TORTORA • FUNKE • CASE
Practical Applications of
Immunology

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

Nature of Ag/Ab Reactions
• Lock and Key Concept
• Non-covalent Bonds
– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds
• Multiple Bonds
• Reversible http://www.med.sc.edu:85/chime2/lyso-abfr.htm
Source: Li, Y., Li, H., Smith-Gill, S. J.,
Mariuzza, R. A., Biochemistry 39, 6296, 2000

• The overall strength of binding between an Ag with many determinants and multivalent Abs
K eq
= 10 4
Affinity
10 6
Avidity
10 10
Avidity

• 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
Cross reactions
Anti-A
Ab
Anti-A
Ab
Anti-A
Ab
Ag A Ag B
Shared epitope
Ag C
Similar epitope

• Affinity
• Avidity
• Ag:Ab ratio
• Physical form of Ag
Ab excess Ag excess
Equivalence – Lattice formation

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

TORTORA • FUNKE • CASE
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.

• MAbs produced from a single clone of B cells
• Monoclonal antibodies all have identical antigenbinding 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

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

1975 Kohler and Milstein
Fusions generated by Sendai virus or PEG

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

PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
•
1) Immunize animal (mouse or rabbit)
•
2) Isolate spleen cells (containing antibodyproducing 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.

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)

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 too low
Titre High
2 weeks

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

PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY
Step 4: - Fusion of Myeloma Cells with Immune
Spleen Cells &
Selection of Hybridoma Cells
PEG
FUSION
SPLEEN CELLS
MYELOMA CELLS
Feeder Cells
Growth Medium
HYBRIDOMA CELLS
ELISA PLATE
HAT Medium
1.
Plating of Cells in
HAT selective
Medium
2.
Scanning of Viable
Hybridomas

PRODUCTION OF MONOCLONAL ANTIBODY
HYBRIDOMA TECHNOLOGY

 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
-useful for the production of a specific antibodies to impure or mixed immunogenes.

Uses
M easuring protein and drug levels in serum
T yping tissue and blood
I dentifying infectious agents
I dentifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas
I dentifying tumor metastasis
I dentifying and quantifying hormones
I mmunoaffinity Purification

Serological Tests

Precipitation :
( Continued )
Precipitation curve :

Precipitation Reactions

Precipitation :
( Continued )
- Precipitation reactions

Precipitation :
( Continued )
Ouchterlony (double diffusion)

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

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

Agglutination
: ( continued )
- Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens.
-
Cellular/molec ular view of agglutination and
Precipitation reactions that produce visible
Ag-Ab complexes.

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

Neutralization Reactions
• Eliminate the harmful effect of a virus or exotoxin

Agglutination Tests
Lattice Formation

•
Definition - tests that have as their endpoint the agglutination of a particulate antigen
– Agglutinin/hemagglutinin
•
Qualitative agglutination test
– Ag or Ab
+ 

Agglutination/Hemagglutination
• Quantitative agglutination test
– Titer
– Prozone
Patient
1
2
3
4
5
6
7
8
Titer
64
8
512
<2
32
128
32
4

Agglutination/Hemagglutination
• Definition
• Qualitative test
• Quantitative test
• Applications
–
Blood typing
– Bacterial infections
–Fourfold rise in titer
• Practical considerations
–
Easy
– Semi-quantitative

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

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

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

Complement Fixation
• Methodology
– 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
Patient’s serum
Ag

Complement Fixation

Complement Fixation

Complement fixation test

Fluorescent Antibody Techniques (Direct)
Figure 18.10a

Fluorescent Antibody Techniques
(Indirect)

Enzyme-Linked Immunosorbent Assay
(Direct ELISA)

Enzyme-Linked Immunosorbent Assay
(Indirect ELISA)

Enzyme-Linked
Immunosorbent Assay

Test antigen Test antiserum

Visualization of reaction

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

The Enzyme
•
The enzyme choice based on three parameters:
1- the enzyme should have high turnover rate of substrate to product.
2the 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:
1) Alkaline phosphatase. 2) β-Galactosidase.
3)Horseredish peroxidase.

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.

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.

Serological Tests

Tests for Cell Associated Antigens
Lattice formation not required

Immunofluorescence
• Direct
– Ab to tissue Ag is labeled with fluorochrome
Fluorochrome
Labeled Ab
Ag
Tissue Section

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

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

Immunofluorescence
• Flow Cytometry cont.
– Data displayed
One Parameter Histogram Two Parameter Histogram
Unstained cells
FITC-labeled cells
Red Fluorescence Intensity
Green Fluorescence Intensity