Worm Breeder's Gazette 8(3): 74

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 Against the Levamisole Receptor

J.A. Lewis, J. Skimming, J. Prenger, T. Malisch, S. McLafferty

In a single fusion using the spleen of one mouse, we have raised 
three hybridomas producing IgG antibodies directed against the native 
levamisole receptor.  We took 1 to 3% pure preparations of the mutant 
receptor of lev-1(x61) prepared as described in the last issue of the 
C.  elegans Newsletter and we injected a mouse twice intraperitoneally 
with an estimated 5 to 10  g of receptor per injection with a four 
week interval in between.  The first injection was in Freund's 
complete adjuvant and the second was just straight receptor solution.  
Receptor solution, initially containing 0.5 M alpha-methylammoside and 
1% Triton X-100, was prepared for injection by dialysis to remove 
alpha-methylammoside and rapid passage over a small column of 
Amberlite XAD-4 to remove ~95% of the Triton X-100.  Triton X-100 
removal allowed us to concentrate the receptor solution ~30 fold with 
sodium carboxymethylcellulose and end up with a final solution for 
injection containing ~1 to 2% Triton X-100.
Supernatants were originally tested for anti-receptor activity by 
first rotating 5  l of goat anti-mouse IgG derivatized Sepharose beads 
(anti-heavy and light chains, 1 mg/ml coupled antibody, Cappel Labs) 
with 50  l of hybridoma supernatant in 0.25 ml total volume for ~6 
hours at room temperature (the beads also react with mouse IgM through 
light chains).  The coated beads were spun down, washed with 1 ml of 0.
5% Triton X-100 buffer, and resuspended with 0.2 ml of a crude 
detergent extract of x61 mutant receptor in 0.5% Triton X-100 (~10 
femtomole of receptor).  The beads were rotated overnight at 4 C with 
the receptor solution.  The following day, the beads were spun down 
and the supernatant solution was assayed for loss of receptor activity 
to the beads.  We have recently found the goat antibody on the beads 
is sufficiently in excess of the mouse antibody in the hybridoma 
supernatant that the assay can be done in one step, rotating hybridoma 
supernatant and receptor solution together and then assaying directly 
for loss of receptor binding activity to the beads.
Using this screening procedure, we found that four of 392 wells 
produced antibody that absorbed more than 90% of the receptor to the 
beads.  The supernatants from five other wells weakly absorbed 
receptor (~50 loss of activity).  On subculturing, three of the four 
strong positives and none of the three weak positives were not 
subcultured).  Two of the three good monoclonals cross-react with the 
wild-type receptor in the bead assay.  The third apparently does not.
Hybridoma supernatants were also screened histochemically.  
Nematodes were squashed between two slides, frozen and broken by 
cracking the slides apart (method of Albertson, Sulston and White with 
modifications by Culotti, Siddiqui and Francis).  The slides were then 
dipped in methanol and then acetone at -20 C and, with appropriate 
washes in between, incubated first in hybridoma supernatant and then 
in fluorescein isothiocynate-conjugated goat anti-mouse IgM and IgG.  
By this histochemical method, none of the very receptor-positive 
hybridomas showed any noteworthy specific staining.  Roughly half of 
the 392 wells were screened by this method and 21 wells were 
identified as containing antibody that recognized regionally localized 
antigens in the worm.  Partly through the inexperience of a technician,
only eight of these hybridomas survived subculturing.  Among the 
survivors are several clones with antibody staining an antigen 
distributed in regularly spaced patches along the hypodermal and/or 
muscle cell surface, much as desmosomes are known to be.  Another 
survivor appears to stain, amongst other things, a few spots behind 
the first bulb of the pharnyx, in front of the nerve ring.  One of the 
more interesting lost clones stained only the region of the sensory 
endings in the worm's nose.  The idea of the histochemical screen was 
to find what sort of other interesting proteins might be co-purifying 
with our receptor.
A more sensitive but less convenient histochemical staining method 
utilized biotinylated second anti-mouse antibodies (5  g/ml) followed 
by avidin-peroxidase (10  g/ml, Vector Labs) and subsequent reaction (
20 minutes at RT) with 0.5 mg/ml diaminobenzidine, 0.04% nickel 
chloride, 0.01% H2O2, freshly made up in 10 mM Tris-HCl, pH 7.4.  With 
this protocol, we see marvelously patchy staining along the length of 
the dorsal and ventral nerve cords.  No mutants have yet been tested 
to prove it's for real.