We describe a rapid, simple HPLC method routinely used in our clinical laboratory for determining amiodarone and its metabolite desethylamiodarone. These compounds are released from serum proteins by pretreatment with an acidic solution and then extracted onto a C2 reversed-phase clean-up column. After elution from the extraction column, the compounds are separated and quantified by HPLC with a C18 reversed-phase column and spectrophotometric detection. The standard curves for the drug and metabolite are linear up to 20.0 mg/L, with a lower limit of detection of 0.16 mg/L. The CVs for intra-assay precision were 5.0% at 0.58 mg/L and 2.9% at 5.96 mg/L; for inter-assay precision, they were 9.6% at 0.52 mg/L and 6.1% at 2.09 mg/L. Lipemia, hemoglobin, and bilirubin up to 300 mg/L do not interfere with this assay. None of > 550 cardiac patients' samples tested contained a compound that interferes with this assay.
Autoantibody of an immortalized human lymphocyte cell line, Su-2E4, derived from peripheral lymphocytes of a patient with mixed connective tissue disease, showed specific binding of the 68K polypeptide of U1 small nuclear RNP (snRNP) and immunoprecipitation of U1 RNA. The reaction patterns of Su-2E4 and a murine monoclonal anti-(U1)snRNP line, 2.73, and results of a competition assay with the 2 antibodies suggest similar, but not necessarily identical, epitope recognition.Autoantibodies to nuclear and cytoplasmic components are frequently found in patients with systemic rheumatic diseases (1-3). The autoantigens are often nucleic acid-associated proteins, such as small nuclear RNP (snRNP) (4). Circulating autoantibodies to some of these antigens are characteristic of Presented by Jintao Chen in partial fulfillment of the requirements for a PhD degree,
TNF-alpha has been shown to be associated with macrophage cell membranes in such a way as to retain cytolytic activity despite fixation of the macrophage effector cells with paraformaldehyde. In this paper we report that, similar to cytotoxic macrophages, natural cytotoxic (NC) cells also use cell-associated TNF to lyse sensitive target cells. However, in contrast to fixed cytotoxic macrophages, NC cells do not retain cytolytic activity after fixation with paraformaldehyde. Additionally, the cytolytic activity of paraformaldehyde-fixed NC cells is not increased by incubation with LPS or by incubation with rTNF before fixation. Western blot analysis indicates that, unlike macrophages, NC cells use a smaller (17 kDa) constitutively active form of TNF. These results indicate that, although both macrophages and NC cells use effector cell-associated TNF to mediate lysis of sensitive targets, the way in which TNF is associated with these two types of effector cells must be different.
We tested by Western blot several thousand antibody-secreting human cell lines immortalized by hybridoma fusion or Epstein-Barr virus transformation of peripheral blood lymphocytes from patients with systemic lupus erythematosus or mixed connective tissue disease. The blots utilized total human Jurkat cell extract as the antigen. More than 20% of these established lines produced antibodies which recognized multiple bands on the blots, frequently 50 bands or more. Experiments were performed to rule out the possibility of the bands being the result of mixed cell populations or nonspecific antibody-antigen binding. Cloning of these cell lines failed to alter the Western blot patterns produced, indicating that the populations were monoclonal. Antibody eluted from a number of the different single blot bands showed the same ability to reproduce the multiple band pattern, thus revealing the presence of only one antibody. Western blots performed in the presence of specific and nonspecific inhibitors demonstrated the ability of the antibody to specifically recognize and bind to certain antigens. Binding did not result from indiscriminate sticking of IgM molecules to the nitrocellulose paper. The patterns of multiple antigen recognition were not due to antigen degradation. Additionally, enzyme linked immunosorbant assays revealed binding of the monoclonal antibodies to specific antigens, and the antibodies failed to recognize commonly crossreactive antigens such as DNA, histone, poly-L-lysine, glycophorin, and serum glycoproteins. The patterns of multiple antigen binding to a large number of polypeptides are therefore due to single antibodies, and the binding is specific.
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