含有十二烷基磺酸钠的生物制剂的内毒素检测不能采用鲎试验

重组人ＩＬ－２在大肠杆菌表达时形成包含体，需经过变性和复性才具有生物学活性，在分离提取过程中常加一定量的ＳＤＳ，最低浓度每毫升不小于０．２５ｍｇ；低于此浓度会发生沉淀。鲎试验（ＬＡＬ试验）是一种灵敏度很高的检查生物制剂中致热源的常规方法。我们发现，当样本中ＳＤＳ的浓度＞０．０２５ｍｇ／ｍｌ时，可抑制尝试制的酶反应，使阳性结果转变为阴性。据此，我们建议，凡生物制剂中含有ＳＤＳ或其他酶抑制剂的产品，以尝试验来检查内毒素含量是没有意义的。     

Evidence that the major surface proteins of three Leishmania species are structurally related

Promastigotes of Leishmania major LRC-L137, L. donovani LEM 75, and L. tropica LRC-L32 were surface radioiodinated. The proteins of the parasites were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and labeled molecules were revealed by fluorography. A single major iodinated protein of Mr 63 000 (p63) was identified in each of the three species. These proteins were partially purified by phase separation in Triton X-114 solution, demonstrating that the p63 of each of the three species is the most abundant integral membrane protein in the promastigote. Peptide maps were obtained by partial proteolysis with N-chlorosuccinimide or Staphylococcus V8 protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The maps of L. major and L. donovani were identical, but only partially homologous to the maps of L. tropica p63. Finally, immunological crossreactivity among the three p63s was demonstrated with the serum of a mouse immunized with purified L. major p63, and the serum of a dog naturally infected with L. donovani. The data show that the major surface proteins found on promastigotes of three Old World Leishmania species are structurally related.     

Purification and characterization of a macromolecular weight cofactor for C3b-inactivator,C4bC3bINA-cofactor,of human plasma

The macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, was purified by an improved method and its protein nature was investigated. The purification procedures were composed of the removal of cold insoluble globulin with gelatin-Sepharose, polyethylene glycol fractionation, gel filtration on Bio-Gel A-15m, heparin-Sepharose chromatography, and the removal of IgM with anti-IgM-Sepharose. The molecular weight of C4bC3bINA-cofactor was estimated to be 450,000 by SDS-polyacrylamide gel electrophoresis.¹ The reduced cofactor gave a single band of 75,000 daltons upon SDS-polyacrylamide gel electrophoresis, suggesting that the cofactor is a disulfide-linked polymer (probably a hexamer) of similar or identical polypeptide chains, each with a molecular weight of 75,000. The cofactor is a glycoprotein and the isoelectric point of the cofactor was estimated to be 6.7. No N-terminal amino acid was detected by the dansylation technique, suggesting that the N-terminal of the cofactor would be blocked. Upon immunoelectrophoresis, the cofactor migrates as a slow β-globulin, and its mobility was shifted towards the anodal side when C4b was added to the cofactor. This result suggested that the cofactor was functionally the same as the C4-binding protein.     

Blotting methods applied to investigations of the mitogenic activity of phytohaemagglutinin (PHA)

PHA polypeptides were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filters which were then cut into identical discs and used to perform blastogenic stimulation on cultured human peripheral blood lymphocytes. This method offers the following advantages: the high resolution of SDS-polyacrylamide gel electrophoresis, the sensitive immunochemical identification of the polypeptides involved in the mitogenic responses of lymphocytes, the determination in the same experiment of the molecular weight, antigenicity and biological activities of the proteins.     

Immunoblotting and dot immunobinding. Emerging techniques in protein immunochemistry

Immunoblotting (also known as Western blot) and dot immunobinding (also known as dot blot) immunoassays, used extensively in immunochemical research, have great potential significance for diagnostic testing in the clinical laboratory. Immunoblotting has distinct versatility in immunochemical test applications. Immunoblotting combines the power of gel electrophoresis for resolving the electrophoretic variants of immunologically cross-reacting proteins with the ease and sensitivity of immunodetection on a solid-phase immunoassay. The potential for diagnostic applications includes: assay of antigens from pathologic serum samples, body fluids, and tissue; assay of patient serum samples for antibody against known antigen; and separation and assay of patient immunoglobulin. The usefulness of these applications is enhanced by the possibility of simultaneous use of nonantibody ligand, reversible protein staining, or, in the case of enzyme proteins, the use of substrate to detect activity. Dot immunobinding, in a similar fashion, permits assays of multiple specimens simultaneously on single strips of blotting media using sample sizes as small as 0.1 microL. Also, both immunoblotting and dot immunobinding techniques permit sequential probing of antigens with different antisera, with subsequent elution and recovery of specific antibody probes.     

Molecular characterization of the complement activating protein in the venom of the Indian cobra (Naja N. siamensis)

The cobra venom factor (CVF) from Naja n. siamensis was isolated from crude freeze-dried venom by a combination of ion-exchange chromatography and gel filtration (Bio-Rex 70, Sephadex and QAE-Sephadex). The yield of isolated product was 6–8 mg per g starting material. CVF appeared as a homogenous protein band in polyacrylamide gel electrophoresis with and without SDS present. In equilibrium sedimentation analysis the protein was homogenous with a molecular weight of 133,000. Under reducing conditions three protein bands appeared in polyacrylamide gel electrophoresis in SDS with molecular weights of 71,000, 48,000 and 28,000, corresponding to a total molecular weight of 147,000. All of the bands stained with basic fuchsin, suggesting the presence of carbohydrate. Gel filtration in 6 M quanidine hydrochloride on Sepharose 4B of reduced and alkylated material gave three peaks, each corresponding to one of the bands visible on polyacrylamide gel electrophoresis in SDS. The amino acid composition and N-terminal amino acid sequence of each peak were determined. Using a monospecific antiserum to CVF, molecules immunologically related to CVF were detected in several other elapid venoms.     

Purified HLA antigens to probe human alloantibody specificity

HLA class I antigens were purified in sufficient quantities to probe the antigen specificity of lymphocyte and platelet antibodies. Purification of HLA was achieved by affinity chromatography using the monoclonal W6/32 antibody that recognizes a nonpolymorphic determinant of HLA-A,B,C molecules. Pooled platelets from a large number of donors were used as source of antigen. A highly purified HLA preparation was obtained that produced a dose-dependent inhibition of the binding of the W6/32 antibody to lymphocytes as measured by flow cytometry. The binding of monoclonal antibodies to T-lymphocyte antigens or to HLA class II antigens was not affected. The purified HLA also inhibited the binding of lymphocyte alloantibodies from renal transplant patients, providing a direct and definitive way to probe HLA specificity. HLA also inhibited the binding of the same antibodies to platelets but it did not interfere with the binding of alloantibodies to the platelet-specific PLA1 antigen. This preparation, therefore, can conclusively probe the HLA specificity of both alloantibodies for clinical investigation purposes and monoclonal antibodies for screening purposes, and has the potential of becoming a reagent for routine use in clinical and research laboratories.     

Complement C3b fragment covalently linked to tetanus toxin increases lysosomal sodium dodecyl sulfate-stable HLA-DR dimer production

Processing and presentation of covalently linked C3b-tetanus toxin (TT) complexes, as compared to unlinked C3b + TT, lead to increased T cell proliferation. The aim of this study was to analyze the effect of coupling C3b to TT on the efficiency of TT peptide loading on HLA-DR1 molecules. In the Epstein-Barr virus-transformed B cell line HOM 2, we detected a significant increase of sodium dodecyl sulfate (SDS)-stable major histocompatibility complex (MHC) class II molecules after exposure to C3b-TT as compared to unlinked C3b and TT. The ratio of compact form/unbound form (C/U ratio) obtained with C3b-TT as antigen (Ag) is about twice that obtained with uncomplexed TT + C3b as Ag. Similar results were obtained using HLA-DR1-transfected fibroblasts that do not express C3b complement receptors, indicating that the SDS-stable HLA-DR1 increase did not result simply from C3b opsonization but rather from a direct effect of C3b-TT linkage on peptide generation. Exposure of HOM 2 cells to C3b-TT resulted in an increase in concentration of SDS-stable HLA-DR molecules in lysosomes but not in endosomes. Thus, C3b attachment to Ag induces a redistribution of peptide/MHC complex which results in a higher efficiency of Ag presentation by MHC class II molecules.     

Levels of type I and type III collagen in the bile duct of rats infected with Fasciola hepatica

Collagen types and their levels were compared between bile ducts from Fasciola infected rats and bile ducts from uninfected animals. Both collagen types I and III were shown to be increased in infected animals but, levels of type I increased less than type III. These results indicate that fascioliasis produces changes in the collagen composition of the bile duct that are similar to those produced in cirrhosis of the liver and other pathologic conditions including wound healing. Such observations suggest that a study of the chronology of collagen deposition in fascioliasis might provide information on the sequence of molecular events which result in bile duct hyperplasia.     

Monoclonal antibody (Tü48) defining alloantigenic class I determinants specific for HLA-Bw4 and HLA-Aw23,-Aw24 as well as -Aw32

A monoclonal antibody (Tii48) was prepared against human lymphocytes. Immunochemical analysis indicated that Tii48 binds to a fraction of HLA molecules. Tii48 was tested on 398 HLA-A,B,C/DR-typed normal blood donors in the microcytotoxicity assay and was found to detect a “supertypic” determinant on molecules bearing the HLA specificities -Bu4. Au23(9). -Au24(9), and -Au32. Among the HLA-Bu4-positive individuals, negative or weak reactivity of Tii48 was found with about one-fourth of normal donors heterozygous for the HLA antigen Bw44. This points to the possibility that the antigenic determinant detected by Tii48 is not expressed on all HLA-molecules carring the HLA-Bu4 specificity on the cell surface. Alternatively, weak or no expression of the Tii48 antigenic determinant on some HLA-Bu44 bearing molecules might be explained by the existence of molecular variants of glycoproteins with this HLA specificity. The concept of “supertypic” HLA specificities is discussed with regard to the expression of a monoclonal antibody-defined epitope on both certain HLA-A and B molecules.     

Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

Changes in the organization of bromegrass mosaic virus in response to cation binding as probed by changes in susceptibility to degradative enzymes

The swelling at alkaline pH and the sensitivity of bromegrass mosaic virus to degradative enzymes are less in the presence of moderate concentrations of KC1 than at low salt concentration. The virus can be efficiently stabilized above neutral pH by divalent metal cations, especially Ca2+, but this effect is antagonized by KC1. Polyamines also can stabilize the virus, but the salt concentrations for optimal protection of the viral RNA and protein moieties are then different, as judged by their susceptibility to degradative enzymes. These changes in the structural organization of the virus revealed by proteases and pancreatic ribonuclease as accessibility probes complement the data from neutron-scattering studies. On the basis of these studies, a tentative model is proposed to account for the polymorphism of BMV in various ionic environments.     

Trypanosoma rhodesiense: Chemical and immunological characterization of variant-specific surface coat glycoproteins

Soluble surface coat glycoproteins were purified by concanavalin A affinity chromatography from variant populations of Trypanosoma rhodesiense (Wellcome strain). Each variant yielded a glycoprotein consisting of a single polypeptide chain. The apparent molecular weights of the different glycoproteins ranged from 58 000 to 67 000. Charge heterogeneity analyses resolved from 1 to 3 closely spaced components with isoelectric points that were considerably different from variant to variant. Amino acid analyses revealed notable variations in amino acid compositions. Immunization of mice with purified glycoprotein protected them from homologous variant trypanosome infection. Hyperimmune sera raised to purified glycoproteins were obtained from rabbits and produced single precipitin lines in immunoelectrophoretic or immunodiffusion tests with homologous glycoproteins. No interaction could be detected in heterologous antiserum-glycoprotein combinations. Only variant homologous trypanosomes were agglutinated by antisera. Surface coat glycoproteins prepared from clone populations of variants were chemically and immunologically indistinguishable from the glycoproteins of original uncloned variants. The observed immunogenic specificity and chemical uniqueness of the glycoprotein preparations identify them as variant-specific surface coat antigens responsible for antigenic variability in T. rhodesiense.     

A novel molecule expressing HLA-DR antigenic determinants

Our previous studies suggested that the polymorphism of HLA-DR antigens (the human equivalent of murine I-E antigens) was a result of structural variation in the small (beta) subunit. In order to more accurately define this polymorphism we have expanded these studies to include HLA-DR antigens isolated with monoclonal cells derived from genotypically HLA-homozygous DRw2, DR2w5, and DRw7 lymphoblastoid cells derived from offspring of consanguineous relationships. Our results indicate the large (alpha) subunits of DRw2 and DRw7 antigens are nearly identical, while their beta subunits show many differences. In contrast, both the alpha and beta subunits of the DRw5 antigen differ strikingly from the respective subunits of the DRw2 and DRw7 antigens. The significance of the variability of the DRw5 alpha subunit is in question at this point. One intriguing possibility is that DRw5 actually represents the human counterpart of the mouse I-A subregion antigen and that the monoclonal antibody is reacting with a determinant which is shared by the human equivalents of murine I-A and I-E antigens.     

Cell-free and cellular synthesis of chromogranin A and B of bovine adrenal medulla

We have studied the cell-free and cellular synthesis of chromogranins A and B, two immunologically distinct protein families of adrenal chromaffin granules. Two cell-free systems (wheat germ and reticulocyte lysate) were used for translating messenger RNA isolated from bovine adrenal medulla. Two primary translation products could be immunoprecipitated in case of chromogranin A. In the presence of microsomes the two chromogranin A precursors (pre-chromogranins A) were converted into a single protein, apparently by the removal of different signal peptides. For chromogranin B only one precursor (pre-chromogranin B) was translated. In isolated chromaffin cells only one chromogranin A protein was synthesized which corresponded to the processed cell-free translation product. During prolonged incubation this protein became slightly larger and more acidic, probably due to glycosylation in the Golgi region. Chromogranin B is post-translationally converted to a significantly more acidic protein. It is concluded that proteolytic breakdown of newly synthesized chromogranin A and B in chromaffin granules is a slow process comparable to that of the enkephalin precursors. It is not yet known what function these chromogranins have and whether breakdown to smaller subunits is necessary for any function to evolve.     

Panhypogammaglobulinemia in systemic lupus erythematosus: in vitro demonstration of multiple cellular defects

Classically, systemic lupus erythematosus (SLE) is a disease of antibody overproduction, whereas the hallmark of acquired immune deficiency is antibody underproduction. Two patients are presented in whom panhypogammaglobulinemia developed during the course of SLE. In both patients, the levels of the major immunoglobulin (Ig) classes did not fall simultaneously. Anti-DNA antibodies were present, and exacerbations of SLE nephritis occurred in both cases 6 to 8 yr after Ig levels became subnormal. One patient still requires immunosuppressive therapy for renal disease; both patients are experiencing recurrent sinopulmonary bacterial infections. In the pokeweed mitogen-stimulated Ig biosynthesis assay, both patients showed abnormal Ig production due to defective function of three cell types: hyporesponsive B cells, excessive T suppression, and subnormal T help. The latter defect is rare in common variable hypogammaglobulinemia. One patient also showed extreme suppression of Ig production by phagocytic mononuclear cells. Thus, despite the similarity in the histories, the cellular function of these two patients was not identical in vitro.