1. Monoclonal vs. Polyclonal Antibodies

2. Review - B cell maturation
Antigen independent maturation of B cells
A light chain and a heavy chain IgG gene in the DNA in each of thousands of immature B cells undergo rearrangement.
The rearrangements in each cell are different from the rearrangements in any other cell.
The result is thousands of cells, each able to recognize a different antigenic determinant.
Cells migrate to secondary lymphoid organs, including spleen.
The next slide shows the antigen dependent maturation of one of these cells.

3. Rearrangement during antigen- independent maturation of two different B cells.

4. Antigen dependent maturation of B cells
4/17/2017
Antigen dependent maturation of B cells
A second B cell in which the Ig genes had undergone a different rearrangement could interact with a different antigenic determinant on the same antigen and likewise undergo clonal proliferation.

5. Antigen dependent maturation of B cells
Another view of antigen-dependent B cell maturation

6. Remember
Each different antigenic determinant on a protein stimulates expansion of a different clone of B cells
Each cell within a single clone of B cells secretes numerous copies of identical antibodies
Antibodies specific to different epitopes are secreted by different clones.
One individual animal may expand several clones in response to several epitopes on the same antigen.

7. Notice the different regions of the p53 molecule that have been identified as epitopes, each recognized by anti-p53 antibody from a different B cell clone.

8. Definitions: polyclonal vs. monoclonal antibody preparations
Polyclonal antibody preparation =
Preparation containing antibodies secreted by cells of numerous different plasma B cell clones
The IgG fraction from blood of any vertebrate is a polyclonal preparation.
Example: the blood of an animal immunized with p53 from another species will contain circulating antibodies against several epitopes of p53 as well as Abs against other antigens to which the animal has been exposed.
Representative cells of the different antibody-producing clones will be found in the animal’s spleen.

9. Definitions: polyclonal vs. monoclonal antibody preparations
Preparation containing antibodies secreted by cells of only one plasma B cell clone
The IgG fraction from blood of any vertebrate is not a convenient source of a monoclonal antibody preparation.
An individual B cell from the spleen of an animal, if separated from the other B cells, immortalized, and expanded as a clone, is a source of a monoclonal antibody preparation.

10. Remember . . .
If someone says, “It’s a polyclonal antibody,” they mean . . .
“It’s a polyclonal preparation of antibodies.”

11. General strategy for preparation of monoclonal antibodies
Immunize animal with antigen (e.g., p53).
Collect clonally expanded B cells from spleen.
These cells are mortal; i.e., they will die in culture.
Immortalize antibody-secreting B cells.
mortal antibody-secreting B cell + immortal myeloma cell + fusogen  immortal Ab-secreting hybridoma cell
cont’d 

12. General strategy for preparation of monoclonal antibodies (cont’d)
Separately expand each of the hybridoma cells to a permanent clonal cell line.
Collect Ab from the growth medium of a single line.
Test the Ab against the immunogen (e.g., p53).
If possible, identify its epitope specificity. 

13. Monoclonal Ab production
Inoculate with denatured p53
Monoclonal Ab production

14. Requirements for myeloma cells used in hybridoma production
Must not be secreting antibody of their own.
Must be unable to make active hypoxanthine phosphoribosyl transferase (HPRT-)
Pre-established HPRT- myeloma lines are available.
Because they are HPRT -, they are unable to survive in HAT medium.
In HAT medium they can’t sythesize DNA (see next slide).
HAT medium contains
Hypoxanthine, Aminopterin, Thymidine

15. HAT Selection 
Note that if the dihydrofolate pathway is blocked, the only option for DNA synthesis of dA,G, and TTP is to use the thymidine pathway and the hypoxanthine pathway. With no HPRT, the hypoxanthine pathway won’t work! Cells die!

16. So how are hybridoma cells made?
Dilute B cells from spleen to one B cell/well. Allow the cell to go through a few rounds of cell division.
Short-term expansion in culture is possible before the mortal cells die.
Allow cells of an antibody negative, HPRT- myeloma line to fuse with these clonal, normal B cells.
A fusogen such as polyethylene glycol is used to make the cells fuse.
Place the culture under selection.

17. What happens under selection?
Myeloma/B cell fusions have genes from
myeloma cells, so they will be immortal, and from
B cells, so they will
be HPRT+ and will survive in HAT,
and will secrete Ab.
Unfused myeloma cells will die in HAT.
Unfused B cells are mortal and will die after a few more divisions.
What’s left? Antibody secreting, immortal hybridoma cells!

18. To screen for epitope specificity of a monoclonal Ab against a protein for which the sequence is known:
Synthesize overlapping oligopeptides representing the immunogen protein (e.g., p53).
Coat different wells of a 96-well plate with different peptides.
Perform ELISA with your monoclonal Ab.
Identify peptide to which Ab is specific.

19. A given monoclonal antibody may or may not work well in a Western, depending in part on the epitope it recognizes. Using what you now know about polyclonal and monoclonal Ab preparations, see if you can find the reasoning behind each of the observations in the chart below.