Worm Breeder's Gazette 10(2): 71

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 with Specificity for the L1 Surface

Steven G. Donkin and Samuel M. Politz

In a screen for monoclonals with specificity for embryonic antigens, 
H. B.  and E. H.  fortuitously found two hybridoma lines that produce 
antibodies that bind specifically to the L1 cuticle in squashes of 
mixed stages.  S. D.  and S. P.  have partially characterized the 
binding to live worms and antigens in extracts of L1 cuticles.
Antibodies were elicited by in vitro immunization of a primary mouse 
splenocyte culture with a mixture of embryonic antigens.  Hybridomas 
were screened by indirect immunofluorescence of Bristol strain worms 
in squashes fixed on microscope slides (Albertson, Sulston, and 
Hedgecock, WBG Vol.  7, #1, p.  73).  Two hybridomas were found that 
stained L1 cuticles specifically.  Only small larvae with L1 type alae 
stained; other structures or stages never stained.  After cloning by 
limiting dilution and rescreening, these cell lines were grown in 
quantity and immunoglobulins isolated from culture supernatants by 0-
50% saturated ammonium sulfate precipitation.  Resuspended and 
dialyzed ammonium sulfate fractions are the standard antibody 
solutions used in the experiments described here.
Immunoglobulin class was determined by immunodiffusion (Ouchterlony).
Both antibodies (M37 and M38) precipitated specifically with sheep 
antimouse IgM serum.  In subsequent indirect antibody binding 
experiments, goat anti-mouse IgM secondary antibodies were used.
Binding of the antibodies to live L1's in immunofluorescence tests 
is strongly temperature-dependent.  When antibody incubations with 
live animals are done at room temperature, little binding is observed. 
If antibody incubations are done at 4 C or at 0 C, binding is quite 
uniform initially, but the antibody stain 'flakes off' in big flakes 
like paint flaking off a wall as the slide warms up.  After flaking 
has occurred, the same sample can be washed and restained, indicating 
that some antigen is still present on the surface.  We do not know 
whether the flaking involves dissociation of the antibody without 
antigen removal, or whether antigen comes off too.  However, heat-
killed L1's can also be restained after flaking, indicating that 
restaining does not require active replacement of the antigen by 
living worms.  Antibody binding to worms fixed in squashes exhibited 
no similar temperature dependence.
L1's were obtained in large numbers (up to 10+E6) for isolation of 
cuticle proteins.  Eggs obtained by Cloroxing were hatched overnight 
in M9.  L1's were harvested and cuticles and cuticle proteins were 
isolated after sonication by standard procedures.  Sonication 
supernatant, SDS extract, and SDS- ME extract were tested for antibody 
binding in a 'dot blot' assay.  Cuticle extracts were dotted onto 
nitrocellulose and incubated sequentially with M37 or M38, alkaline 
phosphatase- or horseradish peroxidase-conjugated goat anti-mouse IgM, 
and enzyme substrate.  Antigen was detected predominantly in the SDS 
extract of L1 cuticles.  Antigen was not detected in similar extracts 
of adult cuticles.  Antibody binding to the SDS extract showed a 
distinct optimum of pH 5-6 for the primary antibody incubation.
Cuticle extracts were separated on 12% Laemmli SDS-PAGE slab gels 
and electrotransferred to nitrocellulose.  Gels were incubated with 
antibody using conditions optimized in the dot blot.  As before, 
antibody binding was observed only in an SDS extract of L1 cuticles.  
The predominant antigenic species formed a 'ladder' of about ten sharp,
equally spaced bands centered around 20,000 daltons in MW.  Less 
reproducibly, higher molecular weight antigenic bands appear.  
Appearance of the lower molecular weight ladder is insensitive to 
predigestion of the sample with protease K.  Currently, our working 
hypothesis is that the antigen recognized by M37-M38 (both produce 
similar patterns in the Western blot experiment) is a stable, non-
protein moiety that may be attached to a labile cuticle surface 
protein molecule.