Antigen Structure and Interactions with Antibody III

When does cross-reactivity happen?

When antibodies in a polyclonal antibody preparation raised against one antigen cross-react with a partially related antigen that bears one or more identical or similar epitopes

What was cross reactivity originally seen in?

ABO blood types

What do individuals possess in terms of ABO blood types and cross reactivity?

Cross reactive antibodies to missing blood type antigens due to intestinal microbe surface structures that closely resemble blood antigens

What happens when an antigen cross reacts with a self antigen?

It can cause autoimmunity

If you have blood type A, what antigens do you possess on RBCs? What are the serum antibodies? What structural component that makes it different from the others does it contain?

Antigens: A

Serum antibodies: Anti-B

Structural component: NAG

If you have blood type B, what antigens do you possess on RBCs? What are the serum antibodies? What structural component that makes it different from the others does it contain?

Antigens: B

Serum antibodies: Anti-A

Structural component: Extra galactose

If you have blood type AB, what antigens do you possess on RBCs? What are the serum antibodies?

Antigens: A and B

Serum antibodies: Neither

If you have blood type O, what antigens do you possess on RBCs? What are the serum antibodies?What structural component that makes it different from the others does it contain?

Antigens: Neither

Serum antibodies: Anti-A and Anti-B

Structural component: Neither

What do we have natural antibodies against even though we’ve never received a blood transfusion?

A, B, and O

Why do we make antibodies against A, B, and O antigens?

They look a lot like polysaccharides in your gut and your body makes antibodies against these gut microbe polysaccharide structures and look like the A and B antigen and cross react

What are precipitation reactions?

They are when you mix an antigen in an aqueous solution with specific antibodies in the correct ratio which results in the formation of a visible precipitate

What are the antibodies involved in a precipitation reaction called?

Precipitins

What is the precipitate?

It is a 3D lattice structure formed by the cross-linking of multivalent soluble antigen with divalent (IgG) or pentameric antibody molecules (IgM)

Are precipitates formed under conditions of antibody or antigen excess?

No you need the right ratio of both in solution

What can precipitation reactions be visualized in?

Semi-solid media such as an agarose gel

When would radial immunodiffusion be used?What did it used to be used for

If you wanted to see if you had an antibody against a certain antigen. History of infection in the blood

Describe how radial immunodiffusion would work

1. Would isolate serum and put it in agar

2. Take the antigen that is immunodominant and put it in agarose and let it diffuse out naturally

3. As it diffuses out, it will create a clear ring of precipitation when it reaches the right concentration

What is an agglutination reaction?

It is the cross-linking of particulate multivalent antigens such as cells by antibodies that are at least divalent leads to agglutination of the particles

What are the antibodies involved in an agglutination reaction called?

Agglutinins

What does antibody excess inhibit? What is this called?

Agglutination and is called the prozone effect

What must occur during agglutination reactions?

Sufficient links must be formed at the greatest possible distance to overcome mutual repulsion by particles with the same charge such as red blood cells

Is IgM or IgG a more effective agglutinin?

IgM

Describe the agglutination reaction but with sheep red blood cells

1. Add same number of sheep red blood cells to each well

2. Increase the concentration of the antibodies from the serum of the person who was vaccinated against this

3. If you have reactive antibodies then at some concentration there will be an agglutination reaction of the red blood cells

4. If there is too much antibody then it will inhibit the production of the complex

What does indirect ELISA detect? What about sandwich ELISA?

Indirect: Detects a specific antibody (good at telling you if you’ve been infected by a certain pathogen)

Sandwich: Detects a specific antigen (good at detecting things in your body that you don’t make antibodies towards)

Describe an indirect ELISA

1. Antigen is adsorbed to the wells of a plastic plate in a monomolecular layer

2. Excess antigen is removed by washing and an irrelevant protein is used to block the remaining free sites on the plastic

3. Excess blocking protein is removed by washing and the patients serum is added to the well (add source of the antibodies which is from the person you want to test if they have antibodies against the pathogen)

4. After another wash the enzyme conjugated anti-immunoglobulin (isotype-specific) is added (secondary antibody)

5. Unbound antibody is removed by washing and the colourless substrate is added

6. The color reaction generated by the enzyme is quantified by absorbance using an automated plate reader

Describe the sandwich ELISA

1. A capture antibody is adsorbed to the well before the antigen-containing preparation is added and everything else stays the same

What is the secondary antibody typically conjugated with?

Something that is able to fluoresce or generate color

What do the two antibodies used in ELISA have to be?

Two different antibodies that bind to that hormone but at different epitopes or else they will compete with one another

What is an example of indirect ELISA?

1. HIV test detects antibody to HIV antigens

2. COVID-19 and other infectious diseases

What is an example of sandwich ELISA?

Pregnancy test which detects pregnancy hormones. Good for detecting antigens when host does not make antibodies to them

What are ELISPOT assays?

They are a modification of ELISA that allows measurement of molecules secreted by individual T cells

Describe what happens in ELISPOT assays

1. Wells are coated with detection antibodies (anti-interferon gamma)

2. They are washed and blocked

3. The cell population being studied is then added and cultured for a period of time to allow secretion of the molecule of interest

4. Cells are then washed away and detection of the antibody is added followed by washing and substrate addition

Describe how ELISPOT analysis works for an antibody made against a cytokine

1. Coat a well with anti-interferon gamma

2. Add the T cells

3. Add secondary antibody linked to an enzyme

4. Add a substrate that the enzyme can cleave and then you have color formation

5. The substrate that is cleaved will bind to the bottom of the well

6. Each dot tells you that a specific T cell was activated by that specific antigen you put in there and made that particular cytokine

What can ELISPOT be used for?

Cancer

What is immunofluroresence? What is it used to assess?

An antigen-antibody complexes that can be visualized under UV light if fluroscent dyes are first coupled to the antibody molecules. Used to assess antigen distribution on cells or tissues

What are the 2 methods for immunofluorescence?

1. Indirect

2. Direct method

What is the direct method?

It is when the primary antibody is directly labelled (conjugated) with a fluorochrome. These fluorochromes are activated by certain wavelengths of light and then emit a different wavelength

What is the indirect method? Is indirect or direct better at amplifying the signal?

This is when you come back with a secondary antibody that sees the primary antibody. Indirect

Can some antibodies be labelled with a microbe proteins that have an affinity for primary antibodies instead of a secondary antibody? What is an example

Yes they can be and an example is protein A

Describe the steps for immunoflurscence

1. Antigen is on the cell

2. Primary antibody will bind which has a fluorochrome conjugated to it

3. Have a laser in the microscope that will activate the fluorochrome and it will emit light that can be detected

4. Can amplify this signal by binding a secondary antibody linked to a fluorochrome

5. They bind to the primary antibodies

6. Can use protein A instead to bind to the Fc portion of an antibody and can have it conjugated to a flurochrome

What is immunohistochemical staining?

It is when you add a color substrate and is a form of immunoflurscence

What is flow cytometry?

It is a sensitive method to detect specific cell populations using antibodies

Describe how flow cytometry works

1. Take a mixture of cells from the blood of a person

2. Mix them with antibodies from an antigen which is coupled to a fluorescent protein

3. Can do multiple antibodies coupled to different fluroscent proteins

4. Force them through a flow cytometer which will squeeze them through one at a time at a fast rate

5. Wavelengths of light are given off by the fluorochromes and are detected by a detector

Out of all the immunoassays which one is the most sensitive?

Flow cytometry

Are earlier immunoassays such as precipitation and agglutination less sensitive?

Yes