Consistent and continual source of identical mab b

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consistent and continual source of identical MAb B cells do not reproduce in culture Hybrid  cells B  cell - genetic components for producing Ab Cell division function of  myeloma  cells (not producing Ab) –  enable growth in culture
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Fig. 9.3 HAT procedure for selecting hybrid spleen- myeloma ( hybridoma ) cells hypothxanthine-guanine phosphoribosyl-transferase polyethylene glycol
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The  HAT  Procedure Myeloma cells Defective for hypoxanthine-guanine phosphoribosyltransferase  ( HGPRT - ) After fusion with splenic cells medium containing  hypoxanthine aminopterin , and  thymidine   (HAT) hypoxanthine is a precursor for the biosynthesis of guanine and  adenine (purines) aminopterin  inhibits  dihydrofolate reductase ( both  purine and  pyrimidine productions) thymidine provided to overcome the block in pyrimidine production
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Fig. 9.4  Screening for mAb production
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Identification  of Specific Antibody- Producing Hybridoma culture medium containing secreted antibodies add to microtiter wells pre-coated with the target  antigen Add  2nd  Ab recognizing mouse Ab conjugated to an enzyme that converts a colorless  substances to a colored compound
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m Ab serial dilution  to establish cell lines from single cells  ( clones ) against same Ag determinant? maintain indefinitely in culture or store in liquid nitrogen for future use mAb or parts (Fab or Fv fragments) selected and produced in E. coli
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Table. 9.2  Targets for Diagnostic mAb
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Clinical Example - Multiple Myeloma Multiple myeloma, plasma cell myeloma A type  o f  c anc e r  o f   plas m a c e lls  -  im m une  s ys te m  c e lls   in bo ne  m arro w that pro duc e   antibo die s Diag no s is :  pro te in e le c tro pho re s is  o f the   blood  and  urine . + is  the  pre s e nc e  o f a  parapro te in  (m o no c lo nal  pro te in, o r M pro te in) band, with o r witho ut re duc tio n o f  the  o the r (no rm al) im m uno g lo bulins .  O ne  type  o f parapro te in is  the   Be nc e  Jo ne s  pro te in  a  urinary parapro te in c o m po s e d o f fre e  lig ht c hains . Parapro te in: an abno rm al  im m uno g lo bulin  pro duc e d by  the  tum o r c lo ne .  Multiple  m ye lo m a c an pro duc e  all c las s e s  o f  im m uno g lo bulin. Ig G  parapro te ins  are  m o s t c o m m o n.  Lig ht and o r he avy c hains  m ay be  s e c re te d in is o latio n:  - o r  -lig ht c hains  o r any o f the  five  type s  o f he avy  κ λ c hains  ( -,  -,  -,  - o r  μ -he avy c hains ). α γ δ ε
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Figure 9.5 GFP and reporter construct
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Adapted from Shaner et al., Nat. Biotechnol. 22: 1567–1572, 2004.
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  • Fall '08
  • staff
  • DNA, Trigraph, multiple myeloma, ro te, rid iza tio

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