Agglutination

·         This is the interaction between Ab and a particular Ag, that results in physical clumping.

·         This clumping is agglutination

·         Agglutination rxns depend on X-linking of polyvalent antigens.

Clinical uses for MC Ab’s

Problems with monoclonal therapy

• Why are there so few monoclonals being used in human therapy a quarter century after their discovery?
• The main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti-mouse antibodies").
• These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys.
• (Monoclonal antibodies raised in humans would lessen the problem, but few people would want to be immunized in an attempt to make them and most of the attempts that have been made have been unsuccessful.)

Recap

Monoclonal Ab recognize one epitope

Polyclonal Ab recognize many epitopes (eg insulin)

Affinity = strength of interactions between single epitope on an Ag and a single binding site on an Ab

Avidity = Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants   and  antibodies

• These represent the strength of binding.
• Strength of binding will increase over time as these cells are selected for
• If there is binding at one site, this will increase the chances of binding at another site
• In other words: the greater the affinity/avidity the faster the B cell divides --- selection of better binders

Precipitation Expts

·         Antibodies and Ag form lattice structures that will develop into visible precipitate.

·         Formation of Ab-Ag lattice depends on

1.      Ab must be bivalent ( a precipitant will not form with fab fragments)

2.      Ag must be bivalent or polyvalent: that is it must have at least 2 copies of the same epitope or have different epitopes that will react with different Ab in a polyclonal antisera

Factors affecting precipitation:

·         Levels of Ab and Ag

·        

Immunodiffusion

Immune precipitates can also form on and agar matrix

·         A ring of precipitation will occur in the zone of equivalence

·         No visible ring will form in the zone of excess.

2 Types of Immunodiffusion:

1.      Radial

·         As Ag diffuses into agar, precipitant ring will form at optimal Ab-Ag concentrations

·         Comparing the area of the ring with that of a standard sample of known  Ag concentration, the test sample's Ag concentration can therefore be determined.

2. Double Immune Diffusion

·        

·         Both Ab and Ag diffuse radially from the wells

·         As equivalence is reached a visible ring of precipitation is formed

Advantages of these types of assays

• Cheap

• Reliable

• Repeatable

• Automated

Real life examples of these tests

    Home Pregnancy Test

·         It is a modification of an agglutination experiment

·         Called agglutination inhibition

·         Good because it is sensitive to small amounts of Ag

·         Based on competition

Radioimmune Assay

1000 times more sensitive than agglutination

Concept

Principle of RIA involves competitive binding of radiolabeled Ag and unlabeled Ag to a high affinity Ab

·         The increase in the concentration of the Ag in the unlabeled test sample, the more radiolabeled Ag will be displaced from the Ag binding sites

·         Therefore the concentration of the test sample Ag is a measure of the decrease in the amount of radiolabeled Ag bound to the Ab

Immunofluorescence

You are trying to determine that the mitochondrial protein cytochrome C is localized in the mitochondria

Use and Ab (red label) that is specific to cyto C

Ex

You are trying to determine if cytochrome c and caspase 9 are localized in the mitochondria

ELISA

• Enzyme linked immunosorbent assay
• Is similar in principle to RIA but depends on an enzyme rather than a radioactive label

Concept

• An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product.
• This substrate is called a chromogenic substrate.

Such enzymes include:

• Horseradish peroxidase
• β-galactosidase

Advantages of the Elisa are that they are safer and cheaper.

Sensitive, reliable, automated, easy to quantitate.

Common uses

• HIV testing (initial test)

• Drug testing (workplace, athletic events)

Q. What do you think the limitations of the ELISA would be regarding HIV presence/absence?

Reasons for labeling the secondary Ab

• Increased sensitivity
• Amplifies signal
• Can use secondary Ab that is species specific (anti-mouse)

Direct ELISA

• The direct ELISA uses the method of directly labeling the antibody itself.
• Microwell plates are coated with a sample containing the target antigen, and the binding of labeled antibody is quantitated by a colorimetric, chemiluminescent, or fluorescent end-point.
• Since the secondary antibody step is omitted, the direct ELISA is relatively quick, and avoids potential problems of cross-reactivity of the secondary antibody with components in the antigen sample.
• However, the direct ELISA requires the labeling of every antibody to be used, which can be a time-consuming and expensive proposition.
• In addition, certain antibodies may be unsuitable for direct labeling. Direct methods also lack the additional signal amplification that can be achieved with the use of a secondary antibody.

Indirect ELISA -  Ab detection

This is the method of choice to detect the presence of serum Ab against HIV.

• 96 well plate used
• Individuals infected with HIV will be producing serum Ab to epitopes on these viral proteins.
• The amount of colored reaction produced is measured via spectrophotometry
• Since the labeled secondary antibody is directed against all antibodies of a given species (e.g. anti-mouse), it can be used with a wide variety of primary antibodies (e.g. all mouse monoclonal antibodies). The use of secondary antibody also provides an additional step for signal amplification, increasing the overall sensitivity of the assay.

Sandwich Elisa - Ag detection

• This technique is used when trying to detect and measure the amount of Ag.
• Ex. drug testing.  Ag (drugs) in the urine could be detected in this fashion
• Again color reaction product is formed

Radiolabeled Ab  used before ELISA

Problems:

• Correct disposal of ¹²⁵I costly

Research Applications

If you were to ask the question.......

Does a patient express Class II MHC?....1st choice would be FACS

• Trap B cells (giving you a purified sample)
• Use specific Ab, one for the α chain (green) and one for the β chain(red)
• Use FACS machine to sort out chains

If patient is expressing Class II MHC then you would see equal amounts of green and red.

If you were to ask the question..............

Are the α and β chains the correct size? (28 and 33 kD)

You could use a radiolabeled membrane protein.  (¹²⁵I)

1. Take B cells and label membrane proteins (¹²⁵I)
2. Solubilize all membrane proteins (now have 1000's of labels)
3. You want to pull out Class II
4. Use an Ab (1^o) that is specific for Class II
5. You then want to pull the 1^o out
6. Use a protein (protein A) that is specific for the Fc region on the primary Ab
7. Beads trap Ab
8. Run on Western to visualize.

Beads act to trap the primary Ab

The purified sample to Ag-Ab complex can then be run on Western Blot to see the size of the fragments

• Transfer to Nylon membrane and then expose to X ray film
• Where ever there was a protein that was radiolabeled, there will be a band on the film

Western blotting

Western Blotting allows you to determine the molecular weight of the protein of interest.

Other uses for Westerns

Parp cleavage - detection of apoptosis

Enhanced Chemiluminescence

The ECL system is probably the most sensitive western detection system currently available.

Protein bands are very sharp, clean, and the exposure time is extremely short.

For many antibodies, the blots can be stripped and reprobed multiple times

Gives off light that can be exposed to film

Chemical used is Luminol

• Oxidation of the compound Luminol by H₂O₂ and the enzyme HRP produces light.

• Main advantages over chromogenic assays is the enhanced sensitivity.

Disadvantages

• Labor intensive

• Not setup for mass production

Applications

Tumor Cells in peripheral blood/tissues

If you  are looking for a tumor cell within a population of normal cells, you could make and Ab specific for the tumor cell in question

ex 66-cl-4 GFP cells

Localization assays

Immunoelectron Microscopy

• In this technique an electron dense label can be directly conjugated to the Fc region of a specific Ab for direct staining.

• Because the electron dense label absorbs electrons, it can be viewed with an EM as small black dots

Ex

Q. Do B cells in question express Class I and II?

Use an Ab conjugated to a small gold fragment that is specific to Class I

Use an Ab conjugated to a large gold fragment that is specific to Class II

Problems

Very expensive