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.

Monoclonal Antibodies to Ascaris Neurons

C.D. Johnson, A.O.W. Stretton

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