Monoclonal Antibody Technology

Question 1

What is are polyclonal antibodies?

Answer 1

Many B cells with different specificities for pathogens with different Ags leads to an increased chance the pathogen will be neutralized

Question 2

What are monoclonal antibodies?

Answer 2

A single type of antibody that targets a specific protein/antigen

Question 3

What is a hybridoma?

Answer 3

a B cell fused with a cancerous B cell (myeloma) that will grow indefinitely in culture

Question 4

How is a hybridoma formed?

Answer 4

Immunize mice with the Ag of interest.
Mice develop Ab.
Remove B cell of interest and fuse it with a myeloma to create the hybridoma

Question 5

How are hybridomas analyzed?

Answer 5

Flow cytometry and ELISA to identify hybridomas that are secreting Abs that bind Ags

Question 6

What are chimeric monoclonal antibodies?

Answer 6

Molecules made up of domains from different species. For example, the Fc region of a mouse mAb may be replaced with those of a human or (any other species) antibody. Entire murine variable region is maintained

Question 7

What are humanized monoclonal antibodies?

Answer 7

Only the hypervariable murine regions are maintained: CDR1, CDR2 and CDR3. Other sequences are swapped for human

Question 8

What are two categories of humanized monoclonal antibodies?

Answer 8

Knockout/transgenic mice and fully humanized

Question 9

How are KO/transgenic humanized monoclonal antibodies created?

Answer 9

In KO mice, the murine Ig locus is removed and replaced with human Ig genes. Mice can correctly rearrange and assemble human antibody genes to express them as normal

Question 10

How are phage display fully humanized monoclonal antibodies created?

Answer 10

No mice used. DNA library of heavy and light chain regions that are transferred into a bacteriophage where Fab fragments are are on the surface of the phage.
screening of the CDRs exhibiting the strongest antigen binding. Once the best CDRs are identified, they are then grafted onto a human antibody scaffold