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#20-OR
ANALYSIS OF MOUSE MONOCLONAL ANTIBODIES WITH SINGLE ANTIGEN BEADS.
Nadim El-Awar , Paul I. Trasaki , Jar-How Lee , Rui Pei , Chris Macaraeg and Karen Ng . Canoga Park CA, One Lambda Inc., 91303, Research .

Specificities determined in antisera by single antigen (SA) beads appear to be more accurate than that determined using cell panels and various statistical software programs. However, the SA beads tend to produce a long list of specificities present in the typical allo-antisera. We examined 170 mouse monoclonal antibodies (MOABS) with the SA beads to study the ‘extra’ reactivity of the beads. All MOABS were tested at several dilutions. Some MOABS directly labeled with either fluorescein isothiocyanate (FITC) or were biotinylated. Biotinylated antibodies were labeled with Allophycocyanin (APC) conjugated to Streptavidin (SA).
There were 100 MOABS that produced clear reactions with one to five SA beads. However, there were 70 others that produced several ‘extra’ reactions (table 1). Most of these extra reactions disappeared upon dilution (1:100 to1:20,000). They also disappeared upon absorption with the extra specificity. To determine whether the reactions were on the same epitope of different specificities, we used blocking or two-antibody/two-color experiments. Several sets of two antibodies that react with the same SA bead were shown to inhibit each other’s reactivity by blocking the binding epitope on the antigen. In some cases, both antibodies were shown to be present on the same SA bead indicating that the antibodies are recognizing two different epitops on the single antigen.
We conclude from studies of monoclonal antibody reactions to single antigen beads that the beads can detect antibody specificity accurately. Also, the 'extra' reactions produced by the beads are for the most part cross reactivities inherent in the HLA system, and not stray false positives.

Reactivity of SA beads with 170 monoclonal antibodies
# of specificities12345>5
# of MOABS40261651370