Worm Breeder's Gazette 7(2): 54
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
With the ultimate goal of isolating neuron specific and/or synapse specific labels which can be used to facilitate the identification of neurons and synapses in light micrographs, we have begun to screen monoclonal antibodies prepared against Ascaris nervous tissue. Balb/c mice have been immunized with ventral and dorsal nerve cords ( prepared by pulling the ventral and dorsal hypodermal ridges off of animals which had been injected with collagenase) or with the anteriormost 2mm of the head (also after collagenase treatment to remove muscle cells). Injections were usually homogenates of nerve cords pooled from the anterior 40% of 10-12 adult females (ca. 200 cm of nerve cord) or 40-60 heads . Initial injections were followed by one or more booster injections, after which the mouse's spleen was removed, dissociated into cells, fused with myeloma strain NS-1 by exposure to polyethylene glycol, diluted and aliquoted into ten 96- well plates (ca. 1 fusion product/well). After two weeks growth, aliquots of the culture media were tested for binding to nerve cord or head homogenates by an ELISA using HRP conjugated goat-anti-mouseIgG as the second antibody. All ELISA positive (and some ELISA negative) cultures were examined anatomically for binding to neurons in a whole mount preparation of the anterior 20-30mm of the head (after removal of muscle with collagenase, fixation and extraction with either organic solvents or detergents). This preparation contains sensory neurons, nerve ring and surrounding ganglia and nerve cords including 14-21 commissures of identified motorneurons ) . In the anatomical screens, Rhodamine conjugated goat-anti-mouseIgG was used as the second antibody. Some cultures were analyzed further by ELISA against separated Ascaris tissues and against homogenates of C. elegans. ( J. Culotti and S. Siddiqui have screened some of the cultures against whole mounts of C.e.; they find ca. 20% cross reaction.) Using these procedures, we have recovered 20-30 hybridomas (from three mice) which reproducibly label neurons in the whole mount preparation, although none of these as yet demonstrate the desired specificity to single or small groups of neurons. Current efforts involve streamlining the immunization and screening procedures and examining neuron-binding antibodies for synapse specificity in frozen sections.