Antibody-Antigen Interactions/Immunoassays

Question 1

What is antibody affinity?

Answer 1

expression of the sum of all the interactions between an Ab binding site and its homologous Ag, only precise in a monovalent Ag-Ab system with monoclonal Ab

Question 2

What is antibody avidity?

Answer 2

strength of binding of multivalent antiserum to multivalent Ag, (antiserum contains many Ab species which can bind to multiple epitopes on the Ag)

Question 3

Why does cross-reactivity for Ag exist in antiserum? (multiple Abs exist in antiserum for a specific Ag)

Answer 3

impurity in immunizing agent that may be better Ag than the target Ag, similarity in structures/epitopes of Ags

Question 4

What two ways are used to eliminate cross-reactivity?

Answer 4

absorption, affinity chromatography

Question 5

Describe how absorption is used to eliminate cross-reactivity.

Answer 5

immobilizing the protein/Ag that unwanted Ab is specific for and exposing antiserum to it, keep what washes off/doesn’t absorb

• eliminates activity that have been there (to bind to the proteins)
• remove contaminating Ab

Question 6

Describe how affinity chromatography is used to eliminate cross-reactivity.

Answer 6

• purify Ab you want
• immobilize protein specific for the target Ab and keep only the Ab that binds to it, wash the others away, acid release/detergent wash of Ab-Ag to release the Ab of interest
• purifies for certain specificity

Question 7

Why are monoclonal antibodies important?

Answer 7

binds only one epitope for specificity, clone can survive indefinitely, huge amount of Ab can be produced, less crossreactivity

Question 8

Describe the preparation of monoclonal Ab

Answer 8

1. Immunize animal
2. Isolate spleen cells from immunized animals (Bcells)
3. Fuse spleen cells to plasmacytoma tumor cells that are nonsecretors, but produce tons of Ab once get the gene from the B cell.
4. Select for only those cells that are hybrids of tumor cells and B cells.
5. Clone hybridomas so each cell grows up independently.
6. Select the individual clone with the specificity of interest.

Question 9

What are the negatives of monoclonal Ab?

Answer 9

some assays need higher affinity than monoclonals can provide, no avidity in response

Question 10

What are chimeric monoclonal Ab?

Answer 10

variable regions are mouse monoclonal, constant regions are human

Question 11

What are humanized monoclonal Ab?

Answer 11

only the points of contact with the Ag remain mouse

Question 12

What causes serum sickness?

Answer 12

hypersensitivity response caused by Ab against non-human Ab forming immune complexes in serum, secondary exposure can be deadly

Question 13

What does immunofluorescence allow the identification of?

Answer 13

specific cell type, cell structure, pathogen, enzyme activity based on antibody specificity or enzyme specificity