Monoclonal vs. Polyclonal Antibodies

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.

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

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.

Antigen dependent maturation of B cells

Another view of antigen-dependent B cell maturation

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.

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.

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.

Definitions: polyclonal vs. monoclonal antibody preparations

Monoclonal antibody preparation = 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.

Remember . . .

If someone says, “It’s a polyclonal antibody,” they mean . . .

“It’s a polyclonal preparation of antibodies.”

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

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.

Monoclonal Ab production

Inoculate with denatured p53

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

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!

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.

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!

•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.

To screen for epitope specificity of a monoclonal Ab against a protein for which the sequence is known:

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.