Monoclonal antibodies to rabbit liver cathepsin B

Three stable hybridoma cell lines (AF8, BC11, CE2) have been produced that secrete antibodies specific for cathepsin B. These have been characterized by ELISA, SDS-PAGE immunostaining, immunoprecipitation and immunofluorescent staining. CE2 immunoprecipitated native cathepsin B with retention of enzymic activity, but failed to cross-react with the alkali-denatured enzyme. BC11 bound only to the denatured form of cathepsin B and AF8 cross-reacted with both native and denatured cathepsin B. However, unlike CE2-immunoprecipitated enzyme, activity could be detected only after dissociation of the antigen-AF8 antibody complex. No cross reaction was found with any lysosomal protein includihg the cysteine proteinases, catbepsins H and L.Abbreviations ELISA Enzyme-linked immunoadsorbent assay - EIP Enzyme immunoprecipitation - PAGE polyacrylamide gel electrophoresis - Ep-475 L-trans-epoxysuccinyl-leucylamido (-methyl) butane - Z benzyloxycarbonyl - NMec N-methylcoumarin - PEB phosphate-EDTA-Brij 35 - IAA iodoacetic acid - PBS phosphate-buffered saline - DMEM Dulbecco's Minimal Essential Medium - FITC fluorescein isothiocyanate     

Immunocytochemistry of magnocellular neurons of supraoptic and paraventricular nuclei of normal and Brattleboro rats with vasopressin anti-idiotype antibody

Summary A vasopressin anti-idiotype antibody was generated by immunization with purified IgG of a primary vasopressin antiserum. The anti-idiotype antibody immunostained neurons in the supraoptic and paraventricular nuclei of the hypothalamus of normal and Brattleboro rats. The distribution of immunostained perikarya in these hypothalamic nuclei together with the staining of fibers in median eminence and neural lobe was similar to that observed in normal rats with anti-vasopressin and suggests strongly that vasopressinergic neurons are being stained. Absorption studies with vasopressin and a vasopressin-binding receptor protein further indicate that a receptor associated with vasopressinergic neurons is recognized by the anti-idiotype antibody.Supported by NIH grants ES03239, NS18626 and NSF grant BNS-8310914. D.T.P. is the receipient of RCDA award NS00869     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.     

Monoclonal antibodies modify acetylcholine-induced ionic channel properties in cultured chick myoballs

Summary Monoclonal antibodies directed against the cholinergic binding site of the acetylcholine receptor were found to alter the ion channel properties in cultured chick myoballs. Time and dose dependent reduction in acetylcholine sensitivity was observed. Noise analysis experiments indicated a decrease in the mean single channel conductance and an increase in the mean single channel open time.     

Anisakiasis: preparation of a stable antigen for indirect fluorescent antibody test

By coupling Anisakis larval hemoglobin with cyanogen bromide-activated Sepharose, a stable and sensitive antigen for the indirect fluorescent antibody test for anisakiasis was prepared. Using this antigen, cross-reaction with other helminth infections was minimized and the test was greatly simplified.Furthermore, the results obtained indicate that this technique may be useful not only in the sero-diagnosis of anisakiasis but may also be applicable to the diagnosis of other helminthic diseases.     

A practical method for rescuing desired hybridomas during monoclonal antibody production

Summary We present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hydridoma cell line that is relatively rare in a population. This work was supported in part by grants EY 06225 and EY 06226 from the National Eye Institute of the National Institutes of Health, Bethesda, MD and by an unrestricted departmental award from Research to Prevent Blindness, Inc.     

Novel redistribution of myosin-containing filaments in cultured keratinocytes identified by a human monoclonal autoantibody

Summary A time-dependent redistribution of microfilaments was observed in cultured human keratinocytes using a human monoclonal autoantibody specific for myosin. Immunofluorescent staining revealed that 5 days after plating keratinocytes in either 0.1 mM or 2.0 mM Ca⁺⁺, myosin was distributed uniformly throughout the cytoplasm. At day 6, parallel arrays of myosin-containing microfilaments were prominent in the cell peripheries. At day 7 the microfilaments formed circumferential rings. The distribution of the microfilaments was disrupted by cytochalasin but not by colchicine, indicating that this novel distribution of myosin was not dependent on colchicine-sensitive vimentin intermediate filaments. The time-dependent redistribution of myosin was not influenced by cell population density, cell shape or cell cycle phase, except for mitotic cells in which myosin was distributed diffusely through the cytoplasm. If, as suggested by Kolega (9), microfilaments align parallel to the direction of applied tension, the redistribution of myosin-containing microfilaments in cultured keratinocytes may reflect the increased tension between cells resulting from increasing strength of cell-cell junctions over time. In sectioned human skin, myosin was localized in the peripheral cytoplasm of stratified epidermal cells. Tensions arising from the numerous desmosomal junctions between cellsin vivo could account for this distribution of myosin. Supported by grant NS-23537 (V. A. L.) from the National Institutes of Health, Bethesda, MD, and by the Mayo Foundation. C. L. W. is recipient of the Kermit E. Osserman and Blanche McClure Fellowship, 1987, National Myasthenia Gravis Foundation.     

Production of monoclonal antibodies against avian LHRH-I

Summary Production of antibodies against peptides or poorly antigenic proteins by conventional methods often requires either large quantities of the native immunogen or some chemical modification to increase their antigenicity. In this study an in vivo and in vitro immunization protocol has been used to generate monoclonal antibodies against the decapeptide luteinizing hormone-releasing hormone (LHRH). Two injections of 100 μg of avian LHRH-I into BALB/c mice were given 7 d apart. Dissociated splenocytes were collected under sterile conditions. They were incubated with 100 μg of the immunogen in 75-cm² tissue culture flasks in thymocyte-conditioned media. After 5 to 8 d exposure to the antigen, splenocytes were fused with SP2/O myeloma cells by polyethylene glycol. The cells were plated into 24 wells and then incubated in hypoxanthine aminopterin and thymidine selective media. After 14 d an initial screening was done by enzyme immunoassay. The positive wells (6/24) were expanded into 96-well plates and rescreened. Selected lines were cloned out 3 times by limiting dilution and the most positive expanded for ascites production. The antibody was affinity purified in a protein A column. The antibody cross-reacted with LHRH-I and II but preferentially to LHRH-I, as shown by competitive assay. A hypothalamic extract from a mature chick showed a higher response than preparations from whole brain explants of 1- to 3-d posthatched chicks, mature quail, and mature mouse. This work was funded by the Maryland Agricultural Experiment Station artical no. A4975, contribution no. 8019.     

恒河猴B病毒相关抗体检测方法的比较

以NT方法为基础比较了ELISA和FIA方法,共检测84份猴B病毒相关抗体的敏感性,结果ELISA和EIA阳性各50份(59.5％),NT阳性45份(53.6％)。三种方法相符者71份,符合率84.5％。ELISA和FIA均较NT敏感,而且快速,简便,经济,可应用于大批标本的检查。