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.
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.