1. Analytical and preparative methods antigen-antibody binding
based on primary
antigen-antibody binding
Immunoaffinity chromatography,
ELISA,
immunoblot,
immunohistochemistry

2. column polymer beads Covalently bound antigen
Affinity purification of antibodies using an antigen-sorbent column
column
polymer beads
Column content
Covalently bound
antigen

3. Addition of antibodies to be purified polyclonal antibodies
Binding
Washing
elution
Antigen-specific
polyclonal antibodies

4. The reverse setup is also used
Purification of antigen on antibody-immunosorbent column
column
polymer beads
Loading the antigen mixture
Column content
Binding
Wash
Elution
Covalently bound
antibodies

5. Enzyme Linked Immune Sorbent Assay
ELISA
Enzyme Linked Immune Sorbent Assay
ELISA plate
Well

6. Antigen/antibody adsorbed to solid surface Antibody conjugated with
enzyme linked
immune sorbent
enzyme
Antigen/antibody adsorbed to solid surface
Antibody conjugated with
enzyme

7. 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

8. Basic setups in ELISA, immunohistochemistry, flow cytometry
Direct method
Indirect method
Label
Secondary antibodies
Label
Primary antibodies
Antigen

9. Basic setups in ELISA, immunohistochemistry, flow cytometry
Enzyme/anti-enzyme system
PAP – peroxidase / anti-peroxidase
APAAP – alkaline phosphatase / anti- alkaline phosphatase
Enzyme
Enzyme-specific antibody, Same isotype as the primary antibody
Secondary
antibody
Primary
antibody
Antigen

10. Basic setups in ELISA, immunohistochemistry, flow cytometry
Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity )
Basic
ABC
Avidin-biotin enzyme complexes
Avidin-enzyme complexes
Biotin-enzyme complex
Avidin
Biotinylated antibody
Antigen

11. Examples of direct, indirect and competitive ELISA systems

12. Steps of the basic indirect ELISA
Detection of antigen or specific antibody
Adsorption of antigen (coating)
Removal of excess antibody
Removal of excess antigen
Saturation 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
Addition of chromogenic substrate

13. Steps of the combined sandwich ELISA
For antigens present at low concentration in complex biological samples
Coating with Ag-specific „capture” antibody
Removal of excess enzyme
Removal of unbound material
Removal of unbound protein
Blocking free plastic surface with inert protein
Removal of unbound material
Addition of antibody- containing sol.
Addition of biotinylated antibody specific to a different epitope on target protein
Addition of avidin-conjugated enzyme
Addition of substrate

14. Competitive ELISA
Highly 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

15. Nonspecific binding can be controlled by:
ELISA
Background due to non-specific binding to plastic
Labeled antibody bound to plastic directly or indirectly gives the same signal as those that bind specifically
Nonspecific binding can be controlled by:
Saturation of free plastic surfaces
Using detergents

16. The chromogens used for detection are CARCINOGENOUS!
ELISA plates - results
The chromogens used for detection are CARCINOGENOUS!

17. Characterization of antigens by
Western blotting

18. Western Blot Western blot SDS-PAGE Membrane
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
Protein sample
Standard
Antibody recognizes epitope in specific protein
Western blot
SDS-PAGE
Membrane

19. 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

20. 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)
Immobilized proteins
Primary antibody
Fab Fc
Horseradish peroxidase
luminol
H2O2 h
Epitope on protein surface
luminol
enhancer
Detection
H2O
Secondary antibody
Membrane

21. 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
Immunfluorescence
Fluorescent dye coupled to antibody
FITC – fluorescein isothiocyanate
PE – phycoerythrin
Immunoenzyme method
enzyme-coupled antibody
P – peroxidase
PA – alkaline phosphatase
(Substrates converted into an insoluble compound)

22. Immunohistochemistry Section before staining
Fixation
Sectioning
Tissue sample
Section before staining
Freezing

23. Immunohistochemistry ABC Method
Enzim X
Avidin
Biotin
Secondary antibody
Primary antibody
Slide
Cells
Tissue sample

24. Immunohistochemistry
Acute bronchopneumonia (hematoxilin- eozin staining)

25. Immunohistochemistry (CD68+ macrophages and lymphocytes, granuloma)

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

27. Immunohistochemistry using fluorescent detection (TRITC)
Detection of actin microfilaments

28. A fixed and permeabilized skin fibroblast
A fixed and permeabilized skin fibroblast. Mitochondria were labeled with mouse
IgG (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

29. 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.

30. Acquired Immune Deficiency Syndrome – AIDS
Certain infectious microorganisms can supress or subvert the immune system.
At the beginning of this century, when tuberculosis was the leading cause of death
and 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 run
rampant and result in death. The mechanism responsible is now known to be the
supression of IL-2 synthesis after binding of measles virus to CD46 on macrophages.

31. 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
rapidly changes. When the rate at
which CD4+ cells are being destroyed
exceeds the capacity of the host to
replenish them, their number decreases
to a point where cell-mediated
immunity falters.
The failure of cell-mediated
immunity renders to the host
susceptible to fatal opportunistic
infections.

32. The Pinkerton-family: infected blood caused tragedy
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 japan woman during his service, who gave birth two healthy girls in 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 fell 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;

33. Laboratory: normal amount of leukocytes (6500/ml);
normal rate of leukocytes (neutrophyl 62%;
lymphocyte 30%; monocyte 5%; eosinophyl 2%; basophyl 1%);
normal serum immunoglobulin levels:
serum IgG: 997 mg/dl (phys.: mg/dl);
serum IgM: 73 mg/dl (phys.: mg/dl);
IgA: 187 mg/dl (phys.: mg/dl);
normal amount of CD8 + T-cells, but the rate of CD4+ T-cells
is very low, only 85/ml (phys.: /ml);
intradermal Candida-antigen did not evoke late-type
hypersensitvity reaction;
results of ELISA and Western-blot analysis:
HIV-antibodies in the serum;

34. ELISA Western blot METHOD EXAMINED PROTEINS SAMPLE DETECTION serum
Anti-HIV IgG and IgM antibodies
serum
(sometimes saliva
or urine)
with secondary (conjugate-) anti-IgG antibodies
Western blot
HIV capsid-proteins: p17, p24 , p6, p7
Envelop:
gp41, gp120, gp160;
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 must
be verificated: usually a Western blot or another ELISA is performed.
After this finding they verified the parents:
Both of them were HIV-positive.

35. While Pinkerton was healthy, his wife was feeling unwell and complained
about 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 baby got two severe infections in turn: Pneumocystis carinii- and
Pseudomonas 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 respiratory
failure, the lieutenant – in spite of his high serum HIV-antibody level – has
not had symptoms yet.