Bioinformatics for Vet. Part XXVBioinformatics for Vet. Part XXV

Sung Youn Lee, PhD. StudentSung Youn Lee, PhD. StudentVeterinary collage, Room 320Veterinary collage, Room 32002 450 3719, 016 293 605902 450 3719, 016 293 6059

leevet@paran.comleevet@paran.com

MCA: Monoclonal Ab

• MIX and Fusion• Screen• Production

Step 1.

• Mix – spleen cells from a mouse that has been immunize

d with the desired antigen with – myeloma cells.

• Use an agent to facilitate fusion of adjacent plasma membranes. (PEG-polyethylenglycol or Sendai virus). Even so, the success rate is so low that there must be a way to select for the rare successful fusions.

Step 2.

• So use myeloma cells that have: – lost the ability to synthesize hypoxanthine-guanine-

phosphoribosyltransferase (HGPRT). This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate 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 synthesize any antibody molecules of their own (so as not to produce a hybridoma producing two kinds of antibody molecules).

Step 3.

• The first property is exploited by transferring the cell fusion mixture to a culture medium - called HAT medium because it contains:

– hypoxanthine – aminopterin – the pyrimidine thymidine

Step 4.

• The logic: – Unfused myeloma cells cannot grow because they l

ack HGPRT. – Unfused normal spleen cells cannot grow indefinite

ly because of their limited life span. However, – Hybridoma cells (produced by successful fusions) a

re able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal.

Count

• 1mm * 1mm * 0.1mm• 0.1cm * 0.1cm * 0.01cm = 104/cm3

• 104/ml

8F2-This monoclonal reacts with antigenic group II viruses of the family Coronaviridae, thus reacting with bovine coronavirus and elk coronavirus nucleoproteins. However, this monoclonal does not recognize antigenic group I virus such as transmissible gastroenteritis virus (TGEV).

Thank you for your attention ~Thank you for your attention ~