1. Diagnostic immunology
324 PHT
Dr. Sarah I. Bukhari
PhD in Clinical Microbiology
Department of Pharmaceutics
Office: rd floor

2. Diagnostic immunology
Tests based on the interactions of antibodies and antigens
These tests determine the presence of antibodies or antigens in a patient.
Specificity
Determined by the percentage of false positive results it gives
Sensitivity
Determined by the percentage of positive samples it correctly detects
Essential elements of
diagnostic tests are sensitivity and specificity. Sensitivity is the
probability that the test is reactive if the specimen is a true positive.
Specificity is the probability that a positive test will not be
reactive if a specimen is a true negative.

3. Immunologic-based diagnostic tests

4. Detect Ag from patient sample Detect Ab from patient sample
Serological Tests
Direct
Indirect
Detect Ag from patient sample
Detect Ab from patient sample

5. Serological Tests Agglutination: Particulate antigens
Hemagglutination: Agglutination of RBCs
Precipitation: Soluble antigens
Fluorescent-antibody technique: Antibodies linked to fluorescent dye
Complement fixation: RBCs are indicator
Neutralization: Inactivates toxin or virus
ELISA: Peroxidase enzyme is the indicator

6. Tests Based on Ag/Ab Reactions
All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes
All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

7. Factors Affecting Measurement of Ag/Ab Reactions
Ab excess
Ag excess
Affinity
Avidity
Equivalence – Lattice formation
Ag:Ab ratio
Physical form of Ag

8. Avidity
The overall strength of binding between an Ag with many determinants and multivalent Abs Y
Keq =
104
Affinity Y
106
Avidity Y
1010
Avidity

9. Cross Reactivity Cross reactions
The ability of an individual Ab combining site to react with more than one antigenic determinant.
The ability of a population of Ab molecules to react with more than one Ag
Anti-A Ab
Ag C
Similar epitope
Cross reactions
Anti-A Ab
Ag A
Anti-A Ab
Ag B
Shared epitope

10. Antibody-based Diagnostic Tests
problem that must be overcome in antibody-based diagnostic tests:
antibodies cannot be seen directly
Antibodies produced in an animal were mixed and limited quantity.
Even at
magnifications of well over 100,000×, they appear only as fuzzy,
ill-defined particles

11. Nobel prize in 1975 Neils Jerne César Milstein Georges Köehler
antibodies were produced by
specialized cells (B cells), it was understood that these were a potential
source of a single type of antibody. If such a B cell producing
a single type of antibody could be isolated and cultivated, it
would be able to produce the desired antibody in nearly unlimited
quantities and without contamination by other antibodies.
Unfortunately, a B cell reproduces only a few times under the
usual cell culture conditions. This problem was largely solved
with the discovery of a method to isolate and indefinitely cultivate
B cells capable of producing a single type of antibody.
Neils Jerne
César Milstein
Georges Köehler

12. Two identical light chains
Antibody Structure
Two identical light chains
Two identical heavy chains
Linked by S-S bonds

13. Monoclonal Antibodies (Mabs)
Pathogens contain many different antigens (and many more epitopes)  Polyclonal antibody response
Monoclonal antibody: single type of antibody directed against one specific epitope. Produced by single B cell clone.

14. ANTIBODIES Polyclonal antibodies Monoclonal Antibodies
Produced by: Many B cell clones A single B cell clone
Bind to: Multiple epitopes of all A single epitope of a single
antigens used in the antigen
immunization
Antibody class: A mixture of different All of a single Ab class
Ab classes (isotypes)
Ag-binding sites: A mixture of Abs with All Abs have the same antigen
different antigen-binding binding site
sites
Potential for cross-reactivity: High Low

15. Monoclonal antibodies
MAbs produced from a single clone of B cells
Monoclonal antibodies all have identical antigen- binding sites. Thus they all bind to the same epitope with the same affinity
Mostly produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture

16. Production of MAbs

17. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
1) Immunize animal (mouse or rabbit)
2) Isolate spleen cells (containing antibody-producing B cells)
3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)
4) Allow unfused B cells to die
5) Add HAT culture to kill unfused myeloma cells
6) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells)
7) Screen supernatant of each clone for presence of the desired antibody (ELISA)
8) Grow the chosen clone of cells in tissue culture indefinitely.
9) Harvest antibody from the culture supernatant.

18. Production of MAbs Hybridoma Production in the laboratory
by fusing a cancerous cell
with an antibody-secreting
plasma cell
The hybridoma cell culture
produces large quantities of
the plasma cell’s antibodies
(Mabs)
Scientists have long observed that antibody-producing B cells
may become cancerous. In this case, their proliferation is unchecked,
and they are called myelomas. These cancerous B cells can
be isolated and propagated indefinitely in cell culture. Cancer cells,
in this sense, are “immortal.” The breakthrough came in combining
an “immortal” cancerous B cell with an antibody-producing normal
B cell. When fused, this combination is termed a hybridoma.
When a hybridoma is grown in culture, its genetically identical
cells continue to produce the type of antibody characteristic
of the ancestral B cell. The importance of the technique is that
clones of the antibody-secreting cells now can be maintained
indefinitely in cell culture and can produce immense quantities
of identical antibody molecules.

19. Monoclonal Antibodies (Mabs)
Serological identification (tissue and blood typing)
As immunotoxins to treat cancer
Measure serum protein and drug levels
Identify infectious agents
Identify and quantify hormones
Treatment of specific diseases
But the therapeutic use of these monoclonal antibodies has been limited because they were produced in mouse

20. Development of New Generations of MAbs

21. Development of New Generations of MAbs
Chimeric mabs (66% human): Genetically modified mice produce Ab with a human constant region.
Humanized mabs (90% human): MAbs that are mostly human, except for mouse antigen-binding.
Fully human antibodies
Bacterial, plant and animal systems under investigation to increase production volumes.

22. Serological Tests

23. Precipitation Reactions
Interaction of soluble antigens with
IgG or IgM antibodies to form large,
interlocking molecular aggregates called
lattices.
When optimal proportions of antigens and antibodies  Lattice formation.
Excess of either component decreases lattice formation and subsequent precipitation.
Precipitin ring test performed in small tube.
Immunodiffusion procedures: precipitation reactions carried out in agar gel medium

24. Precipitation

25. Precipitation
- Precipitation reactions

26. Precipitation :
Ouchterlony (double diffusion)

27. Agglutination Reactions
Interaction of particulate antigens and antibodies.
Antigens may be
On a bacterial cell:
direct agglutination.
Attached to latex spheres:
indirect or passive agglutination. E.g.: syphilis test
Diseases may be diagnosed by combining the patient’s serum with a known antigen

28. Agglutination Reactions
Involve particulate antigens and antibodies
Antigens may be:
On a cell (direct agglutination)
Attached to latex spheres (indirect or passive agglutination)

29. Antibody Titer Is the concentration of antibodies against a particu
Agglutination reactions are used
in blood typing, the diagnosis of certain diseases, and the identification of viruses.
Figure 18.5

30. Agglutination
- Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens.
- Cellular/molecular view of agglutination and
Precipitation reactions that produce visible Ag-Ab complexes.

31. Neutralization Reactions
Harmful effect of a bacterial exotoxin or virus is eliminated by a specific antibody (antitoxins and virus neutralization)
Some viruses (mumps, measles, influenza) agglutinate RBCs in vitro.
Antibodies against these viruses can be detected by their ability to interfere with viral hemagglutination.

32. Viral Hemagglutination-Inhibition Tests
Hemagglutination involves agglutination of RBCs
Some viruses agglutinate RBCs in vitro
Antibodies prevent hemagglutination

33. Fluorescent Antibody Techniques
Use antibodies labeled with fluorescent dyes.
Direct fluorescent-antibody tests: identify specific microorganisms.
Indirect fluorescent-antibody (IFA) tests: demonstrate the presence of antibodies in serum.
FACS =Fluorescence-activated cell sorting

34. Enzyme-Linked Immunosorbent Assay
ELISA
Enzyme-Linked Immunosorbent Assay

35. Thank you