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An introduction
Chalee Sienes-Reyes, RMT, MS-MT Jasmen S. Pasia, RMT, MS-MT Aileen Grace Ang, RMT, MAST-Bio Francis Ian L. Salaver, RMT
Antigen-Antibody Interactions
3 categories: 1. Primary or Initial Reactions 2. Secondary interactions 3. Tertiary interactions
Secondary Interactions
Ag-Ab interactions which involve building a lattice to form visible aggregates of precipitates or agglutinates Less sensitive but do not require sophisticated instruments
Tertiary Interactions
Biologic expressions of AgAb interaction that may either be beneficial or harmful to the body These consequences of immune response in vivo are measured by tertiary binding tests
Effect of temperature
Equivalence point:
(suitable proportion between the virus particles and RBCs)
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Precipitation Curve
Prozone antibody excess, many antibodies coat all antigen sites- results in false negative Postzone antigen excess, antibody coats antigen but cannot get lattice formation, results in false negative Zone of Equivalence antigen and antibody present in optimal proportions to bind and give visible reaction
Agglutination
When the antigen is particulate, the reaction of an antibody with the antigen can be detected by agglutination (clumping) of the antigen. The general term agglutinin is used to describe antibodies that agglutinate particulate antigens.
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All antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good agglutinating antibody.
Agglutination
Qualitative and quantitative Quantitation of agglutination tests to detect specific Abs is done by titration The reciprocal of the highest dilution giving a positive reaction is known as titer and provides a measure of the amount of antibody in the serum A fourfold or more increase in the antibody titer between a paired sera is significant
Agglutination
Tube 1
SALINE SERUM FINAL DILUTION
Tube 2 1 1:4
Tube 3 1 1:8
Tube 4 1 1:16
Tube 5 1 1:32
Tube 6 1 1:64
Tube 7 1 1:128
1 1 1:2
Agglutination
Agglutinins
Antibodies that produce such reactions
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SENSITIZATION
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Secondary Phenomenon
Lattice Formation
The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation
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Agglutination
Direct Agglutination
Slide Agglutination Test Plate Agglutination Test Tube Agglutination Test
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Bacterial Agglutination
Widal test VDRL, RPR
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Agglutination Techniques Slide Test If (+) reactivity is obtained using undiluted serum, serial dilution of serum is done to determine Ab titer False (-) can occur when Ab titer in undiluted serum is too high
Reading/Grading Agglutination Reactions Pseudo agglutination or the Rouleaux Formation also occurs
Red blood cells appear as stacks of coins.
Addition of physiologic Nacl will disperse pseudo agglutination Saline Replacement is done after pseudo agglutination is observed so that it may not give false negative result due to the dilution effect of the saline
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Agglutination Techniques Microtiter Plate Tests serial dilution of serum sample to determine the Ab titer
Constant amount of Ag is added to each serum containing well, rotated and titer is read
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1/10
1/20
1/40
1/80
1/160
1/320
Neg. ctrl
GRADE
Negative (-)
DESCRIPTION No aggregates
Appearance
Weak (+/-)
Tiny aggregates that are barely visible macroscopically; turbid and reddish supernatant
1+
A few small aggregates just visible macroscopically; turbid and reddish supernatant
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2+
3+
4+
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Passive hemagglutination:
The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes with a soluble antigen and use the coated red blood cells in an agglutination test for antibody to the soluble antigen. This is called passive hemagglutination. The test is performed just like the agglutination test. Applications include detection of antibodies to soluble antigens and detection of antibodies to viral antigens.
latex agglutination passive hemagglutination (treated red blood cells made resistant)
Examples of tests - agglutination for leptospirosis Widal test (typhoid fever)
Example
detecting cholera toxin
Reverse Agglutination Ab is attached to the particles and the biological sample is tested for the presence of Ag Used to detect the presence of CRP
A protein secreted into the blood during a inflammatory response
Anti-CRP antibodies are affixed to to the latex particles Mixed with the serum of patients to detect the presence of CRP
Co-agglutination
Co agglutination is similar to the latex agglutination technique for detecting antigen. Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions
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Co agglutination Test
Agglutination test in which inert particles (latex beads or heatkilled S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria.
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Haemagglutination
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Example
influenza virus binds to fowls red blood cells
Haemagglutination
RBC
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Viral Haemagglutination
Some viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together
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Viral Hemagglutination
the attachment of viral particles by their receptor sites to more than 1 cell. As more and more cells become attached in this manner agglutination becomes visible
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virus
serial dilution 8 mix with red blood cells side view 16 32 64 128 256
top view
Titer = 32 HA units/ml
One HA unit :minimum amount of virus that causes complete agglutination of RBCs
VIRUSE
SERUM
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Virus
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Virus
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Coombs (Antiglobulin)Tests
Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes
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Patients RBCs Coombs Reagent (Antiglobulin)
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Coombs Test Direct ant globulin test (also called the Coombs test,
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Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
Step 1 Patients Serum Step 2
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Target RBCs
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Applications
Detection of anti-Rh Ab Autoimmune hemolytic anemia
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WASHING RBCs
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Washing process
Take place 4-5 time . Until get clear solution above the RBCs after centrifugation . Using PBS or normal saline .
Note :(avoid using water to wash RBCs because it will definitely lead to RBCs lyses)
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Procedure
Obtain blood samples in tubes, spin at 1500 RPM for 5 minutes. Draw off the supernatant using Pasteur pipette. Add 2ml PBS to each tube and move to a clean test tube. Centrifuge again. Each time draw off the washing solution and add 10 ml PBS until the solution above the RBCs layer becomes clear.
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Neutralization Test
Ag-Ab reaction in which the harmful effects of a bacterial exotoxin or a virus are blocked by specific antibodies (antitoxins).
Precipitation Reactions
- is the reaction between a soluble antigen and its specific antibodies -soluble antigens are smaller and in solution; complexing with antibodies make these bigger and they fall out of solution as a precipitate visible to the eye.
IgG is a much better precipitating Ab than IgM (the reverse is true of agglutination) 2 types:
Precipitation in liquid medium Precipitation in gel medium
Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.
Nephelometry indirect measurement, measures amount of light scattered by the antigenantibody complexes.
Turbidimetry
Measures turbidity or cloudiness of a solution by measuring the amount of light PASSING THROUGH the solution. Soluble antigen and antibody join and once they join in sufficient amounts precipitate, results in cloudiness. The more cloudy the solution, the less light can pass through.
Nephelometry
Measures SCATTERED light bouncing off antigenantibody complexes.
Nephelometry
Antibody reagent is combined with patient sample. If antigen is present in the patients sample, Ag/Ab complexes will form and precipitate out of solution.
Nephelometry
When light is passed through the solution, the precipitates cause the light to scatter at various angles. The light that is scattered at a particular angle is measured. This corresponds to the level of antigen in the sample.
Light source
Detector
Passive Immunodiffusion
Reactions in gels/liquids Migrate towards each other and where they meet in optimal proportions form a precipitate.
Four Methodologies
Single diffusion, single dimension Single diffusion, double dimension Double diffusion, single dimension Double diffusion, double dimension
Immunodiffusion Tests
Single diffusion test: if only one reactant (usually Ag) is moving Double diffusion test: if both Ag and Ab are moving through the gel medium Single dimension: if theres only one effective dimension for Ag or Ab migration, like up and down Double dimension: if circular holes or wells are cut in a gel on a flat surface like petri dish,Ag or Ab diffuses from the wells radially
Oudin Precipitation
Solution of antibody is carefully layered on top of a solution of antigen, such that there is no mixing between the two. With time at the interface where the two layers meet, antigen-antibody complexes form a visible precipitate.
Radial Immunodiffusion
Ab is fixed to the agar in a petri dish Circular wells are cut in the gel and are loaded with Ag A ring of precipitate is formed as the Ag diffuses radially towards its equilibrium concentration
Radial Immunodiffusion
1. Fahey Method
Also known as Kinetic Method or Incomplete Immunodiffusion Diameter of precipitin ring measured at 24 hours of diffusion Diameter proportional to log of antigen concentration
2. Mancini Method
Also known as End Point Method or Complete Immunodiffusion Area of precipitin ring formed at 48 hours of diffusion measured Area proportional to antigen concentration
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Ouchterlony Immunodiffusion
Ouchterlony - Identity
The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity.
Ouchterlony Non-Identity
If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig. 3
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Immunodiffusion Test
Disadvantages of ordinary immunodiffusion tests
incubation of several hours or even days are needed to ensure equilibrium of the system has occurred possibility that 2 or more precipitation bands may form in the same plate
Electrophoretic Techniques
Electrophoretic Techniques
Immunodiffusion can be combined with electrical current to speed things up.
Rocket Immunoelectrophoresis
Antigen is electrophoresed into gel containing antibody. The distance from the starting well to the front of the rocket shaped arc is related to antigen concentration.
Rocket Electrophoresis
Immunoelectrophoresis
Immunoelectrophoresis
Two-dimensional immunoelectrophoresis. Antigens are separated on the basis of electrophoretic mobility. The second separation is run at right angles to the first which drives the antigens into the antiserum-containing gel to form precipitin peaks; the area under the peak is related to the concentration of antigen.
Immunoelectrophoresis
Immunofixation Electrophoresis
Immunofixation Electrophoresis (IFE) combines zone electrophoresis with immunoprecipitation. This technique may be used to identify and characterise serum proteins. In IFE, proteins of sample are first separated by electrophoresis on a support (agarose) according to their charge and after that the medium is overlaid with monospesific antiser reactive with specific protein antigen. If the antigen is present a characteristic immunoprecipitin band will be formed.
Immunofixation Electrophoresis
Western Blot
1) Proteins are separated by gel electrophoresis. 2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.
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Western Blot
3) An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time. 4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.
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Immuno-chromatography: Principle
Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line Either antibody specific for the labelled antibody, or antigen, is bound at the control line
Bound AB Free labled AB Nitrocellulose strip Lysing agend Labled AB. Test band Control band (bound AB) (bound AB)
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Immuno-chromatography: Principle
If antigen is present, some labelled antibody will be trapped on the test line Excess-labelled antibody is trapped on the control line
Captured Ag-labelled Ab-complex Captured labelled Ab
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Limitations
Cost Concern validated data
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