Generating a phage display antibody library against an identified neuron.
The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones. As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron. Mice were immunized with isolated leech Retzius (R) neurons. The spleen poly A+ RNA was isolated and first-strand cDNA was prepared. The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants. Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened. Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron. The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells. However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity.
Merz
D C
DC
Department of Biology, McGill University, Montreal, Quebec, Canada.
Dunn
R J
RJ
Drapeau
P
P
D000818:Animals; D000911:Antibodies, Monoclonal; D000917:Antibody Formation; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005820:Genetic Testing; D007150:Immunohistochemistry; D007865:Leeches / Q000166:cytology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009474:Neurons / Q000276:immunology*
D000911:Antibodies, Monoclonal