A Quick Method for Identifying the Molecular Targets of Potentially Diagnostic New Monoclonal Antibodies Using Protein Array Technology.

During the course of raising monoclonal antibodies, reagents are often produced that are not directed against the immunising antigen. These may pass unnoticed unless a screening step based on immunostaining of human tissue is included. Many of these reagents are auto-antibodies, often directed against intracellular targets (e.g. nuclear components), and the process of hybridoma production serves to “rescue” these self-reactive B cell clones. Such unexpected antibodies of this sort may prove of interest if their distribution is analysed on normal tissues and within the spectrum of leukaemia/lymphoma samples showing specific patterns of staining. However, before they can be used diagnostically or therapeutically, their target molecule needs to be identified, and this can be technically demanding. We describe a novel approach that can be used to define the targets of new monoclonal antibodies to intracellular molecules. The technique involves screening against protein arrays comprising thousands of recombinant human proteins expressed in bacteria. In our initial experiments we showed that a control monoclonal antibody against nucleophosmin identified 22 clones encoding this ubiquitous protein in a human foetal brain cDNA library. Monoclonal antibody JC1 (first published in 1992) was known to recognise an unidentified proliferation-associated nuclear molecule, and screening on the recombinant protein array defined nuclear factor I/X (NFIX) as its probable target. A second antibody (JJ166), reactive with cell nuclei, was shown by the same technique to recognise the mitotic spindle-associated molecule NUMA1 (the gene for which is involved in rare chromosomal translocations). To confirm these putative specificities, the antibodies were shown to react with Cos-1 cells that had been transiently transfected with the corresponding clones from the array (after transfer into a mammalian vector pcDNA4HisMaxC, incorporating an Xpress tag). This new method represents an effective and quick strategy for defining the protein targets of new monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.