免疫磁珠分离技术纯化和检测泰泽氏病原体的研究

目的建立泰泽氏病原体(Ty)纯化方法,获得纯的菌体供抗原研究,为ELISA诊断抗原的制备提供依据;并尝试建立Ty隐性感染血清学抗原检查方法。方法选择特异性抗体包被磁珠,从感染大鼠肝脏中富集和纯化Ty;用SDS-PAGE和Western blot技术考察纯化Ty的蛋白和抗原图谱;同时用免疫磁珠分离技术(IMS)直接检查隐性感染大鼠肠道上皮细胞内的Ty。结果用辛酸-硫酸铵纯化的Ty单克隆抗体M5以0·5μg/107beads以上浓度包被抗IgG抗体预结合的磁珠4h,可最大效率地富集Ty;分离反应进行1h,敏感性达到103菌体/mL;吖啶黄染色镜检法可以直接、快速地观察到结合于磁珠上的细菌;抗原分析表明,IMS较好地去除了肝组织和真核细胞成分,纯化的Ty RJ株具有3个免疫优势的抗原成分,相对分子质量(Mr)分别为160×103、116×103、55×103;此外,IMS法可直接从隐性感染大鼠盲肠上皮细胞中检测到少量寄生的Ty。结论用IMS技术可有效地富集和纯化Ty,并可以作为Ty隐性感染血清学抗原检查的候选方法。     

Kidney Transplantation in Patients with Antibodies against Donor HLA Class II

The immunologic risk associated with donor-specific antibodies (DSA) against Class II human leukocyte antigens (HLA) in kidney transplant (KTx) recipients is unclear. The aim of this study was to determine the outcome of KTx when DSA was detected only against HLA Class II. To isolate the impact of anti-Class II DSA, we retrospectively analyzed 12 KTx recipients who at baseline had a positive B-cell flow cytometric crossmatch (FXM) and a negative T-cell FXM. Using alloantibody specification analysis, 58.3% (7/12) had DSA against donor Class II and 41.7% had no demonstrable DSA. Biopsy-proven AMR occurred in 57% (4/7) in the Class II(+) group and 0% in the Class II(-) group (p > 0.05). Peritubular capillaries stained positive for C4d in 86% (6/7) of the Class II(+) patients and in 40% (2/5) of the Class II(-) patients (p > 0.05). One patient in the Class II(+) group lost their graft at 3 months to accelerated transplant glomerulopathy, while all other grafts were functioning 3-37 months posttransplant despite the persistence of anti-Class II DSA. We conclude that KTx recipients with clearly defined anti-Class II DSA are at risk for humoral rejection suggesting that desensitization and/or close posttransplant monitoring may be needed to prevent AMR.     

A new method for application of force to cells via ferric oxide beads

 We describe a new method that uses straightforward physics to apply force to substrate-attached cells. In this method, collagen-coated magnetic ferric oxide beads attach to the dorsal surface of cells via receptors of the integrin family, and a magnetic field gradient is applied to produce a force. In this paper we present a complete characterization of the method in a configuration that is easy to use, in which a permanent magnet provides a fairly uniform gradient over a relatively large area. This allows a fairly uniform average force that can be controlled in magnitude, direction, and duration to be applied to a large number of cells. We show how to determine the applied force per cell by measuring the force per unit volume of magnetic bead, the distribution of bead diameters, and the distribution of beads per cell. We also show how to calculate the force per unit volume of bead in a three-dimensional region near the permanent magnet on the basis of field measurements, and present results for three of the magnets. An upward force applied to fibroblasts by this method produces a measurable time-dependent increase in attachment of cytoskeletal actin filaments to the force application points, and an increase in actin cross-linking. This is accompanied by an actin-dependent retraction of the force-induced upward movement of the dorsal surface of the cells. Received: 27 February 1997 / Received after revision: 10 August 1997 / Accepted: 1 September 1997     

Chemiluminescence Immunoassay to Detect Anti-HTLV-I Antibodies

A fully automated chemiluminescence immunoassay was developed for the detection of antibodies to HTLV-I. We used partially purified viral antigens coated on small polystyrene beads together with acridinium ester-labeled anti-human immunoglobulin G mouse immunoglobulin G in this method. A good agreement was observed between our proposed method, the indirect immunofluorescence assay, the particle-agglutination test and the enzyme immunoassay. This new method, which is simple, sensitive, specific and rapid, should be useful for mass screening of anti-HTLV-I antibodies.     

Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of ₂-mircoglobulin (2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to 2M mRNA, to CEA mRNA/2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–10⁴ IS174T cells) of cDNA and 2M mRNA external standards prepared with tenfold serial dilutions (10²–10⁷ leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.     

Suitability of dyed latex bead agglutination test for immunodiagnosis of Karnal bunt (Tilletia indica) teliospores in a single seed of wheat

The immunodiagnosticum for this test was prepared extempore by mixing blue color dyed latex beads (1% suspension) with equal volume of diluted anti-teliospore serum. This test was considered to be better for the detection of solubilized teliosporic antigens over intact teliospores of Karnal bunt. The teliosporic antigens solubilized using sonication and detergent extraction were used for the standardization of the test by optimizing the dilution of latex bead suspension and determining the detection limits. For determining the sensitivity of test, antigen concentration kinetics analysis was performed by adding 15 µl of antibodies sensitized latex beads to 15 µl of different concentrations of solubilized antigens on glass slide. The detection limit of this test was 7.5 µg solubilized teliosporic antigens equivalent to 750 teliospores and suitable for single seed analysis. Small agglutinin formation with solubilized antigen of Puccinia recondita and T. barclayana interpreted on the basis of partial cross reactivity of immunodignosticum with these pathogens. However, no cross reactivity was found with teliosporic antigen(s) of spores of Curvularia lunata, Ustilaga tritici, Helminthosporium sativum, Ustilaginoidea vircns and Alternaria triticina.     

人外周血CD4~+树突状细胞的纯化及鉴定

目的　建立人外周血树突状细胞 (dendriticcell,DC)的分离方法 ,观察其形态学和免疫组织化学特点 ,为下一步细胞融合提供DC来源。方法　以免疫磁珠分选法从人外周血单个核细胞中分离CD4 + DC ,流式细胞仪检测所得细胞的纯度 ,光镜、电镜和激光共聚焦扫描显微镜观察其形态 ,SP免疫细胞化学方法检测DC的分子表达。结果　此纯化方法所得细胞纯度可达到 80 %以上 ,形态学观察可见纯化细胞具有典型的DC特征 ,该细胞能高表达HLA DR和S 10 0分子。结论　免疫磁法可获得较高纯度典型DC ,为进一步进行DC与肿瘤的融合实验及临床应用提供了可能     

The incorporation and release of glucose oxidase and interleukin 2 from a bead formed macroporous hydrophilic polymer matrix

Freeze-thaw photopolymerization at low temperature of a mixed solution of 2-hydroxyethyl methacrylate (HEMA), ethylene glycol dimethacrylate (EDM), and either glucose oxidase (GOx) or interleukin 2 (IL-2) around frozen ice crystals has been used to generate a bead-formed macroporous hydrophilic matrix with potential for immobilization and sustained release. The mean equilibrium acetate buffer content (EBC) of unloaded p-HEMA beads at room temperature and controlled humidity was ~72%. The incorporation of GOx into beads significantly increased the EBC to ~76%. The release of GOx was characterized by a short initial burst release which declined rapidly until by day 14 no further biologically active enzyme release could be detected. Bead size had no significant effect on the total mean cumulative release of GOx at room temperature. Since only ~ 4% of the original therapeutic load of GOx was released over 14 days a substantial proportion of biologically active enzyme had become associated with the hydrogel matrix surface generating a bead formed immobilised enzyme system. Total cumulative release profiles for IL-2 were almost linear and maintained for at least 16 days. In absolute terms, the proportion of the original theoretical incorporated load subsequently released over this period was low. However, such a low level sustained release of IL-2 may lend itself therapeutically to a reduction in unwanted non-specific systemic activity.     

Detection of IgG-class antibodies to measles,mumps, rubella,and varicella-zoster virus using a multiplex bead immunoassay

Serologic testing for measles, mumps, rubella, and varicella (MMRV) IgG is traditionally performed by immunofluorescence assay or enzyme immunoassay (EIA). Although sensitive and specific, these methods are labor intensive, time consuming, and require separate assays for each analyte. This study evaluated the performance of the MMRV IgG AtheNA Multi-Lyte® assay using nonclinically characterized serum specimens submitted to our laboratory for routine MMRV IgG testing. Mumps (n = 492) or rubella (n = 500) IgG were initially tested by enzyme-linked fluorescent antibody (ELFA), whereas measles (n = 494) or varicella (n = 497) were analyzed by EIA. Each sample was also tested by the AtheNA Multi-Lyte assay. Discordant results were retested by the predicate method and the multiplex assay, with further discrepancies being arbitrated by a third test. Compared to EIA/ELFA for MMRV IgG, the AtheNA assay demonstrated an overall agreement of 97.4%, 98.2%, 97.6%, and 100%, respectively. Use of this multiplex assay allows for the simultaneous detection of MMRV IgG, potentially decreasing cost, sample volume requirements, aliquot errors, and hands-on testing time.