Evaluation of a new commercial method for von Willebrand factor multimeric analysis.

INTRODUCTION: Evaluation of von Willebrand factor (VWF) multimeric distribution is useful for subclassification of von Willebrand disease (VWD). Multimer analysis has historically been a manual, labor-intensive laboratory-developed test. The first commercial method for multimeric analysis was recently developed that utilizes a single instrument for gel electrophoresis, staining, and densitometry. The current study was undertaken to evaluate the performance characteristics of the new commercial method. METHODS: Studies performed with the commercial method included evaluation of accuracy (method comparison), reference intervals (establishment of normal migration patterns in normal donor specimens), precision (multimer pattern reproducibility), and analytical sensitivity. RESULTS: In the method comparison studies, concordant interpretations were obtained in 19 of 24 comparisons, including normal and abnormal specimens. The 5 specimens with discordant interpretations all involved slight differences and none were considered clinically significant. Thirty-eight normal donor specimens demonstrated normal multimer patterns. Multimer pattern reproducibility was demonstrated in normal and abnormal controls tested on each gel. In the sensitivity studies, adequate visualization of multimers was determined to require VWF protein concentrations of approximately 5%-10% of normal. CONCLUSION: The commercial multimer method is a streamlined test that demonstrates comparable performance characteristics to our current laboratory-developed method and that provides the advantage of both electrophoresis gels and densitometry scans to aid interpretation.

Environmental induction and genetic control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes can alter their surface coat protein compositions at the molts between developmental stages or in response to environmental changes; such surface alterations may enable parasitic nematodes to evade host immune defenses during the course of infection. Surface antigen switching mechanisms are presently unknown. In a genetic study of surface antigen switching, we have used a monoclonal antibody, M37, that recognizes a surface antigen on the first larval stage of the free-living nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans can be induced to display the M37 antigen on a later larval stage by altering the growth conditions. Mutations that result in nonconditional display of this antigen on all four larval stages fall into two classes. One class defines the new gene srf-6 II. The other mutations are in previously identified dauer-constitutive genes involved in transducing environmental signals that modulate formation of the dauer larva, a developmentally arrested dispersal stage. Although surface antigen switching is affected by some of the genes that control dauer formation, these two process can be blocked separately by specific mutations or induced separately by environmental factors. Based on these results, the mechanisms of nematode surface antigen switching can now be investigated directly.

Altered expression of an L1-specific, O-linked cuticle surface glycoprotein in mutants of the nematode Caenorhabditis elegans.

Mouse mAb M38 was used in indirect immunofluorescence experiments to detect a stage-specific antigen on the surface of the first larval stage (L1) of the free-living nematode Caenorhabditis elegans, and to detect alterations in the apparent expression of this antigen in two distinct classes of C. elegans mutants. In previously described srf-2 and srf-3 mutants (Politz S. M., M. T. Philipp, M. Estevez, P.J. O'Brien, and K. J. Chin. 1990. Proc. Natl. Acad. Sci. USA. 87:2901-2905), the antigen is not detected on the surface of any stage. Conversely, in srf-(yj43) and other similar mutants, the antigen is expressed on the surface of the first through the fourth (L4) larval stages. To understand the molecular basis of these alterations, the antigen was characterized in gel immunoblotting experiments. After SDS-PAGE separation and transfer to nitrocellulose, M38 detected a protein antigen in extracts of wild-type L1 populations. The antigen was sensitive to digestion by Pronase and O-glycanase (endo-alpha-N-acetylgalactosaminidase), suggesting that it is an O-linked glycoprotein. This antigen was not detected in corresponding extracts of wild-type L4s or srf-2 or srf-3 L1s, but was detected in extracts of srf-(yj43) L4s. The antigen-defective phenotype of srf-3 was epistatic to the heterochronic mutant phenotype of srf-(yj43) in immunofluorescence tests of the srf-3 srf-(yj43) double mutant, suggesting that srf-(yj43) causes incorrect regulation of a pathway of antigen formation that requires wild-type srf-3 activity.