对硫磷人工抗原合成及其鉴定

目的 制备对硫磷人工抗原和抗血清，为建立酶联免疫吸附法提供技术储备。方法 还原对硫磷硝基成为氨基，合成半抗原氨基对硫磷；重氮化偶联大分子载体牛血清白蛋白制备人工抗原；免疫新西兰白兔。进行多抗制备。结果 合成的半抗原核磁共振图谱鉴定结果为6：6．9～7(m，2H，CH2)6．4～6．5(m，2H，CH2)4．1～4．2(q，4H-CH2)3．4(s，2H，NH2)1．3～1．4(t，6H，CH3)；氨基对硫磷与牛血清白蛋白的结合比为35～36：1；抗血清经间接酶联免疫吸附法检测，效价达1：128000，双向免疫扩散实验测得抗血清与人工抗原形成沉淀；间接竞争酶联免疫吸附法测得抗血清对对硫磷最低抑制浓度为0．16ng／mL；经间接酶联免疫吸附法检测人工抗原与羊抗人血清、兔抗人血清、正常兔血清无交叉反应。结论 合成对硫磷半抗原及人工抗原纯度高、结合比高。具有较好的免疫原性。     

Clonality of cold agglutinins in patients with hemolytic anemia: an analysis by high-resolution two-dimensional gel electrophoresis.

High-resolution two-dimensional gel electrophoresis (2-DGE) was used to analyse plasma samples and partially purified cold agglutinins (CA) obtained from two selected patients. Both presented an acute hemolytic anemia with CA of high thermal amplitude, normal immunoglobulin levels, no detectable paraproteinemia, and no clinical evidence of a malignant B-cell disorder. The electrophoretograms of their plasma showed evident alternations of the "normal" protein profile, which were directly related to hemolysis (absence of the spots of haptoglobin and in one case of those of hemopexin), but no monoclonal gammopathy. The electrophoretograms of their purified CA revealed two clearly different spot patterns respectively corresponding to a monoclonal IgM and to polyclonal IgM. These results show that the clonality of CA associated with hemolytic anemia can be easily determined by 2-DGE. This technique may be very useful to discriminate chronic cold agglutinin disease in the early phase from "parainfectious" CA.     

Use of monoclonal antibodies against myosin light chains 1 to detect the corresponding antigen in blood of patients with myocardial infarction

Research Institute of Human Genetics, All-Union Medical Genetics Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 10, pp. 416–417, October, 1991.     

An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibodies to different parts of the gram-negative lipopolysaccharide core region

Bovine serum albumin was complexed with the core antigens of either Escherichia coli J5 LPS, Salmonella minnesota R595 LPS or E. coli lipid A. These core-BSA complexes were used for solid-phase coating in ELISAs for anti-core antibodies. Antibodies, binding to various parts of the core region were easily quantified in a single experimental set-up, which was hitherto not possible. The ELISA has only 3 incubation steps and is not costly as only moderate amounts of the core antigens (i.e., 1 microgram per test) were needed for coating. The sensitivity proved to be excellent and the complexes were biologically fully active (compared to native, smooth LPS), which make them suitable for the screening (after fusion) of monoclonal anti-core antibodies. Another possible application is the large-scale screening of blood-bank sera in order to find samples with a high anti-core antibody content.     

Elevation of CD8+ CD11b+ Leu-8− T cells is associated with the humoral immunodeficiency in myeloma patients

Recurrent bacterial infections due to humoral immunodeficiency are an important cause of death in myeloma patients. Recent data indicate that CD8⁺ T lymphocytes and a reduction of T helper type 1 cells with disease progression may be involved in the regulation of polyclonal immunoglobulin secretion. In mixed lymphocyte cultures derived from peripheral blood mononuclear cells (PBMC) of 24 myeloma patients with reduced immunoglobulin serum levels we investigated the association of CD4⁺ and CD8⁺ T cell subsets and immunoglobulin-secreting B cells (ISC) upon mitogenic stimulation with pokeweed mitogen (PWM) and concanavalin A (Con A). In supernatants of cultured PBMC of myeloma patients the spontaneous secretion of the type 1 cytokine interferon-gamma was reduced. After PWM stimulation reduced numbers of polyclonal ISC were found in 79% of patients, and monoclonal ISC were observed in 12% of patients. After Con A stimulation, again formation of polyclonal ISC was reduced, but monoclonal ISC were found in 41% of patients. Elevation of monoclonal and reduction of polyclonal ISC after stimulation with Con A were associated with an increase of CD8⁺ CD11b⁺ Leu-8⁻ T cells (P < 0.05). We conclude that the elevated numbers of CD8⁺ CD11b⁺ Leu-8⁻ T cells play a role in the stimulation of monoclonal and suppression of polyclonal immunoglobulin secretion in myeloma patients.     

Guidelines for the production of polypeptide specific antisera using small synthetic oligopeptides as immunogens

The production of antisera to specific proteins using as immunogens short, synthetic oligopeptides corresponding in sequence to regions of the proteins is analysed. Of 103 oligopeptides used for this purpose and reported in the literature before the end of 1983 all those corresponding to N or C terminal sequences produced antisera reacting with the complete protein. Of 69 oligopeptides corresponding to internal sequences only 71% were successfully used to prepare antisera. An analysis of these 69 oligopeptides showed that peptides of less than 10 amino acids were unlikely to produce useful antisera and that the more hydrophilic peptides were marginally more useful than those less hydrophilic.     

Preferential expansion of Ly-1 B and CD4 CD8 T cells in the polyclonal lymphocyte responses to murine T.cruzi infection

Acute murine infection with T.cruzi results in polyclonal lymphocyteresponses manifested by blast transformation of a large fractionof B, CD4+, and CD8+ cells. We describe here the finding ofsignificant increases in the splenic representation of minorpopulations, Ly-1+ B cells and CD4-CD8- T cells. These lymphocytepopulations might play an important role in the host response,as shown by T.cruzi infection of hosts that had been lethallyirradiated and reconstituted with autologous bone marrow. Underthese conditions, the splenic polyclonal PFC responses are nearlyabrogated, and not restored by the transfer of syngeneic peritonealcells which, however, reconstitute T15 idiotype production inthe same hosts. Control levels of PFC responses, however, arereconstituted by transfer of syngeneic splenic T cells. Sincebone marrow-reconstituted animals contain normal numbers ofCD4+ and CD8+ T cells which are actually activated by infection,these results suggest the participation of other T cell populationsin the host response to infection, as also suggested by themarked increases in T cell receptor and messages detectedin the spleen of infected animals. The implications of thesefindings in immunopathology of Chagas' disease are discussed.     

Penicillin-specific antibodies: Monoclonals versus polyclonals in ELISA and in an optical biosensor

Two penicillin-specific monoclonal antibodies mAb 19C9 and mAb 9H3 and the penicillin-specific polyclonal antibodies pAb K2 were evaluated for their use in a competitive ELISA and in the BIAcore™ optical biosensor. In the ELISA, an ampicillin-protein conjugate was used as a coating molecule. For the biosensor assay, ampicillin was immobilized on a CM5 chip. With both monoclonal antibodies and in both test systems, ampicillin, amoxicillin and benzylpenicillin were better recognized than oxacillin, cloxacillin and dicloxacillin. Because the reproducibility was better in the biosensor (CV = 1.6%) than in the ELISA (CV = 8.9%), the limit of detection for ampicillin in buffer solution using mAb 19C9 was lower in the biosensor (46 ng ml^-1) as compared to the ELISA (356 ng ml^-1). Ampicillin can thus be detected below the MRL (50 ng ml^-1) in the biosensor assay but not in the ELISA. Both the ELISA and biosensor assay using the polyclonal antibodies pAb K2 were more sensitive as compared to the assays with the monoclonals. The ELISA using pAb K2 allowed the detection of all tested penicillins below the MRL. In the biosensor assay, ampicillin was also detected below the MRL (IC50 = 10 ng ml^-1). In contrast to the binding of the monoclonals, no spontaneous dissociation was observed after injection of the polyclonal antibodies in the biosensor. Whereas the monoclonals were completely removed from the sensor surface using ampicillin in the buffer solution as regeneration solution, stronger conditions were necessary for the pAb binding.     

兔BK通道β亚基多克隆抗血清的制备

目的:用小鼠制备抗兔BK通道β亚基的抗血清。方法:采用RT-PCR扩增编码兔BK通道β亚基胞内段的基因。在大肠杆菌中表达GST-β亚基融合蛋白。以从PAGE凝胶上切下的融合蛋白免疫BALB/c小鼠制备多克隆抗血清。用ELISA和Westernblot鉴定抗血清的特异性。结果:用RT-PCR扩增出约300bp的兔BK通道基因。序列分析显示,扩增的序列与已发布的新西兰大白兔骨骼肌BK通道的序列完全一致。在E.coliDH5α成功地表达Mr约为37000GST-β亚基融合蛋白。ELISA和Westernblot检测证明,针对GST-β亚基融合蛋白的抗血清,不仅可特异性地识别GST-β,也可识别源于兔组织的β蛋白。抗血清的最高滴度达1∶128000。结论:克隆了编码兔BK通道β亚基胞内段的基因,并成功地获得可特异性识别新西兰大白兔BK通道β亚基的高滴度抗血清,为BK通道的进一步研究提供了物质基础。     

Some studies on the mechanism of action of antibodies to nerve growth factor.

The effects of antibodies to the nerve growth factor from mouse salivary gland were examined in vitro and in vivo. Treatment of explants of receptive ganglia with antibody and complement did not produce cell damage as judged by the ability of the tissue to respond to nerve growth factor. New-born mice experimentally depleted of or genetically deficient in key complement components were susceptible to the action of the antiserum.These results show that the effect of the antibody is independent of complement and are consistent with the view that it acts by neutralization of endogenous nerve growth factor.