SOS BIOTECHNOLOGY MSc II SEM By Dr Richa Sijoria
54
SOS BIOTECHNOLOGY MSc II SEM By Dr Richa Sijoria
Transcript of SOS BIOTECHNOLOGY MSc II SEM By Dr Richa Sijoria
SOS BIOTECHNOLOGY
MSc II SEM
By Dr Richa Sijoria
Techniques of cellular immunity
1. Precipitation
2. Agglutination
3. Radioimmunoassay
4. Enzyme-linked Immunosorbent Assay
5. Western Blotting
6. Immunostaining
•Immunofluorescence
•Immuno-gold EM
7. Flow Cytometry and Fluorescence
8. Immunoelectron Microscopy
Serology refers to using antigen-antibody
reactions in the laboratory for diagnostic
purposes. Its name comes from the fact that
serum, the liquid portion of the blood
where antibodies are found is used in testing.
Definition:
a. To identify unknown antigens (such as microorganisms). This is called direct serologic testing. Direct serologic testing uses a preparation known antibodies, called antiserum, to identify an unknown antigen.
b. To detect antibodies being made against a specific antigen in the patient's serum. This is called indirect serologic testing. Indirect serologic testing is the procedure by which antibodies in a person's serum being made by that individual against an antigen associated with a particular disease are detected using a known antigen.
Serologic testing may be used in the clinical laboratory in two distinct ways:
Laboratory use of antibodies
• Quantitation of an antigen
– RIA, Elisa
• Identification and characterization of protein antigens
– Immunoprecipitation
– Western blotting
• Cell surface labelling and separation
• Localisation of antigens within tissues or cells
• Expression librairies
• Phage display
Classes of antibodies
Isotype Structure Placenta
transfert
Activates
complementAdditional features
IgMNo Yes First Ab in development and response
IgDNo No B-cell receptor
IgGYes Yes Involved in opsonization and ADCC.
Four subclasses; IgG1, IgG2, IgG3,
IgG4
IgENo No Involved in allergic responses
IgANo No Two subclasses; IgA1, IgA2. Also found
as dimer (sIgA) in secretions.
Commercial production of antibodies: polyclonal vs monoclonal
Polyclonal monoclonal
• A Monoclonal antibody, by contrast, represents antibody from a single antibody producing B cell and therefore only binds with one unique epitope. Each individual antibody in a polyclonal mixture is technically a monoclonal antibody; however, this term generally refers to a process by which the actual B-cell is isolated and fused to an immortal hybridoma cell line so that large quantities of identical antibody can be generated.
• A Polyclonal Antibody represents a collection of antibodies from different B cells that recognize multiple epitopes on the same antigen. Each of these individual antibodies recognizes a unique epitope that is located on that antigen.
Antigen-antibody interaction
• Antigen: foreign molecules that generate antibodies or any substance that can be bound specifically by an antibody molecule– Proteins, sugars, lipids or nucleic acids
– Natural or synthetic
• Antibody: molecules (protein) responsible for specific recognition and elimination (neutralization) of antigens– Different structures (7-8 classes in mammals)
– Powefull research tools for basic research, clinical applications and drug design
4 types of noncovalent forces
Nature of binding forces
• Hydrogen bonding
– Results from the formation of hydrogen bridges between appropriate atoms
• Electrostatic forces
– Are due to the attraction of oppositely charged groups located on two protein side chains
• Van der Waals bonds
– Are generated by the interaction between electron clouds (oscillating dipoles)
• Hydrophobic bonds– Rely upon the association of non-polar, hydrophobic groups so that contact with water molecules is
minimized (may contribute up to half the total strength of the antigen-antibody bond)
Factors affecting measurement of antigen-antibody reactions
1. Affinity The higher the affinity of the antibody for the antigen, the more stable will be the interaction. 2. Avidity Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.3. Antigen to antibody ratio The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody.4. Physical form of the antigen The physical form of the antigen influences how one detects its reaction with an antibody.
• In terms of infectious diseases, the following may act as antigens:
1.Microbial structures (cell walls, capsules, flagella, pili, viral capsids, envelope-associated glycoproteins, etc.).
2. Microbial exotoxins
• Certain non-infectious materials may also act as antigens if they are recognized as "nonself" by the body. These include:
1. Allergens (dust, pollen, hair, foods, dander, bee venom, drugs, and other agents causing allergic reactions).
2. Foreign tissues and cells (from transplants and transfusions).
3. The body's own cells that the body fails to recognize as "normal self" (cancer cells, infected cells, cells involved in autoimmune diseases).
Antigenic determinants
• An antibody will recognize– Epitope: defined segment of an antigen
– Immunoreactivity of epitopes may depend on primary, secondary, tertiary or quaternary structure of an antigen
– Define the possible applications
– Variability of epitopes depends on the species
• Antibodies are antigen themselves
Antigen-antibody affinity
The affinity with which antibody binds antigen results from a balance
between the attractive and repulsive forces. A high affinity antibody implies
a good fit and conversely, a low affinity antibody implies a poor fit and a
lower affinity constant
Antigen-antibody interaction: concentration dependence
Concentration of unknown samples are determined from a standard curveSTD concentration values are obtained when the interaction between
• General equation for a dose response curve
• It shows response as a function of the logarithm of concentration
• X is the logarithm of agonist concentration and Y is the response
• Log EC50 is the logarithm of the EC50 (effective concentration, 50% of maximal response)
• IC50 (inhibitory conc.)
Sigmoidal dose response curve
10%
90%
Detection principles
• Radiolabelled isotopes (antigen)
– 125I, 32P, 35S
• Enzymes (Ab)
– Peroxydase
• Chromophores (Ab)
– Fluorogenic probes (UV, visible or IR)
Agglutination reaction vs Precipitation reaction
• Agglutination reaction and precipitation reaction have
great importance in immunology as they are serological
reactions that help in the detection of bacterial infection
in the serum of a patient.
• Major difference between precipitation and
agglutination is the size of antigens involved.
• Antigens are soluble in case of precipitation while they
are insoluble in agglutination
• Agglutination is more sensitive than precipitation.
• Precipitation in fluids: Known antiserum is mixed with soluble test antigen and a cloudy precipitate forms at the zone of optimum antigen-antibody proportion. • Precipitation in gels
- radial immunodiffusion (Mancini method)- double immunodiffusion (Ouchterlony method)
Immunoelectrophoresis
Precipitation Reactions:
Precipitation Reactions
Single diffusion technique. Semi-quantitative
immunoprecipitin reaction. Ag is loaded into single well of
agarose gel. Gel has been impregnated with specific Ab
solution. Ag will diffuse out from well in radial fashion.
Diameter of immunoprecipitin ring is proportional o the
concentration of the Ab or Ag present
Ab and Ag specific to each other will form immune complex that will
precipitate as a visible line in a gel. Ab and Ag placed in separate,
opposing wells inside of agarose gel. Solutions diffuse toward one another
in a radial fashion normal at RT.
Shape and positioning of precipitation line is proportional to concentration
of reactants. A precipitation line is formed at the zone of equivalence
where diffusing Ab and Ag fronts complex
Immunoelectrophoresis:
A complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs.This tests is used for the qualitative analysis of complex mixtures of antigens, although a crude measure of quantity (thickness of the line) can be obtained. This test is commonly used for the analysis of components in a patient' serum. Serum is placed in the well and antibody to whole serum in the trough. By comparisons to normal serum, one can determine whether there are deficiencies on one or more serum components or whether there is an overabundance of some serum component (thickness of the line). This test can also be used to evaluate purity of isolated serum proteins.
Immunoelectrophoresis
Known antiserum causes bacteria or other particulate antigens to clump together or agglutinate. Molecular-sized antigens can be detected by attaching the known antibodies to larger, insoluble particles such as latex particles or red blood cells in order to make the agglutination visible to the naked eye.
HemagglutinationBacterial AgglutinationPassive AgglutinationAgglutination Inhibition
Agglutination Reactions:
Haemagglutination
Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the presence of viral particles.The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells.
-Influenza and other viruses-Two spike proteins: NEURAMINIDASE , HAEMAGGLUTININ ( Binds specifically to red blood cells)
Steps to haemagglutination:1. Dispense diluent.2. Add red blood cells and mix by gently shaking.3. Allow the red blood cells to settle and observe the pattern.4. Observe if the cells have a normal settling pattern and there is no auto-agglutination. This will be a distinct button of cells in the micro test and an even suspension with no signs of clumping in the rapid test.
Hemagglutination
Agglutination No Agglutination No Ab
Agglutination Inhibition
Complement-fixation
• Known antiserum is mixed with the test antigen and complement is added. Sheep red blood cells and hemolysins (antibodies that lyse the sheep red blood cells in the presence of free complement) are then added. If the complement is tied up in the first antigen-antibody reaction, it will not be available for the sheep red blood cell-hemolysin reaction and there will be no hemolysis.
• A negative test would result in hemolysis.
Radioactive binding techniques
• Test antigens from specimens are passed through a tube coated with the corresponding specific known antibodies and become trapped on the walls of the tube. Known antibodies to which a radioactive isotope has been chemically attached are then passed through the tube where they combine with the trapped antigens. The amount of antigen-antibody complex formed is proportional to the degree of radioactivity.
Solid-phase Radioimmunoassay (RIA)
Test antigens from specimens are passed through a tube (or a membrane) coated with the corresponding specific known antibodies and become trapped on the walls of the tube (or on the membrane). Known antibodies to which an enzyme has been chemically attached are then passed through the tube (or membrane) where they combine with the trapped antigens. Substrate for the attached enzyme is then added and the amount of antigen-antibody complex formed is proportional to the amount of enzyme-substrate reaction as indicated by a color change.
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA
- Chemiluminescence
Enzyme-linked Immunosorbent Assay (ELISA):
Elispot Assay
Details required
Peroxydase reaction
Western Blot
The western blot (alternatively, protein immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
Western Blotting
Immunostaining- Immunoflourescence
-Immuno-gold EM
Immunoflourscence: A laboratory technique to identify specific antibodies or antigens. Antibody identification is usually performed on blood (serum).
1. Antibody tagged with flourescent dye2. Antibody attached specifically to antigen 3. View specimen under exciting light 4. Flourscence microscope
Immunofluorescence
Immunofluorescence
Immunogold Electron Microscopy :
Same principle as immunoflourscenceGold particles attached to antibodies (nanometer size particles)Viewed under EM to localise specific proteins or antigens
Flow Cytometry
• is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a fluorescent tag by either direct or indirect immunofluorescence. The cells are then analyzed on the flow cytometer.In a flow cytometer, the cells exit a flow cell and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors.
Flow Cytometry
FACS:
Fluoresence-activatedCell sorter
Immunoelectron microscopy
An electron microscope is a type of microscope that uses electrons to illuminate a specimen and create an enlarged image. Electron microscopes have much greater resolving power than light microscopes and can obtain much higher magnifications.
