutination (Blood grouping)
• Slide agglutination / Tube agglutination
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Dilution & TiterDilution & Titer
Final positive serum dilution = 1:128 (or 1/128)
Ab Titer = 128
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Dilution 1:4
1:8
1:16
1:32
1:64
1:128
1:256
1:512
1:1024
-
Aggltn +/- + + + + + - - - -
Serial Two-fold dilutionSerial Two-fold dilution
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A
B
C
D
E
F
G
H
Hemagglutination in microtiter Hemagglutination in microtiterplateplate
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Dilution & Titer = ?
Ab Titer = ?
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Dilution 1:?
1:?
1: ?
1: ?
1:?
1: ?
1: ?
1:?
1:?
-
Aggltn + + + + + + - - - -
Serial Two-fold dilutionSerial Two-fold dilution
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Dilution & Titer = ?
Ab Titer = ?
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Dilution 1:?
1:?
1: ?
1: ?
1:?
1: ?
1: ?
1:?
1:?
-
Aggltn + + + + + + - - - -
Serial Two-fold dilutionSerial Two-fold dilution
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Indirect (or Passive) Agglutinatio Indirect (or Passive) Agglutinationn• Test Ag --> Soluble Ag• Ag coated inert particle• Inert particles : latex particle, gelatin particle,
human gr. O RBC, sheep RBC (particles that do not react with test serum.)
• Ab detection
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Reverse Passive AgglutinationReverse Passive Agglutination
• Ab coated inert particle
• Ag detection
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Agglutination InhibitionAgglutination Inhibition• Ag coated inert particle + limit amount ofAb
• Ag detection : eg. HCG in urine
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Direct Coombs’ Test Direct Coombs’ Test
• Detect Ab sensitized patient’s RBC
Antiglobulin test (Coombs’ T Antiglobulin test (Coombs’ Test)est)
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Indirect Coombs’ Test Indirect Coombs’ Test• Detect & identify free Ab in Detect & identify free Ab in patient’s patient’s serserumum
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Antiglobulin test in Hemolytic Disease of
Newborn• Maternal bloodMaternal blood
– Direct Antiglobulin test = +ve or –ve?– Indirect Antiglobulin test = +ve or –ve?
• Fetal bloodFetal blood– Direct Antiglobulin test = +ve or –ve?– Indirect Antiglobulin test = +ve or –ve?
• What is the blood group of RBC used in Indirect Antiglobulin test?
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Complement ActivationComplement Activation
Membrane attack pathway (Common or Terminal
pathway)
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Function of Complement
• Opsonization of cellular (bacterial) antigen
• Provoke inflammation• Poke holes in membranes leading
to lysis of bacteria• Clear immune complexes• Activates antigen-specific B cell
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Complememt Fixation (CF) testComplememt Fixation (CF) test
• Detection of Ag or AbDetection of Ag or Ab
• Use Complement (C) and Use Complement (C) and
• Ab sensitized SRBC or EAAb sensitized SRBC or EA
Ag+ Ab +C Ag-Ab-C
+ EA No hemolysis
No hemolysisNo hemolysis = Positive test = Positive test
HemolysisHemolysis = Negative test = Negative test
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CF test ControlsCF test Controls
1. Serum (Ab) control : Ab + C + EA 1. Serum (Ab) control : Ab + C + EA ? ?
2. Ag control 2. Ag control : Ag + C + EA : Ag + C + EA ??
3. C control : C + EA 3. C control : C + EA ??
4. RBC control : EA 4. RBC control : EA ??
? = Hemolysis or No hemolysis
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Labeled Ag-Ab ReactionLabeled Ag-Ab Reaction(Labeled Immunoassay)(Labeled Immunoassay)
• RadioimmunoassayRadioimmunoassay
• Immunofluorescence or Immunofluorescence or Fluorescence Fluorescence ImmunoassayImmunoassay
• Enzyme immunoassayEnzyme immunoassay
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Radioimmunoassay (RIA)Radioimmunoassay (RIA)
• Use r adioisotope : 125 I , 131I• - Radioiotope labeled Ag and limit
amount of specific antibody• Ag detection : eg. hormones• High sensitivity ng/ml , pg/ml• Competitive binding format• Separation of free Ag* (Free form, F)
- from Ag* Ab complex (Bound form, B)• Detect by gamma counting
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Radioimmunoassay (RIA)
AgAg Ag-Ab Ag-Ab
Ab +Ab +
Ag*Ag*(F)(F) Ag*-Ab Ag*-Ab (B)(B)
B/F Ag
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Radioimmunoassay (RIA)
B/F Ag
AgAg Ag-Ab Ag-Ab
Ab +Ab +
Ag*Ag*(F)(F) Ag*-Ab Ag*-Ab (B)(B)
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RIA Standard curveRIA Standard curve
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Separation of Free form (F) from Separation of Free form (F) from Bound form (B)Bound form (B)
1. Salt precipitation of B form
2. Precipitation of B form by Antiglobulin
3.F form precipitation by Dextran or charcoal
4. Ab coatiing on solid phase
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ImmunofluorescenceImmunofluorescence• Fluorochromes :Fluorochromes :
• Fluorescein isothiocyanate (FITC)Fluorescein isothiocyanate (FITC)
• Rhodamine isothiocyanateRhodamine isothiocyanate (RITC)(RITC)
• Fluorochrome labeled Ab Fluorochrome labeled Ab
(or Fluorochrome labeled protein A)(or Fluorochrome labeled protein A)
• Ag -> cells or particulate AgAg -> cells or particulate Ag
• Direct method Direct method (Ag detection)(Ag detection)
• Indirect method Indirect method (Ab detection)(Ab detection)
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ImmunofluoresceImmunofluorescencence
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Flow Cytometric Flow Cytometric CD4+ T cell countCD4+ T cell count
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The parameters of flow cytometric analysis
Laser beam Forward light scatter
FluorescenceSide scatter granularity
Forward angle light scatter, 90° light Forward angle light scatter, 90° light scatter and scatter and FluFluoresorescencecence
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Properties of cells measured Properties of cells measured by Flow Cytometryby Flow Cytometry
• Its relative size Its relative size – Forward Scatter (FSC)Forward Scatter (FSC)
• Its relative granularity or Its relative granularity or internal complexity internal complexity – Side Scatter (SSC)Side Scatter (SSC)
• Its relative fluorescence Its relative fluorescence intensityintensity– FL1, FL2, FL3 and FL4FL1, FL2, FL3 and FL4
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Three-color monoclonal antibody
panel
Tube No. FL3-mAb FL1-mAb FL2-mAb
1 CD45 CD3 CD4
2 CD45 CD3 CD8
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CD4 Count using B-D TriTEST CD4 Count using B-D TriTEST
CD3 FITC/CD3 FITC/ CD4 PECD4 PE//CD45 PerCPCD45 PerCP ReagentReagent
Fig. 1 Ungated CD45 vs SSC dot plot.
Fig. 2 Lymphocyte-gated CD3 vs CD4 dot plot.
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Enzyme Immunoassay
• Enzyme labeled Ag or Ab – Alkaline Phosphatase (AP) – Horse radish peroxidase (HRP, Px)
• Enzyme-Linked ImmunoSorbent Assay (ELISA)
• Ab or Ag coated on solid phase
• Separation of Free from Bound form (Washing)
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ELISA format
• Ag assay – Sandwich format
• Ab assay – Indirect format
– Competitive format
– Sandwich format
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Ag Ag assayassay
Double Antibody Sandwich or Double Antibody Sandwich or Two-Site ELISATwo-Site ELISA
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Double Ab S andwich ELISAfor detecting antigen
EE
Enzyme conjugated Enzyme conjugated Ab2Ab2
AgAg
Ab1Ab1
BB
E
E Biotinylated Biotinylated anti-p24 Abanti-p24 Ab22
Enzyme conjugated avidinEnzyme conjugated avidin
Substrate Color Product
Ab1Ab1
AgAg
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Double Antibody Sandwich or Double Antibody Sandwich or Two-Site ELISATwo-Site ELISA
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ELISA for Ab assay:ELISA for Ab assay:• Indirect ELISAIndirect ELISA
• Competitive ELISACompetitive ELISA
• Double Ag Sandwich Double Ag Sandwich
ELISA ELISA
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Double Ag Sandwich Double Ag Sandwich ELISA for Ab detectionELISA for Ab detection
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Third generation Double Antig Third generation Double Antig en Sandwich ELISA en Sandwich ELISA
PP
Test Ag
Enzyme conjugated Ag
IgGIgG AbAb
PPIgM AbIgM Ab
Substrate Color Product
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Fourth generation SandwichSandwich ELISAELISA
PP PP
AbAg
Enzyme-conjugated Ab
AgAg
Enzyme conjugated Ag
Ab
Substrate Color Product
for detecting HIV Abs and Ag simultaneously
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Substrate of ELISASubstrate of ELISA
• Alkaline Phosphatase (AP) – p-Nitrophenyl phosphate (pNPP) – OD405 nm
• Horse radish peroxidase (HRP, Px)– H2O2 + chromogen
• H2O2 + o-phenylelne diamine (OPD) stop reaction with H2SO4 -> OD492 nm.
• H2O2 + Tetramethyl benzidine (TMB) stop reaction with H2SO4 -> OD450 nm.
• Absorbance or Optical Density (OD) – 0.000 - 2.000 / 3.000 OD.
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Immunoblot (Western blot)Immunoblot (Western blot)
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Immunochromatographic assayImmunochromatographic assay (Strip test)(Strip test)
Ag assay Ag assay (Double Antibody sandwich assay)(Double Antibody sandwich assay)
Test band
Control band
Dye labeled reagent
Sample flow
= Dye-labeled Ab1= Dye-labeled Ab1 (mobile) (mobile)
= Ab2 coated bandAb2 coated band
Ab against Ab1 =Ab against Ab1 =
• Positive = test and control bandsPositive = test and control bands
• Negative = control band onlyNegative = control band only
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Ag Test StripAg Test Strip
Ab against Ab1 Ab against Ab1
Dye-labeled Ab1Dye-labeled Ab1 (mobile) (mobile)
Ab2 coated bandAb2 coated band
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Ab AssayAb Assay: :
Double Antigen sandwich assayDouble Antigen sandwich assay
Control band Control band (Ab against Ag)(Ab against Ag)
Dye-labeled Ag Dye-labeled Ag (mobile)(mobile)
Test band (Ag Test band (Ag coated band)coated band)
Sample flowSample flow
• Positive Positive
= test and control bands= test and control bands
• Negative Negative
= control band only= control band only