ANTIGEN-ANTIBODY INTERACTION

BYSHWETA PATEL
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DEFINITIONS
Antibodies (also known as immunoglobulin's abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

DEFINITIONS
Antigens A substance that when introduced into the body stimulates the production of an antibody

Antigen-Antibody interactions
Characterized as: Non-covalent interaction (similar to lock and key fit of enzyme-substrate) The exact and specific interaction has led to many immunological assays used to: 1. detect Ag or Ab 2. diagnose disease 3. measure magnitude of humoral IR 4. identify molecules of bio and medical interest
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Ag-Ab interactions
Bonds:
Hydrogen Ionic Hydrophobic interactions Van der Waals forces Each bond is weak;

TECHNIQUES
1. 2. 3. 4. 5. Precipitation Agglutination Radio Immunoassay ELISA Immunoflorescence assaya) Florescence activated cell sorter (FACS) 6. Cytotoxicity assay 7. Cytokines assaya)ELISA b)ELISPOT
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IMMUNOLOGIC TESTS:
1. PRECIPITATION REACTIONS: -Abs and Ags in aqueous solutions form a lattice => Precipitin Lattice formation requires: 1) polyvalent Abs 2) Ag must be bivalent, polyvalent

Precipitation Reactions
Reaction of soluble antigens with antibodies

A PRECIPITATION CURVE

 ADVANTAGE
1. Ease to handle

DISADVANTAGE
1. Very slow 2. Not much sensitive 3. Qty of Ag-Ab required more

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PRECIPITATION REACTION IN GEL

1. RADIAL IMMUNODIFFUSION (MANCINI METHOD) 2. DOUBLE IMMUNODIFFUSION (OUCHTERLONY METHOD) 3. ROCKET IMMUNOELECTROPHORESIS

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1. RADIAL IMMUNODIFFUSION
Ag sample placed in well & diffuse into agar containing antiserum Leads to formation of precipitin ring Precipitin ring proportional to Ag conc.

2. DOUBLE IMMUNODIFFUSION
Ag-Ab diffuse radially from wells towards each other
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Precipitation reaction in gels

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 IMMUNOELECTROPHORESIS:
Incorporation of electrophoresis & double diffusion

An Ag mixture is 1st separated by charge Then, troughs are cut parallel to direction of electric field and antisera is added to trough Ags and Abs diffuse towards each other to produce precipitin bands It is a qualitative technique. Detects only high conc. of Ab.
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IMMUNOELECTROPHORESIS:
Ag preparation (orange) is 1st electrophoresed. Ag separates on basis of charge Antiserum (blue) added on troughs Lines of precipitation (colored arcs) is seen

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CLINICAL USAGE
1. Detects presence or absence of protein in serum 2. Determines low amount of isotypes 3. Determines overproduction of serum protein such as albumin, immunoglobulin, or transferrin. E.g.- overproduction of myeloma protein in multiple myeloma disease

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3. ROCKET ELECTROPHORESIS
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.

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LIMITATION
Negatively charged Ag is required Some protein like immunoglobulin cannot be analyzed Amount of several Ag cannot be determined

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2. AGGLUTINATION REACTION
1896: First observed by Gruber and Durham when serum antibody was found to react with bacterial cells

Interaction between Ab & particular Ag

agglutination Abs that produce such reaction are called agglutinins simple, inexpensive, but sensitive
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Agglutination reactions
Several types exist: A) Hemagglutination i) Bacterial Agglutination ii) Passive Agglutination iii) Agglutination Inhibition

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Types of particles that participate in such reactions:

Erythrocytes Bacterial cells Inert carriers such as latex particles

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A) HEMAGGLUTINATION
Detects antibody to erythrocyte antigens - sufficient concentration of antibody present-> antibody cross-link= agglutination - non-reactive/insufficient antibody present= no agglutination

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i) BACTERIAL AGGLUTINATION
Carried out to identify bacterial species & intensity of infection Bacterial infection induce production of serum Ab specific for surface Ag of bacterial cell wall In this case, the Ag-Ab reaction forms an agglutination, which is directly visible. These reactions can be performed on slides (rapid tests) or on microtiter plates or tubes for Antibody titration if required
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 Last tube gives visible agglutination reaction Thus indicate Ab titer of patient Patient suffering from typhoid fever elevate agglutination titer to Salmonella typhi

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ii) PASSIVE AGGLUTINATION

An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces

Particle carriers include:

Red blood cells Polystyrene latex Bentonite charcoal
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ii) Passive Agglutination

Ag-coated red blood cells- soluble Ag + RBC (treated with tannic acid or chromium chloride- promote adsorption of Ag to surface of cell) Serum is diluted & placed into microtiter wells & Ag-coated RBC are added & spread pattern observed Synthetic beads are often used now a days
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 ADVANTAGES1. Uniformity 2. Stability 3. Consistency Used in the detection of :

Rheumatoid factor
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iii) AGGLUTINATION INHIBITION

Based on the competition between particulate and soluble antigens for limited antibody combining site Lack of agglutination is indicator of a positive reaction

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Agglutination Inhibition
Pregnancy Testing
-classic example of agglutination inhibition Human chorionic gonadotropin (hCG) Appears in serum and urine early in pregnancy

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Agglutination Inhibition
urine Antiserum

No hCG in urine Anti-hCG free

hCG in urine Anti-hCG neutralized

Carriers coated with hCG added

Carriers coated with hCG

Agglutination of carriers: Negative test for pregnancy

Agglutination of carriers: Positive test for pregnancy

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IMMUNOASSAY
A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample

Analyte
The sample being analyzed and in immunoasssays the analyte is either Antibody or Antigen
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3) RADIOIMMUNOASSAY (RIA)
Radioimmunoassay (RIA) involves the separation of a protein (from a mixture) using the specificity of antibody - antigen binding and quantitation using radioactivity The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma Involves competitive binding of radiolabeled Ag & unlabeled Ag to high affinity Ab
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THE TECHNIQUE
A mixture is prepared of radioactive antigen the radioactive isotopes 125I or 131I are often used. antibodies against that antigen. Known amounts of unlabeled antigen are added to samples of the mixture. These compete for the binding sites of the antibody

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 At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules. The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured.

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 ADVANTAGE Radioimmunoassay is widely-used because of its great sensitivity. Using antibodies of high affinity, it is possible to detect a few picograms (1012 g) of antigen in the tube. The greater the specificity of the antiserum, the greater the specificity of the assay

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 DISADVANTAGE The main drawbacks to radioimmunoassay are the expense and hazards if preparing and handling the radioactive antigen. Both 125I or 131I emit gamma radiation that requires special counting equipment;

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 CLINICAL USAGE 1. RIA has become a major tool in the clinical laboratory where it is used to assay 2. plasma levels of:
1. most of our hormones; 2. certain abused drugs

3. for the presence of hepatitis B surface antigen (HBsAg) in donated blood;

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4) ELISA TECHNIQUE
Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample OR ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.

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 In ELISA, an unknown amount of antigen is affixed to a surface, and then a specific antibody is applied over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal, most commonly a colour change in a chemical substrate
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 Allows for qualitative or quantitative testing. Each one can be used for qualitative detection of Ag or Ab Also, a std curve based on known *C+s of Ag/Ab can be prepared and an unknown [C} can be assured ELISA has many of the advantages (e.g., sensitivity, ease of handling multiple samples) without the disadvantages of dealing with radioactivity (like in RIA

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 The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used. The test requires:
the antibodies fixed to a solid surface, such as the inner surface of a test tube; a preparation of the same antibodies coupled to an enzyme. This is one (e.g., -galactosidase) that produces a colored product from a colorless substrate.
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Performing the Test

The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich". After washing away any unbound conjugate, the substrate solution is added. After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen. 43

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ELISA

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ADVANTAGE
1. ELISA tests are generally relatively accurate tests. 2. They are considered highly sensitive and specific and compare favorably with other methods used to detect substances in the body, such as radioimmune assay (RIA) tests. 3. They have the added advantages of not needing radioisotopes (radioactive substances) or a costly radiation counter (a radiation-counting apparatus). 4. Freedom from radiation hazards. 5. Non-requirement of specialized laboratories with expensive equipment and cheaper reagents with relatively longer shelf lives. 6. Particularly suitable for use in small laboratories
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 CLINICAL USAGE Literally hundreds of ELISA kits are manufactured for research human and veterinary diagnosis Some examples: screening donated blood for evidence of viral contamination by HIV-1 and HIV-2 (presence of anti-HIV antibodies) hepatitis C (presence of antibodies) hepatitis B (testing for both antibodies and a viral antigen)
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 measuring hormone levels HCG (as a test for pregnancy) LH (determining the time of ovulation) TSH, T3 and T4 (for thyroid function) hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes detecting infections sexually-transmitted agents like HIV, syphilis, and chlamydia hepatitis B and C Toxoplasma gondii

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 detecting allergens in food and house dust measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like lupus erythematosus measuring toxins in contaminated food detecting illicit drugs, e.g.,
cocaine

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The technique is divided into

1- Competitive ELISA 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

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1) COMPETITIVE ELISA
The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal.

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2) SANDWICH ELISA
The ELISA plate is coated with Antibody to detect specific antigen

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 Prepare a surface to which a known quantity of capture antibody is bound. Block any non specific binding sites on the surface

Apply the antigen-containing sample to the plate.

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 Wash the plate, so that unbound antigen is removed. Apply enzyme linked primary antibodies as detection antibodies which also bind specifically to the antigen.

Wash the plate, so that the unbound antibodyenzyme conjugates are removed.
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 Apply a chemical which is converted by the enzyme into a coloured product. Measure the absorbency of the plate wells to determine the presence and quantity of antigen

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 ADVANTAGE 1. Quick because only one antibody and fewer steps are used. 2. Cross-reactivity of secondary antibody is eliminated.

DISADVANTAGE
1. Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. 2. Labeling primary antibodies for each specific ELISA system is time-consuming and expensive. 3. No flexibility in choice of primary antibody label from one experiment to another. 4. Minimal signal amplification

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3) INDIRECT ELISA
The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen
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 Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well. Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it
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 A substrate for this enzyme is then added. Often, this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen. The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength
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 ADVANTAGE 1. A wide variety of labeled secondary antibodies are available commercially. 2. Versatile. 3. Maximum immunoreactivity of the primary antibody is retained because it is not labeled. 4. Sensitivity is increased 5. Different visualization markers can be used with the same primary antibody.
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 DISADVANTAGE
1. Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal. 2. An extra incubation step is required in the procedure

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