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#myeloma cells

3 public questions tagged with this topic.

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 2-9, leading to premature stop codon and nonsense mediated mRNA decay, enzymatic activity undetectable via radiometric assay. In complete medium containing folate, deficiency tolerated because de novo purine synthesis via amidophosphoribosyltransferase using glutamine nitrogen supplies sufficient IMP via ten-step pathway. Once switched to HAT where aminopterin blocks that pathway, reliance shifts entirely to salvage, now impossible in mutants. ATP depletion, accumulation of PRPP, activation of AMPK, inhibition of mTORC1, and caspase 9 mediated apoptosis eliminate myeloma. Clones SP2/0-Ag14, P3X63-Ag8.653 routinely used worldwide ensure background below 1 per million, maximizing hybrid recovery efficiency.

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

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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 generating constitutively active p95 truncated lacking ectodomain and inducing internalization clathrin-mediated requiring c-Cbl E3 ubiquitin ligase ubiquitination lysosomal degradation reducing surface density. Fc human IgG1 engages Fc gamma RIIIa CD16a NK cells triggering perforin granzyme ADCC and macrophage ADCP phagocytosis. Combination taxane docetaxel increased response 32 to 50 percent overall survival prolongation establishing HER2 testing companion diagnostic immunohistochemistry score 3+ FISH ratio >2.0 guiding patient selection achieving paradigm targeted immunotherapy solid tumors effective safe with cardiac monitoring due occasional LV dysfunction.

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

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Immunological Applications

Hybridoma cells survive in HAT medium because they:

Handling hybridoma cultures demands sensitive high-throughput detection secreted immunoglobulin microgram per mL among abundant serum proteins albumin bovine Ig. ELISA screening provides optimal format 96-well polystyrene plates coated purified antigen 1 ug/mL overnight 4 degrees passive hydrophobic adsorption van der Waals blocked 2 percent BSA 5 percent nonfat milk PBS preventing nonspecific binding plastic. Culture supernatant 50 uL added incubates 1h 37 degrees enabling specific binding Fab antigen interaction affinity association rate 10^5 per molar per second. After three washes PBS 0.05 percent Tween removing unbound proteins immune complex remains due high avidity. HRP conjugated goat anti-mouse IgG secondary binds Fc region captured mouse antibody. Substrate tetramethylbenzidine oxidized peroxidase H2O2 generates blue radical 650 nm converting yellow diimine after sulfuric acid stop absorbance 450 nm proportional titer. Purification Protein A Staphylococcus aureus or Protein G Streptococcus binds Fc CH2-CH3 interface neutral pH 7.4 affinity 10^-8 M eluting pure IgG pH 3.0 glycine followed dialysis concentration ultrafiltration yielding clinical-grade monoclonal reagent therapeutically diagnostically used screening high producers early.

Ref: Janeway Hybridoma Screening ELISA Technique Protocol; Alberts Protein A Purification Antibody Methods Chapter 8; Lodish Antibody Purification Immunotechniques Handbook Methods.

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