GENERATING A PHAGE DISPLAY ANTIBODY LIBRARY AGAINST AN IDENTIFIED NEURON

The generation of monoclonal antibodies by conventional hybridoma tech nology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from i solated clones. As an alternative approach, we have investigated the s uitability of phage display libraries for the production of recombinan t antibodies against an identified neuron. Mice were immunized with is olated leech Retzius (R) neurons. The spleen poly A(+) RNA was isolate d and first-strand cDNA was prepared. The variable regions of light- a nd heavy-chain IgG molecules were amplified by the polymerase chain re action (PCR) and separate libraries of each were constructed and combi ned in the pComb8 vector to yield a combinatorial library of similar t o 10(7) transformants. Single R neurons that were plated in culture bo und similar to 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonie s were isolated and used for immunocytochemistry with leech CNS gangli a: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific f or) the R neuron. The phage display library approach thus simplifies t he screening of large libraries with small numbers of (and even single ) cells. However, the combinatorial antibodies with binding activity m ust still be tested individually by immunocytochemistry, which is furt her limited by their apparently low affinity.