1-34 Antibody-Antigen Interactions (Immunoassays)

Question 1

What is the structure of the hypervariable regions, and what is its function?

Answer 1

The heavy and light chain “V” regions contain 3 hypervariable regions each. Although the hypervariable regions are distant from one another in primary sequence, and are in fact on two separate chains, they lie relatively close together in the antigen binding site, forming a continuous surface capable of providing complementarity with a specific antigen. The variability in sequence composition provides the sequence of the very specific antigen binding site.

Question 2

Are antibodies homogenous or heterogenous?

Answer 2

Heterogenous (polyclonal).

Many B cells respond to any one antigen, and serum can contain antibodies for many different antigens. Antibodies have multiple specificities and affinities.

Question 3

What is an epitope?

Answer 3

The site on a complex antigen that is bound by a single antibody. An epitope may represent a few (5-10) amino acids, or carbohydrates, or modified amino acids.

Question 4

What are two techniques that can be used to eliminate cross-reactivity?

Answer 4

1. Adsorption: the use of the cross-reacting material to remove the activity that causes the cross-reaction.
   ex) For a serum that recognizes B cells as well as T cells, adding enough isolated B cells, mixing long enough, and centrifuging out the B cells will leave aqueous antibody that now only binds to T-cells.
2. Affinity chromatography: a powerful technique used to purify many immunologic reagents, including antibodies and antigens.
   ex) You want an antiserum that will recognize CD4-positive T-cells only. You have made a CD4 antiserum, but it’s very cross-reactive.

Obtain some pure CD4 protein and bind it to an insoluble support like agarose. Passing the antiserum over the CD4-agarose will bind the anti-CD4 antibody only; other cross-reacting antibodies will just flow through, leaving pure anti-CD4 antiserum. Adding a mild denaturant (like salt) will elute off the CD4-agarose, yielding purified anti-CD4.

Question 5

What is a monoclonal antibody?

Answer 5

A single clone of one B-cell produced by fusing that cell to a tumor cell, then isolating the clone with the antibody specificity of interest.

• One specificity
• Once produced, the clone can survive forever
• Huge amounts can be produced
• Less cross reactivity
• Lower affinity and little avidity (accumulated strength of multiple affinities)

Question 6

How are monoclonal antibodies prepared?

Answer 6

• Immunize an animal (usu. mouse)
• Isolate spleen cells from the immunized animal
• Fuse the spleen cells to plasmacytoma tumor cells
• Select for only those cells that are hybrids of tumor cells and B-cells
• Clone the hybridomas so that each single cell grows up independently
• Select the individual clone with the specificity that you are interested in

Question 7

What is serum sickness?

Answer 7

A type of hypersensitivity reaction that occurs in the immune system as a reaction to certain medications, injected proteins used to treat immune conditions, or antiserum.

Symptoms (fever, rash swollen lymph nodes) usually do not develop until 7-21 days after the first dose of antiserum or exposure to the medication. However, people may develop symptoms in 1-3 days if they have previously been exposed to the substance.

Question 8

What are the four types of monoclonal antibody?

Answer 8

1. Murine: mouse (-omab)
2. Chimeric: variable regions = mouse, constant regions = human (-ximab)
3. Humanized: mostly human, only the points of contact with antigen = mouse (-zumab)
4. Human: all human, made through molecular biology techniques (-umab)

Question 9

What is an Enzyme-Linked ImmunoSorbant Assay (ELISA)?

Answer 9

An assay as sensitive and specific as the radioimmunoassay, but not radioactive. Can be used to assay for antibody or antigen. The process is as follows:

• Stick antigen to the bottom of a well/tube
• Add antibody #1 and allow it to incubate
• Wash away unbound antibody #1
• Add antibody #2, which has an enzyme molecule covalently bound to it and which itself will bind to antibody #1. Allow it to incubate, then wash away unbound antibody #2
• Detect the amount of antibody #2: add a chemical reagent that turns color in the presence of the enzyme when bound to antibody #2

Question 10

What is immunofluorescence, and how can it be used with regard to immunology?

Answer 10

A technique that can be used on tissue or cells, potentially to identify antigen. Similar to ELISA, except that it uses a fluorescent marker instead of a color-changing dye. The process is as follows:

• React tissue/cells with antisera specific for a cell marker or pathogen
• Remove unbound antiserum via wash
• Add a second antibody specific for the first antibody, and allow it to bind
• View fluorescent second antibody through special microscope

Question 11

What is FACS?

Answer 11

Fluorescence Activated Cell Sorter is special kind of automated flow cytometry (which only scans) that scans and sorts large numbers of cells for immunofluorescence and size.

Question 12

What is a Western immunoblot?

Answer 12

A technique that determines the apparent molecular weight and concentration of an antigen. Similar to ELISA. The process is as follows:

• Separate mixtures of proteins (e.g., cell lysates or serum) via electrophoresis
• The separated proteins are bound to nitrocellulose paper
• React the nitrocellulose with antibody to the protein of interest, allowing the specific antiserum to bind
• Wash away the other, unbound, antibodies
• Detect the specific antibodies