Analytical and preparative methods based on primary

antigen-antibody binding

Immunoaffinity chromatography, ELISA,

immunoblot, immunohistochemistry

Affinity purification of antibodies using an antigen-sorbent column

column polymer beads

Covalently boundantigen

Column content

Addition of antibodies to be purified

Binding

elution

Washing

Antigen-specificpolyclonal antibodies

Purification of antigen on antibody-immunosorbent column

polymer beads

Covalently bound antibodies

column

Column content

The reverse setup is also used

Loading the antigen mixture

Binding

Wash

Elution

ELISA

ELISA plate

Enzyme Linked Immune Sorbent Assay

Well

enzyme linked immune sorbent

Antibody conjugated with enzyme

enzyme

Antigen/antibody adsorbed to solid surface

Enzyme activity in ELISA is directly proportional to the amount of antigen

present

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

Similar principle applies to many other antibody-based detection methods

Basic setups in ELISA, immunohistochemistry, flow cytometry

Direct method Indirect method

Antigen

Primary antibodies

Label

Label

Secondary antibodies

Basic setups in ELISA, immunohistochemistry, flow cytometry

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

Examples of direct, indirect and competitive ELISA systems

Steps of the basic indirect ELISA

Detection of antigen or specific antibody

Adsorption of antigen (coating)

Removal of excess antigenSaturation of

uncovered surface area with protein such

as BSA, casein etc..

Removal of excess protein

Addition of Ag-specific antibodies

Addition of Secondary Ab

conjugated with enzyme

Removal of excess antibody

Addition of chromogenic

substrate

For antigens present at low concentration in complex biological samples

Removal of unbound material

Blocking 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 the combined sandwich ELISA

Competitive ELISAHighly sensitive method used to detect and quantitate small amounts of antigen in complex biological samples. Antigen in solution and on the solid surface compete for the binding site of labeled specific antibody.

- Coat with antigen, blocking- Add experimental sample that contains or lacks antigen -Add labeled antibody, --- Binding- Washes- Add substrate

Background due to non-specific binding to plastic

Nonspecific binding can be controlled by:

•Saturation of free plastic surfaces•Using detergents

ELISA

Labeled antibody bound to plastic directly or indirectly

gives the same signal as those that bind specifically

ELISA plates - resultsELISA plates - results

The chromogens used for detection are CARCINOGENOUS!

Characterization of antigens by Western blotting

• The use of antibodies in molecular biology is widespread• It is probably most often encountered in Western analysis• SDS-PAGE gel resolved into single protein bands (overlap possible)• Presence of a protein is determined by hybridizing the proteins, transferred or applied to a membrane, with the relevant antibody

StandardProtein sample

SDS-PAGE Membrane Western blot

Antibody recognizes epitope in specific protein

Western Blot

Western Blot

• Used to detect specific proteins in a sample

• Proteins separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred to a membrane

• Primary (1st) antibody (monoclonal or polyclonal) used to detect protein

• Enzyme linked 2nd antibody (e.g. horseradish peroxidase-linked) used to detect 1st antibody

Fab

Fc

Epitope on protein surface

Primary antibody

Secondary antibody

Horseradish peroxidase

Immobilized proteins

Membrane

H2O2

H2O

luminol

enhancer

h

Detection

Enhanced chemiluminescence (ECL)

•In the presence of H2O2, horseradish peroxidase (HRP) oxidizes diacylhydrazides such as luminol• Directly after oxidation, luminol is in an exited state, and emits a photon to return to the ground state• This photon can be detected with a film or a camera• Light emission can be enhanced by ~1000-fold with phenolic compounds such as 6-hydroxybenzothiazole (enhancer)

luminol

Immunohistochemistry

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 isothiocyanatePE – phycoerythrin

• Immunoenzyme method• enzyme-coupled antibody

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

Tissue sample

Fixation

Section before staining

Freezing

Sectioning

Immunohistochemistry

Immunohistochemistry ABC Method

Secondary antibody

Avidin

Cells

Slide

Primary antibody

Biotin

Enzim

X

Tissue sample

Histochemistry

Acute bronchopneumonia (hematoxilin- eozin staining)

Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)

Immunohistochemistry using fluorescent detection Anti-nuclear autoantibodies in SLE (FITC)

Immunohistochemistry using fluorescent detection(TRITC)

Detection of actin microfilaments

A fixed and permeabilized skin fibroblast. Mitochondria were labeled with mouseIgG (anti–OxPhos Complex V) and visualized using goat anti–mouse IgG conjugated with orange-fluorescent Alexa Fluor 555. F-actin was labeled with green-fluorescent Alexa Fluor 488 phalloidin (a mushroom toxin), and the nucleus was stained with TO-PRO-3 iodide

Peroxisome labeling in fixed and permeabilized pulmonary artery

endothelial cell. Peroxisomes were labeled using an antibody directed at peroxisomal membrane protein 70 and detected with Alexa Fluor 488–labeled

goat anti–mouse IgG. Mitochondria were stained with MitoTracker Red prior

to fixation; nuclei were stained with blue-fluorescent DAPI.

Acquired Immune Deficiency Syndrome – AIDS

Certain infectious microorganisms can supress or subvert the immune system.At the beginning of the last century, when tuberculosis was the leading cause of deathand fully half the population was tuberculin-positive, it was well-known that an inter-current measles infection would cause a well-contained tuberculosis infection to runrampant and result in death. The mechanism responsible is now known to be thesupression of IL-2 synthesis after binding of measles virus to CD46 on macrophages.

Some of the microorganisms that supress immunity act by infecting lymphocytes.

The human immunodeficiency virus (HIV) presents a chilling example of the consequences of infection and destruction of immune cells by a microorganism.

The T-cell surface CD4 molecule acts as a receptor for HIV. CD4 is also expressed on the surface of cells of the macrophage lineage and they too can be infected by this virus.

The clinical latency is long,usually it means several years.

During this period, the level of CD4+ cells and virus particles in the blood changes. When the rate atwhich CD4+ cells are being destroyedexceeds the capacity of the host toreplenish them, their number decreasesto a point where cell-mediated immunity falters.

The failure of cell-mediatedimmunity renders to the host susceptible to fatal opportunisticinfections.

Case study: The Pinkerton-family: infected blood caused tragedy

Benjamin Pinkerton was a US-navy lieutenant who saw service at Japan. He married with a japanese woman during his service, who gave birth two healthy girls in 1987. She bore a boy four years later, who seemed healthy, as well. The boy got the routine DPT-vaccination and an oral polio-virus immunization.These vaccinations had no side-effect and the boy grew normally.At the age of six months he got sick and started to lose weight. He had severe, chronic diarrhea with fever. Besides a chronic oral candidiasis, the boy got two otitis, one after the other . The navy doctors examined the baby several times and prescribed antibiotic but it proved ineffectual.

Results of somatic examination:- body-temperature 38oC;- candidiasis on the lateral sides of tongue and on the mucosal surface of the oral cavity;- „diaper-pimples”, which is also caused by Candida infection;- at respiration a subtle, slurping noise was heard in each pulmonary lobes;

Oral candidiasis

esophageal candidiasis

Diaper pimples

Laboratory:

- normal amount of leukocytes (6500/l);- normal rate of leukocytes (neutrophyl 62%; lymphocyte 30%; monocyte 5%; eosinophyl 2%; basophyl 1%); - normal serum immunoglobulin levels:- serum IgG: 997 mg/dl (phys.: 800-1000 mg/dl);- serum IgM: 73 mg/dl (phys.: 50-150 mg/dl);- IgA: 187 mg/dl (phys.: 150-300 mg/dl);- normal amount of CD8 + T-cells, but the rate of CD4+ T-cells is very low, only 85/l (phys.: 1000-1200/l);

- intradermal Candida-antigen did not evoke late-type hypersensitvity reaction;- results of ELISA and Western-blot analysis: HIV-antibodies in the serum;

METHOD EXAMINED

PROTEINS

SAMPLE DETECTION

ELISAELISA Anti-HIV IgG and IgM antibodies

serum

(sometimes saliva

or urine)

with secondary (conjugate-) anti-IgG antibodies

Western blotWestern blot HIV capsid-proteins: p17, p24 , p6, p7

Envelop:

gp41, gp120, gp160;

serum with secondary (conjugate-) anti-IgG antibodies

After this finding they verified the parents:Both of them were HIV-positive.

During HIV-diagnostics samples are always analyzed firstly by ELISA-method.In case of reactivity (serum positive) two more measurements are needed(2nd and 3rd analysis). If the 2nd and 3rd measurements show reactivity as well, the subject’s sample mustbe verificated: usually a Western blot or another ELISA is performed.

While Pinkerton was healthy, his wife was feeling unwell and complainedabout the swelling of her cervical lymphatic nodes.

It turned out, that she was pregnant right before the boy’s birth. At the end of pregnancy the fetus had died and had to be removed by caesarean section. The operation was going well but – because of the loss of blood – she needed blood-transfusion (she got two units of blood).

The boy got two severe infections in turn: Pneumocystis carinii- andPseudomonas aeruginosa. He had serious cough with bloody spit (hemoptysis). A week after this he died.

The parents got AZT- (zidovudin) therapy. While his wife died soon in respiratoryfailure, the lieutenant – in spite of his high serum HIV-antibody level – hasnot had symptoms yet.