8th SEMINARLABORATORY METHODS BASED ON

ANTIGEN-ANTIBODY INTERACTIONS I

THE SENSITIVITY OF IMMUNOASSAYS

Sensitive methods:

• precise• expensive• usually used for verification

Less sensitive methods:

• give semiquantitative results• cheap• usually used for screening

IMMUNOAFFINITY CHROMATOGRAPHY

Separation/purification of antigens or antibodies from a mixture

column polymer beads

covalently boundantigen

affinity purified antibody : monoclonal antibodies which can be ordered from catalogues are also purified using this technique

AFFINITY PURIFICATION OF ANTIBODIES USING AN ANTIGEN-SORBENT COLUMN

1) Addition of antibodies to be purified

2) Binding3) Washing4) Elution

STEPS OF PURIFICATION

polymer bead

fixed antigen-specific Abs on the surface of the bead

column

PURIFICATION OF ANTIGENS1) Loading the antigen mixture

2) Binding

3) Washing

4) Elution

Purified antigens

ELISAEnzyme Linked Immune Sorbent Assay

ELISA plate

well

Enzyme Linked Immune Sorbent

Antibody conjugated with enzyme

enzyme

Antigen/antibody adsorbed to solid

surface

Enzyme activity is measured by the color reaction due to conversion of substrate

Similar principle applies to many other antibody-based detection methods

ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF

IMMUNECOMPLEX PRESENT

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY

Direct method Indirect method

Antigen

Primary antibodies

Label

Label

Secondary antibodies

Enzyme/anti-enzyme system

PAP – peroxidase / anti-peroxidaseAPAAP – alkaline phosphatase / anti- alkaline phosphatase

Antigen

Primaryantibody

Secondaryantibody

Enzyme-specific antibody, same isotype as the primary antibody

Enzyme

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY

Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity )

Basic ABC

Antigen

Biotinylated antibody

Avidin

Avidin-enzyme complexes

Biotin-enzyme complex

Avidin-biotin enzyme

complexes

BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY

For antigens present at low concentration in complex biological samples

Removal of unbound materialBlocking free plastic surface with inert protein Removal of unbound protein

Addition of biotinylated antibody specific to a different epitope on target protein

Removal of unbound material

Addition of avidin-conjugated enzyme

Addition of substrate

Coating with Ag-specific „capture” antibody

Addition of antigen- containing solution

Removal of excess enzyme

STEPS OF COMBINED/’SANDWICH’ ELISA

STEPS OF BASIC INDIRECT ELISA Detection of antigen or specific antibody

Adsorption of antigen (coating)

Removal of excess antigen

Saturation of uncovered surface area with proteins

Removal of excess protein

Addition of Ag-specific antibodies

Addition of Secondary Ab conjugated with enzyme

Removal of excess antibody

Addition of chromogenic substrate

Removal of excess antibody

concentration

10

00

50

0

25

0

12

5

62

31

16

7.8

3.9

1.9

0.9

7

0.4

9

0.2

4

0.1

2

0.0

30

0.0

61

0.0

07

0.0

15

0.0

04

0

The sample with unknown concentration

OD

EQUAL ABSORBANCE = EQUAL CONCENTRATION

According to OD: it could be anyone

?

You should also dilute the unknown sample

This region could indicate the concentration

This region could indicate the concentration

PRACTICAL USE OF ELISA TECHNICS

Sandwich ELISAMeasuring the amount of a given

antigen (molecule)

• cytokines, hormones, drugs• viral/bacterial antigens –

diagnosis of infection• tumor antigens – diagnosis of

tumors / screening / follow-up

Indirect ELISAMeasuring the amount of antigen-

specific antibodies

• pathogen-specific antibodies – diagnosis of infection*

• isotype of antibodies – time course, monoclonal antibodies

• autoantibodies – diagnosis of autoimmune disorders

WESTERN BLOT (IMMUNOBLOT)

Steps:

1) Sample preparation (cells, tissues)

2) Gel electrophoresis

3) Blotting

4) Labeling (by primary and secondary antibodies)

5) Detection

Identification of defined components from protein mixtures by antigen specific antibodies

Anode(+)

Cathode(-)

Standard Protein sample

SDS-PAGE Membrane

Primary antibody binds to its epitope in the protein, then the labeled

secondary antibody binds to the primary antibody

Gel-electrophoresis Blotting LabelingLysis of sample Loading

X-ray film

WESTERN BLOT (IMMUNOBLOT)

IMMUNOPRECIPITATION

• Isolation and concentration of a particular protein from a protein mixture

• Detection of protein associations (e.g. members of receptor signalization)

CHROMATIN IMMUNOPRECIPITATION (ChIP)

Identification of molecules (mainly transcription factors) binding to a specific site of the DNA

Provides information about the link between signaling pathways and gene activation

Labeled antibodies added to fixed tissue sections detect the distribution of the chosen antigen within the tissue or within the cells

of a particular tissue

• Immunofluorescence• Fluorescent dye coupled to antibody

FITC – fluorescein isothiocyanate (green)PE – phycoerythrin (orange)

• Immunoenzyme method• enzyme-coupled antibody

P – peroxidase AP – alkaline phosphatase(Substrates converted into an insoluble compound)

IMMUNOHISTOCHEMISTRY

Tissue sample

Fixation

Section before staining

Freezing

Sectioning

IMMUNOHISTOCHEMISTRY

Secondary antibody Avidin

Cells

Slide

Primary antibody Biotin

Enzyme

X

Tissue sample

IMMUNOHISTOCHEMISTRY

Classical histochemistry Acute bronchopneumonia (hematoxylin-eozin staining)

Only few cell types could be identified

Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)

Antinuclear (ANA) autoantibodies from the serum of a SLE patient can be visualized in cell culture (HEp-2) by indirect

fluorescent labeling (immunofluorescence)

A fixed and permeabilized skin fibroblast

Mitochondria F-actin Nucleus

Fixed and permeabilized pulmonary artery endothelial cell

Peroxisomes Mitochondria Nuclei