[18F]Tetrafluoroborate

An essential step in monoclonal antibody (mAb) development is the characterization and final identification of the specific target antigen and its epitope. Antibody validation is rather straightforward when immunization is carried out with peptide or purified protein, but is more difficult when whole cells or other complex antigens are used for the immunization. Determining antigen specificity of a mAb is further complicated, when reactivity of an antibody is not detected in Western blotting and/or immunoprecipitation assay. In addition to protein-based methods used for antibody characterization, a number of gene-based techniques, such as cDNA expression or short-interfering RNA (siRNA) knockdown have been applied for validation of antibodies with restricted reactivities. Earlier we have generated, characterized, but not identified the BF4 mAb that specifically stains viral biofilms on the surface of the Human T-lymphotropic Virus Type I (HTLV-1) infected T cells. In this study, using the recently developed genome-scale CRISPR-Cas9 knockout (GeCKO) library vectors, we have established the CEM T- and the Raji B cell lines with pooled libraries. After immunofluorescent staining of these cells, negative cell sorting, and guide-RNA (gRNA) sequencing, we have identified BF4 as an anti-CD82 mAb. A deep sequence analysis of GeCKO library transferred to the cells shows that the chance to succeed in the selection of antibody-negative cells and, therefore, to identify a mAb depends on the quality of cell library preparation. We believe that the described method is applicable for identification of many other hybridomas and represents a good alternative to the current protein- and gene-based methods used for mAb validation.

p59/HBI, a Heat shock protein (hsp90)-Binding-Immunophilin of approximately 59 kDa, was originally detected in the heterooligomeric, non-DNA binding form of numerous mammalian steroid hormone receptors. P59/HBI belongs to the FKBP family since it binds the immunosuppressants FK506 and Rapamycin. Using immobilized FK506, we purified [3H]Org 2058-progesterone receptor (PR)-hsp90-complexes from chick oviduct cytosol in a species migrating at 9S in density gradient. This led to suppose that a protein present in these complexes is a FK506 binding protein. Following incubation of this FK506-affinity purified 9S-PR with BF4, a specific anti-chick hsp90 monoclonal antibody, a shift of the [3H]Org 2058-PR complexes from 9S to 11S has been observed, indicating the presence of hsp90, hsp70 also is included in the 9S-PR complexes as demonstrated by Western blotting and density gradient experiments. Two lines of evidence support the notion that an immunophilin of 50 kDa also associates to heterooligomeric 9S-PR complexes. Firstly, a shift at 11S of the FK506 eluted 9S-PR occurs in sucrose gradient after incubation with 419, a new polyclonal antibody raised against a peptide corresponding to the Hinge I region [Callebaut I., Renoir J. M., Lebeau M. C., Massol N., Burny A., Baulieu E. E., Mornon J. P. (1992) Proc. Natl. Acad. Sci. USA 89, 6270-6274] of the rabbit p59/HBI (amino acids Phe135 to Gly149). Secondly, in Western blotting experiments, both 419 and another anti-FKBP antibody, raised against purified Streptomyces Crysomallus FKBP12 [Pahl A. and Keller U. (1992) J. Bacteriol., 3, 325-333], allow detection of the same 50 kDa protein in the affinity purified PR.(ABSTRACT TRUNCATED AT 250 WORDS)