1. Practical Applications of Immunology

2. 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

3. Nature of Ag/Ab Reactions
Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000
Lock and Key Concept
Non-covalent Bonds
Hydrogen bonds
Electrostatic bonds
Van der Waal forces
Hydrophobic bonds
Multiple Bonds
Reversible

4. 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

5. 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

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

7. 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

8. . Clone: a strain of cells descended form single cell.
Monoclonal antibody
Definition:
. Mono: one
. Clone: a strain of cells descended form single cell.
. Antibody: a molecule of animal origin that has immunological activity only against the antigen to which it was made.

9. 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

10. HISTORY

11. 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

12. Hybridoma 1975 Kohler and Milstein
Fusions generated
by Sendai virus
or PEG

13. Antibodies Polyclonal Monoclonal recognize multiple antigenic sites
Individual B lymphocyte hybridoma
is cloned and cultured.
Secreted antibodies are collected
from culture media
Antibodies that are collected
from sera of exposed animal
recognize
multiple antigenic sites
of injected biochemical.
recognize
ONE antigenic site
of injected biochemical

14. 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.

15. Ab titre reached in Serum
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells
ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification)
Ab titre reached in Serum
Spleen removed
(source of cells)

16. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 2: - Screening Of Mice For Antibody Production
After several weeks of immunization
Serum Antibody Titre Determined
(Technique: - ELISA / Flow cytometery)
Titre High
Titre too low
2 weeks
BOOST (Pure antigen)
BOOST (Pure antigen)

17. Step 3: - Preparation of Myeloma Cells Immortal Tumor Of Lymphocytes
PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 3: - Preparation of Myeloma Cells +
8 - Azaguanine
Myeloma Cells
Immortal Tumor Of Lymphocytes
Myeloma Cells
HGPRT-
High Viability & Rapid Growth hypothanthin-aminopetrin Medium
HGPRT = Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

18. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &
Selection of Hybridoma Cells
PEG
FUSION
MYELOMA CELLS
SPLEEN CELLS
Feeder Cells
Growth Medium
Plating of Cells in HAT selective Medium
Scanning of Viable Hybridomas
HYBRIDOMA CELLS
ELISA PLATE
HAT Medium

19. PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

20. Disadvantages Need high experience qualification
High risk of contamination
Some mcABs are very liable so activity may be lost on freezing and thawing or long term storage
The frequent need to purify a mcAB from monoclonal culture medium
Advantages
-useful for the production of a specific antibodies to impure or mixed immunogenes.

21. Measuring protein and drug levels in serum Typing tissue and blood
Uses
Measuring protein and drug levels in serum
Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity Purification

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

23. Precipitation : ( Continued )
Precipitation curve :

24. Precipitation Reactions
Involve soluble antigens with antibodies

25. Precipitation : ( Continued )
- Precipitation reactions

26. Precipitation : ( Continued )
Ouchterlony (double diffusion)

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

28. Antibody Titer
Is the concentration of antibodies against a particular antigen
Figure 18.5

29. Agglutination : ( continued )
- 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.

30. Hemagglutination Hemagglutination involves agglutination of RBCs.
Viral hemagglutination inhibition tests for antibodies by the antibodies' ability to prevent viruses from agglutinating RBCs.
Figure 18.7

31. Neutralization Reactions
Eliminate the harmful effect of a virus or exotoxin

32. Agglutination Tests
Lattice Formation 32

33. Agglutination/Hemagglutination
Definition - tests that have as their endpoint the agglutination of a particulate antigen
Agglutinin/hemagglutinin Y + 
Qualitative agglutination test
Ag or Ab

34. Agglutination/Hemagglutination
Quantitative agglutination test
Titer
Prozone
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
1/1024
Pos.
Neg.
Titer 64 8
512
<2 32
128 4
Patient 1 2 3 5 6 7

35. Agglutination/Hemagglutination
Definition
Qualitative test
Quantitative test
1/2
1/4
1/8
1/16
1/32
1/64
1/128
1/256
1/512
Applications
Blood typing
Bacterial infections
Fourfold rise in titer
Practical considerations
Easy
Semi-quantitative

36. Passive Agglutination/Hemagglutination
Definition - agglutination test done with a soluble antigen coated onto a particle Y + 
Applications
Measurement of antibodies to soluble antigens

37. Coombs (Antiglobulin)Tests
Incomplete Ab
Direct Coombs Test
Detects antibodies on erythrocytes +  Y
Patient’s RBCs
Coombs Reagent
(Antiglobulin)

38. Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum anti-rhesus factor (Rh) antibodies Y
Patient’s
Serum
Target
RBCs + 
Step 1 +  Y
Coombs Reagent
(Antiglobulin)
Step 2

39. Y Complement Fixation Methodology Ag No Ag
Ag mixed with test serum (Preheated) to be assayed for Ab
Standard amount of complement is added
Erythrocytes coated with Abs is added
Amount of erythrocyte lysis is determined Ag
No Ag Ag Y
Patient’s
serum Ag Y

40. Complement Fixation

41. Complement Fixation

42. Complement fixation test

43. Fluorescent Antibody Techniques (Direct)
Figure 18.10a

44. Fluorescent Antibody Techniques (Indirect)

45. Enzyme-Linked Immunosorbent Assay (Direct ELISA)

46. Enzyme-Linked Immunosorbent Assay (Indirect ELISA)

47. ELISA Enzyme-Linked Immunosorbent Assay

48. Test antigen
Test antiserum

49. Visualization of reaction

50. ELISA to test MAb production itself
The dark blue spot represents a positive clone.
The light blue spots are positive controls,
the next two wells to the right are negative controls
+ve clone
-ve control
+ve control

51. The enzyme choice based on three parameters:
1- the enzyme should have high turnover rate of substrate to product.
2- the product should be detected with max sensitivity.
3- the enzyme activity should be minimally susceptible to interference from factors may be present in the sample.
The commonly used Enzymes:
Alkaline phosphatase ) β-Galactosidase.
3)Horseredish peroxidase.

52. The developed color is read by eye as qualitative assays
The Enzyme
The developed color is read by eye as qualitative assays
To perform quantitative assays take the sample for serial dilutions ,the highest dilution to give +ve result is taken for potency measurement by spectrophotometer.

53. Microtitre Plate It is a disposable plate with 96 wells.
Ether Antigen or antibody can be adsorbed to the wall of each well.
Subsequent washing and reagent addition become very easy using the microtitre system specially in using of multihead pipettes.
Because of its simplicity ,ELISA has become the method of choice for screening mcAB production cultures.

54. Serological Tests

55. Tests for Cell Associated Antigens
Lattice formation not required

56. Immunofluorescence Direct Y
Ab to tissue Ag is labeled with fluorochrome Ag Y
Fluorochrome
Labeled Ab
Tissue Section

57. Qualitative to Semi-Quantitative
Immunofluorescence
Indirect
Ab to tissue Ag is unlabeled
Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y
Fluorochrome
Labeled Anti-Ig
Tissue Section
Unlabeled Ab
Qualitative to Semi-Quantitative

58. Immunofluorescence Flow Cytometry
Cells in suspension are labeld with fluorescent tag
Direct or Indirect Fluorescence
Cells analyzed on a flow cytometer
Flow
Tip
Laser FL
Detector
Light
Scatter

59. Green Fluorescence Intensity Two Parameter Histogram
Immunofluorescence
Flow Cytometry cont.
Data displayed
Red Fluorescence Intensity
Green Fluorescence Intensity
Two Parameter Histogram
One Parameter Histogram
Unstained cells
Number of Cells
FITC-labeled cells
Green Fluorescence Intensity