1. A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain
- Author
-
Boya Zhang, Anthony Berardi, Colin F. Greineder, Peter M. Tessier, Alec A. Desai, Henriette A. Remmer, and Ghasidit Pornnoppadol
- Subjects
Cell Lines ,Immunoglobulin Variable Region ,Myeloma ,Artificial Gene Amplification and Extension ,Polymerase Chain Reaction ,law.invention ,Hematologic Cancers and Related Disorders ,Mice ,Spectrum Analysis Techniques ,law ,Medicine and Health Sciences ,Cloning, Molecular ,Frameshift Mutation ,Signal Amplification ,Polymerase chain reaction ,Multidisciplinary ,Genes, Immunoglobulin ,Antibodies, Monoclonal ,Hematology ,Flow Cytometry ,Recombinant Proteins ,Oncology ,Spectrophotometry ,Recombinant DNA ,Medicine ,Engineering and Technology ,Biological Cultures ,Cytophotometry ,Antibody ,Immunoglobulin Heavy Chains ,Multiple Myeloma ,Research Article ,Cell Binding ,Cell Physiology ,DNA, Complementary ,medicine.drug_class ,Science ,CHO Cells ,Biology ,Research and Analysis Methods ,Immunoglobulin light chain ,Monoclonal antibody ,Cell Line ,Frameshift mutation ,Cricetulus ,Antigen ,Complementary DNA ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Myelomas and Lymphoproliferative Diseases ,Molecular Biology Techniques ,Molecular Biology ,Immunohistochemistry Techniques ,Cloning ,Hybridomas ,Tetraspanin 30 ,Cancers and Neoplasms ,Biology and Life Sciences ,Cell Biology ,Molecular biology ,Histochemistry and Cytochemistry Techniques ,HEK293 Cells ,Signal Processing ,Immunologic Techniques ,biology.protein ,Immunoglobulin Light Chains - Abstract
The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.
- Published
- 2021