#hybridoma technology

Immunological Applications

Myeloma cells used in hybridoma technology are selected to be:

Establishing stringent selection requires myeloma background auxotrophic for purine salvage, achieved by exposure to 8-azaguanine, purine analogue structurally similar to guanine. Salvage competent cells convert 8-azaguanine via HGPRT to 8-azaguanosine monophosphate incorporated into mRNA and rRNA causing miscoding, translational errors, and lethal proteotoxicity triggering unfolded protein response. Spontaneous HPRT1 mutants survive because they cannot metabolize analogue, accumulating no toxic nucleotides. Surviving subclones harbor frameshift, nonsense, or missense mutations in coding exons

Ref: Szybalski & Szybalska 1962 HGPRT negative mutants 8-azaguanine; ATCC SP2/0 Ag14 datasheet selection mechanism.

View Details

Immunological Applications

Hybridoma cells survive in HAT medium because they:

Survival in HAT environment demands functional purine and pyrimidine salvage enzymes inherited from normal B parent. B lymphocytes express HPRT1 locus on X chromosome encoding 24 kDa hypoxanthine-guanine phosphoribosyltransferase that catalyzes phosphoribosyl transfer from PRPP to hypoxanthine yielding IMP plus pyrophosphate and to guanine yielding GMP, bypassing de novo steps requiring folate. They also express cytosolic thymidine kinase 1 cell-cycle regulated peaking in S phase and mitochondrial kinase 2 constitutive, phosphorylating thymidine supplied in medium to TMP using ATP. Hybridoma c

Ref: Janeway Immunobiology salvage pathway HGPRT; Freshney Animal Cell Culture hybrids survive HAT via HGPRT TK from B cell.

View Details

Immunological Applications

Myeloma cells used in hybridoma technology are selected to be:

Trastuzumab humanized IgG1 kappa mAb 148 kDa targets HER2 proto-oncogene extracellular domain IV juxtamembrane receptor overexpressed due ERBB2 amplification 17q12 20 percent breast gastric cancers poor prognosis. HER2 no known ligand but dimerizes HER1 HER2 HER3 after EGF binding autophosphorylating intracellular tyrosines Tyr1221 Tyr1222 recruiting Grb2 Shc activating PI3K Akt survival phosphorylating Bad Forkhead preventing apoptosis plus RAS RAF MEK ERK proliferation cyclin D1. Trastuzumab binds KD 5 nM sterically hindering dimerization preventing proteolytic cleavage shedding ECD generati

Ref: NEJM Trastuzumab HER2 Mechanism Slamon 2001 Clinical; FDA Herceptin Package Insert MOA Description; Janeway ADCC Trastuzumab Immune Mechanism NK Chapter 14.

View Details

Basic Applied Biology

Hybridoma technology produces:

Hybridoma technology developed by Georges Köhler and César Milstein in 1975 and awarded Nobel 1984 created paradigm shift by immortalizing antibody-producing B lymphocytes through somatic cell fusion. Mice immunized with antigen develop germinal center reaction where B cells undergo VDJ recombination, somatic hypermutation and class switching to generate high affinity plasma cells. Splenic lymphocytes harvested shortly after boost are fused with HGPRT-deficient myeloma partner such as SP2/0-Ag14 or NS0 using polyethylene glycol 1500 that perturbs lipid bilayers promoting membrane fusion formin

Ref: Köhler & Milstein Nature 1975 Continuous cultures of fused cells; Abbas Cellular and Molecular Immunology 10th ed. monoclonal antibody generation.

View Details