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     

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.     

单克隆抗体γ3重链可变区cDNA的合成与克隆

用异硫氰酸胍法从分泌单克隆抗体的杂交瘤细胞中提取总RNA,经oligo(dT)-纤维素柱亲和层析获得poly(A)~ RNA后,用恒定区5′端第122—125号氨基酸密码的互补序列3′A-T-A-G-G-T-G-A-C-C 5′做为引物,进行逆转录酶反应,合成双链cDNA,大小为300bp左右,与重链可变区基因的长度相符。用dC:dG接尾的方法,将ds-cDNA插入pUC19质粒,转化E.coli HB101。分离出重组体之后,经菌落原位杂交,酶切重组质粒DNA及Southern印迹,证明插入片段是重链可变区基因。     

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。     

番木瓜环斑病毒单克隆抗体的制备及在病毒分型上的初步应用

本试验是用番木瓜环斑病毒(Papaya ringspot virus, PRV)提纯制剂免疫的BALB/c小白鼠脾细胞与Sp~2/o-Ag14骨髓瘤细胞融合,获得三个能稳定传代并分泌抗番木瓜环斑病毒的单克隆抗体的杂交瘤细胞系。其中23H1 McAb的效价较高,用ELISA检测,腹水抗体效价高达1:76800,能被PRV兔抗血清所阻断。这3个杂交瘤细胞系产生的单抗与TMV和CMV无血清交叉反应。它们可把PRV四个毒株初步区分为三个血清型。