Monoclonal antibodies to the exon 18 encoded moiety of NCAM

Aim: Exon 18 expression of NCAM has been recognized as a biomarker for small cell lung cancer (SCLC). To use this finding for an improved diagnosis of SCLC and personalized treatment of patients, techniques to identify and quantitate E18, the exon 18 encoded protein moiety of NCAM, are needed. We developed three monoclonal antibodies for this purpose.Methods: The his-tagged E18 antigen was expressed in E. coli and, after purification, used to immunize mice. Hybridoma’s were isolated by standard procedures and tested for their reaction with E18.Results: Three monoclonal antibodies, MUM-1, MUM-4 and MUM-6 were obtained. They reacted with E18 in western blots, with SCLC cell line NCI-H82, but not with unrelated his-tagged proteins. Only permeabilized NCI-H82 cells stained with the antibodies, confirming the intracellular position of E18. Next an enzyme-linked immunosorbent assay was developed using the earlier isolated monoclonal antibody MUMi-21B2, coated on the surface of microtiter wells as capture antibody and biotinylated MUM-6 as second antibody. Using streptavidin conjugated to horse radish peroxidase a linear dose response curve to his-tagged E18 antigen was obtained between 0 and 5 µg/mL with a sensitivity of at least 0.5 µg/mL or 50 ng/well.Conclusion: Four monoclonal antibodies are available to be used in assays for the identification and quantification of SCLC biomarker E18. This will enable the development of liquid biopsies to follow the tumor load in patients

Development and validation of an algorithm to estimate the risk of severe complications of COVID-19: a retrospective cohort study in primary care in the Netherlands

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Temperature-Sensitive Mutants of Mouse Hepatitis Virus Strain A59: Isolation, Characterization and Neuropathogenic Properties.

Twenty 5-fluorouracil-induced temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 were isolated from 1284 virus clones. Mutants were preselected on the basis of their inability to induce syncytia in infected cells at the restrictive temperature (40 degrees) vs the permissive temperature (31 degrees). Of these mutants, only those with a relative plating efficiency 40 degrees/31 degrees of 3 x 10(-3) or smaller were kept. Virus yields at 40 degrees compared to 37 degrees and 31 degrees (leakiness) were determined. Most mutants (16) were RNA-, i.e., unable to synthesize virus-specific RNA at the restrictive temperature. The other four were RNA+. No qualitative differences were detected in the virus-specific RNAs in cells infected with RNA+ ts-mutants, both at 31 degrees and 40 degrees. Virus-specific proteins present in cells infected with ts-171 (RNA-) and the RNA+-mutants (ts-43, ts-201, ts-209, and ts-279) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates. No qualitative differences in the pattern of virus-specific cellular proteins were detected among the mutants except for an additional polypeptide o