Worm Breeder's Gazette 8(2): 17
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.
I am developing stage-specific cuticle antibodies to help identify genes controlling the stage transitions in C. elegans. I want to use these antibodies to select heterochronic mutants expressing stage- specific cuticle antigens at alternative times in development compared to wild-type. Rabbit anti-cuticle antibodies elicited by immunization with whole cuticles or solubilized cuticle collagens bind to the surface of live worms, making it fluoresce uniformly when fluorescein conjugated (FITC) second antibody is applied. Surface immunofluorescence was at first visualized in the compound microscope. Recently, I have developed an arrangement for viewing immunofluorescing worms on agarose plates under a low power dissecting microscope adapted for illumination by an FITC excitation source. This allows me to pick individual live fluorescing worms by the normal method. The basic strategy for using stage-specific antibodies to select heterochronic mutants is to bind antibody to worm populations under conditions of synchrony or genetic background where only mutants expressing stage-specific antigens heterochronically will fluoresce, enabling them to be recognized amongst thousands of nonfluorescent wild-type worms. Approaches involving adult-specific antibodies are in progress; characterization of adult-specific antibodies and surface antigens is described below. For antibody characterization, I devised a quantitative radioimmunoassay to measure surface binding of antibodies. Aliquots containing about 500 worms are incubated with antiserum, washed, incubated with [125I]-labeled Staph. aureus protein A, washed, and counted for total bound radioactivity. Binding is saturable by worms, antiserum, or protein A, indicating stoichiometric reaction. Pre-immune serum gives minimal binding. This assay has been used to study stage-specificity of antibodies and antigens. Rabbit anti-adult cuticle serum cross-reacts with the surface of live L4's. I have made the serum adult-specific by adsorption; after adsorption with saturating numbers of live L4's, serum no longer binds detectably to L4's, but binding to adults is reduced by only 50%. As expected, this adult-specific serum does not bind to adults of strain CB0912(lin-4), a retarded cell-lineage mutant that never develops adult cuticle. After a serum made to L4 cuticles was adsorbed with saturating numbers of adults, no binding to either L4's or adults was observed. These results indicate the existence of at least two distinct classes of surface antigens; i.e., those common to both L4 and adult cuticles, and those restricted to the adult cuticle (adult-specific surface antigens).