9th SEMINARLABORATORY METHODS BASED ON

ANTIGEN-ANTIBODY INTERACTIONS II

THE SENSITIVITY OF IMMUNOASSAYS

Sensitive methods:

• precise• expensive• usually used for verification

Less sensitive methods:

• give semiquantitative results• cheap• usually used for screening

FLOW CYTOMETRY

An immunofluorescent method that is similar to immunohistochemistry but gives rather

quantitative than qualitative results

Investigation of cells travelling in a flow with high velocity

Detects light scatter of particles and fluorescence intensity of the labeled cells

Enormous number of cells can be investigated in short period of time

ADVANTAGES OF FLOW CYTOMETRY

Most cells can be easily suspended and labeled by fluorescent cell surface marker-specific antibodies

The cells’ light scatter and immunofluorescent properties can be analyzed statistically

Rare cell populations can be identified and examined (e.g. antigen specific lymphocytes)

The method provides both qualitative and quantitative data: presence of antigen and the expression levels of these antigens

Changes in the expression of certain molecules can be followed after different treatments (e.g. cell activation, disease progression)

LaserForward angle light

scatter sensor(FSC)

Side light scatter (SSC) represents granularity

and fluorescence detectors

represents size

BASIC STRUCTURE OF THE FLOW CELL

fluid flow

sheath fluid

forward scatter (FSC)(size)

side scatter (SSC)(granularity)

granulocytes

monocytes

lymphocytes

DENSITY PLOT OF PERIPHERAL BLOOD CELLS AFTER LYISING RED BLOOD CELLS

Different cell types characteristic light scattering

CELL TYPES, DIFFERENTIATION STAGES CAN BE IDENTIFIED USING A COMBINATION OF

CELL SURFACE MARKERSUsed in diagnostics:

ratio of different cell types altered expression of cell surface markers

Examples: Inflammatory processes – increased neutrophil numbers increase of CD5+ B cells – typical for some B cell leukemias HIV progression – decrease of CD4+ T cell count

In normal case CD4+ : CD8+ = 1.6Normal CD4+ T cell count = 600 – 1400/l

AIDS = CD4+ T cell count <200/l

BNKTh Tc

Example: Measurement of CD4+ (helper) and CD8+ (cytotoxic) T cell ratio (e.g. monitoring AIDS progression)

Labeling: FITC labeled anti-CD4 antibody(α-CD4-FITC)PE labeled anti-CD8 antibody (α-CD8-PE)

Lymphocytes in the peripheral blood sample:

Fluorescent microscopy:

IMMUNOPHENOTYPING BY FLOW CYTOMETRY

focu

sed

lase

r bea

m

high velocity flow stream(in cuvette or stream in air)

CD4FITC

CD

8P

E

de

tec

tor

detecting CD4-FITC labeled (TH) cell

signal processing unit

screen

a dot representing aCD4+ CD8- cell

increasing fluorescence

intensity

microscopy:

de

tec

tor

detecting the PE labeled cell(CD8-PE)

CD

8P

E

CD4FITC

signal processing unit

increasing fluorescence intensity

de

tec

tor

detecting an unlabeled cell(e.g. B cell) by autofluorescence

CD

8P

E

CD4FITC

Signal processing unit

increasing fluorescence intensity

microscopy: dim (autofluorescent) cell

quadrantstatistics

CD

8P

E

CD4FITC

Signal processing unit

38%

0%

44%

18%

dot-plot

contour-plot

density-plot

GRAPHICAL REPRESENTATIONS I

GRAPHICAL REPRESENTATIONS IIHistogramm

homogenous cell population has normal distribution (Gaussian)Numeral intensity values: ~ 7 ~ 1300

CD antigen cell type function ligand

CD3 T cells TCR signaling -

CD4 helper T cells, (monocytes, pDC)T cell co-receptor,

(HIV receptor) MHC-II, HIV

CD5 T cells, (B cell subset: B1) adhesion, activation signals CD72

CD8 cytotoxic T cells, (NK, T cells) T cell co-receptor MHC-I

CD14monocytes, macrophages,

some granulocytesLPS binding LPS, LBP

CD19 B cells part of CR2, B cell co-receptor C3d, C3b

CD28 T cells co-stimulatory signals to T cells

(B7-1, B7-2)CD80, CD86

CD34 hematopoietic progenitor cell adhesionCD62L

(L-selectin)

CD56 NK cell, (T and B cell subset)homoadhesion

(N-CAM isoform)

CD80, CD86 (B7-1, -2)

APC: DC, B, monocyte, macrophage

co-stimulatory signals CD28, CD152

THE SENSITIVITY OF IMMUNOASSAYS

Sensitive methods:

• precise• expensive• usually used for verification

Less sensitive methods:

• give semiquantitative results• cheap• usually used for screening

FORMATION OF IMMUNECOMPLEXES

Immune complexes are rapidly formed in solutions. In a steady-state equilibrium, determined by the dissociation constant (Kd), they may form precipitates by secondary interactions

PRECIPITATION OF IMMUNE COMPLEXES

antigen excessantibody excess

Increasing antibody amounts

EQUIVALENCEantibody excess antigen excess

Increasing antigen amounts

pre

cip

itat

ed

pro

tein

sp

reci

pit

ated

p

rote

ins

Equivalent amount of Ab and Ag will lead to a visible precipitationExcess in either Ab or Ag will lead to soluble complexes

IMMUNODIFFUSION METHODS I

In gelAntigen

Antibody

Antibody

Antigen

Mancini method / Radial immunodiffusion

2D simple immodiffusion

Applied for quantitative determination of viral antigens or virus specific antibodies in the serum

The size of the precipitation ring is relative to the concentration of the antigen; higher concentrations of antigens will diffuse further until

they reach the equilibrium

IMMUNODIFFUSION METHODS II

antigen

antibody

antigen

antibody

2D double immunodiffusion

Gold standard method for detection of Extractable Nuclear Antigen (ENA)-specific autoantibodies

ENAs: e.g. Ro, La, Sm, RNP, Scl-70, Jo1

Autoantigens located in the nuclei of cells. In case of systemic autoimmune disorders autoreactive B-cells specific to these antigens get activated and continuously produce antibodies against their targets.

Ro

La

Sm

patient’s serum sample

RNP

Scl-70

Jo1

Ouchterlony method

AGGLUTINATION REACTIONS

Agglutination: The clumping of cells such as bacteria or red blood cells in the presence of antibodies.

It differs from precipitation only in size – here the antigen carrying substances are cells, or colloidal structures.

Hemagglutination: When the particles involved are RBCs.

TYPES AND USES OF AGGLUTINATION REACTIONS

DIRECT AGGLUTINATION Aggregation followed by antibody binding (mostly IgM)

(AB0 blood group antigens)

INDIRECT AGGLUTINATIONAggregation is achieved with a secondary antibody recognizing the Fc region

of the primary antibody (mostly IgG) (Rh blood group- D antigen)

PASSIVE AGGLUTINATIONRBCs or latex particles (latex beads) to which haptens or protein antigens artificially

are coupled, aggregate after the addition of the specific antibodies(RF- Rheumatoid Factor detection)

DIRECT AGGLUTINATION

A, B or both AB antigens on the surface of RBCs will bind to the

appropriate anti-A, Anti-B antibodies and agglutinate

AB0 blood group typing Detecting the presence of an antigen

DONORS AND RECIPIENTS FOR BLOOD TRANSFUSION

Only theoretically, recipients must receive their own type of blood!

Quantitative detection of antibodies (antibody-titer) specific to a given antigen.

The antigen is present on the surface of RBCs / bacteria, or fixed to the surface of RBCs / latex beads.

QUANTITATIVE HAEMAGGLUTINATION TEST

Rh blood group determination Diagnosis of autoimmune hemolytic anemia Cross matching donor and recipient; ABO

and Rh compatibility

Primary AB

Secondary AB

INDIRECT AGGLUTINATION

ABO incompatibility intravascular hemolysis(complement mediated hemolysis)

Rh incompatibility hemolytic disease of the newborn (erythroblastosis fetalis)(opsonisation of red blood cells,

which are then phagocytosed by macrophages and granulocytes)

Rh prophylaxis

Administration of Anti-D IgG vaccine within 72 hours after birth of the first Rh-incompatible fetus prevents production of maternal antibodies.

EFFECTS OF AGGLUTINATION IN VIVO

PATHOLOGICAL CONSEQUENCES OF PLACENTAL TRANSPORT OF IgG(hemolytic disease of the newborn)

anti-Rh

Erythropoiesis in lung

Erythroblasts in bone marrow

Both Coombs tests are indirect agglutinations because the usage of secondary antibodies

Direct Coombs test – e.g. detection of autoimmune hemolitic anemia

Indirect Coombs test – e.g. detection of minor blood group incompatibility

PASSIVE AGGLUTINATION

Latex agglutination test for Rheumatoid Factor in Rheumatoid Arthritis

Fixating antigens on the surface of latex beads / RBCs and mixing them with the patient’s serum

Rheuma factor is self IgG/IgM reactive IgM produced by autoreactive plasma cells