MONOCLONAL ANTIBODIES

An antibody produced by a single clone of cells (specifically, a single clone of hybridoma cells ) and therefore a single pure homogeneous type of antibody. The term “monoclonal” pertains to a single clone of cells, a single cell and the progeny of that cell.
Köhler and Milstein found a way to combine
• The unlimited growth potential of myeloma cells with
• The predetermined antibody specificity of normal immune spleen cells.
They did this by literally fusing myeloma cells with antibody-secreting cells from an immunized mouse. The technique is called somatic cell hybridization. The result is a hybridoma.

PROCEDURE

Mix the following spleen cell from a mouse that has been immunized with the desired antigen with myeloma cells.
Use an agent to facilitate fusion of adjacent plasma membrane like PEG. The success rate is so low that there must be a way to select for the rare successful fusions.
• Lost the ability to synthesize hypoxanthine-guanine-phosphoribosyltransferase(HGPRT).
This enzyme enables cells to synthesized purine using an extracellular source of hypoxanthine as a precursor. The absence of HGPRT is not a problem for the cell because cells have an altenate pathway that they can use to synthesize purines.

However, when cells are exposed to aminopterin (a folic acid analog), they are unable to use this other pathway and are now fully dependent on HGPRT for survival.
• Lost the ability to synthesized any antibody molecules of their own (so as not to produce a hybridoma producing two kinds of antibody molecules).

The first property is exploited by transferring the cell fusion mixture ta a culture medium called HAT medium because it contains:
• Hypoxanthine
• Aminopterine
• Pyrimidine thymidine
The logic:
 Unfused myeloma cells cannot grow because they lack HGPRT.
 Unfused normal spleen cells cannot grow indefinitely because of their limited life span.
 Hybridoma cells (produced by successful fusion) are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal.