tortora funke case practical applications of immunology

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

• Reversible

• Multiple Bonds

Source: Li, Y., Li, H., Smith-Gill, S. J.,

Mariuzza, R. A., Biochemistry 39, 6296, 2000

http://www.med.sc.edu:85/chime2/lyso-abfr.htm

Avidity• The overall strength of binding between an Ag

with many determinants and multivalent Abs

Keq = 104

Affinity106

Avidity1010

Avidity

Cross Reactivity• 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 A

Anti-A Ab

Ag B

Shared epitope

Anti-A Ab

Ag C

Similar epitope

Cross reactions

Factors Affecting Measurement of Ag/Ab Reactions

• 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

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.

• 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

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 singleantigens used in the antigenimmunization

Antibody class: A mixture of different All of a single Ab classAb classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same antigendifferent antigen-binding binding sitesites

Potential for cross-reactivity: High Low

Hybridoma 1975 Kohler and Milstein

Fusions generated by Sendai virus

or PEG

Antibodies

Polyclonal Monoclonal

Antibodies that are collected from sera of exposed animal

recognize multiple antigenic sites of injected biochemical.

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 ANTIBODYHYBRIDOMA

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.

PRODUCTION OF MONOCLONAL ANTIBODY

Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of

Hybridoma cells

HYBRIDOMA TECHNOLOGY

ANTIGEN ( Intact cell/ Whole cell

membrane/ micro-organisms ) +

ADJUVANT (emulsification)

Ab titre reached in Serum

Spleen removed

(source of cells)


Step 2: - Screening Of Mice For Antibody Production


After several

weeks of immunizati

onSerum Antibody Titre Determined

(Technique: - ELISA / Flow cytometery)

Titre too low

BOOST(Pure antigen)

Titre High

BOOST(Pure

antigen)

2 weeks


Step 3: - Preparation of Myeloma Cells


Immortal Tumor Of Lymphocytes

+8 - Azaguanine

Myeloma Cells

High Viability & Rapid Growth hypothanthin-aminopetrin

Medium

HGPRT-

Myeloma Cells

HGPRT = Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)


Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells &

Selection of Hybridoma Cells


FUSION

PEG

MYELOMA CELLS

SPLEEN CELLS

HYBRIDOMA CELLSELISA PLATE

Feeder CellsGrowth Medium

HAT Medium

1. Plating of Cells in HAT selective Medium

2. Scanning of Viable Hybridomas

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.

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 hormonesImmunoaffinity Purification

Uses

Uses

Serological Tests

Direct Indirect

Detect Ag from patient sample .

Detect Ab from patient sample

Precipitation : ( Continued )

Precipitation curve :

Precipitation Reactions

• Involve soluble antigens with antibodies


- Precipitation reactions


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 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.

Agglutination : ( continued )

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

Agglutination/Hemagglutination

• Definition - tests that have as their endpoint the agglutination of a particulate antigen– Agglutinin/hemagglutinin

+

• Qualitative agglutination test– Ag or Ab


• Quantitative agglutination test– Titer– Prozone

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

1/10

24

Pos

.

Neg

.

Titer

64

8

512

<2

32

128

32

4

Patient

1

2

3

4

5

6

7

8


• Definition • Qualitative test• Quantitative test

• Applications – Blood typing– Bacterial infections

–Fourfold rise in titer

• Practical considerations– Easy– Semi-quantitative

1/2

1/4

1/8

1/16

1/32

1/64

1/12

8

1/25

6

1/51

2

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 anti-rhesus factor (

Rh) antibodies

Patient’s Serum

TargetRBCs

+ Step 1

+

Coombs Reagent(Antiglobulin)

Step 2

Complement Fixation

– 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

Patient’sserum

Ag No Ag

Ag

• Methodology

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)

ELISAEnzyme-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.

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:

1) Alkaline phosphatase. 2) β-Galactosidase.

3)Horseredish peroxidase.

• 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.

The Enzyme

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

Ag

FluorochromeLabeled Ab

Tissue Section

Immunofluorescence

• Indirect– Ab to tissue Ag is unlabeled– Fluorochrome-labeled anti-Ig is

used to detect binding of the first Ab.

Ag

FluorochromeLabeled Anti-Ig

Tissue Section

UnlabeledAb

• Qualitative to Semi-Quantitative

Immunofluorescence

• Flow Cytometry– Cells in suspension are labeld with fluorescent tag

• Direct or Indirect Fluorescence– Cells analyzed on a flow cytometer

FlowTip

Laser

FLDetector

LightScatter

Detector

Immunofluorescence

• Flow Cytometry cont.– Data displayed

Green Fluorescence Intensity

Nu

mb

er o

f C

ells

Unstained cells

FITC-labeled cells

One Parameter Histogram

Red Fluorescence Intensity

Gre

en F

luor

esce

nce

In

ten

sity

Two Parameter Histogram

tortora funke case practical applications of immunology

Documents