尼帕病毒和亨得拉病毒核蛋白单克隆抗体的制备与鉴定

尼帕病毒（Nipahvirus，NiV）和亨得拉病毒（Hendravirus，HeV）是近年来出现的2种新的高致病性副粘病毒，在我国尚未发现。为防范2种病毒在我国的出现，本研究开展了前瞻性工作，成功研制了针对2种病毒核蛋白（N）的单克隆抗体，可用于病毒监测与诊断。首先利用大肠杆菌表达的2种病毒N蛋白免疫BALB／c小鼠，细胞融合后应用间接免疫荧光的方法对杂交瘤细胞克隆进行筛选，获得了5株N蛋白特异单抗。单抗腹水的抗体效价均超过2×10^5，培养上清抗体效价1：64～1：256。Western—blot和间接免疫荧光试验证明，5株单抗均特异针对N蛋白，其中1株（H4D11）只与HeVN蛋白反应，而不与NiVN蛋白反应，表明它具有鉴别2种病毒的能力。所研制的单抗对建立2种病毒的检测技术，用于动物监测，以防范2种新发传染病在我国流行具有重要意义。     

抗Asia1型口蹄疫病毒单克隆抗体的制备及生物学特性鉴定

用纯化的Asia1型口蹄疫病毒免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经间接ELISA和间接免疫荧光(IFA)筛选,有限稀释法克隆,获得了2株稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为3H6、5G3,其细胞培养上清效价分别为1:64和1:128,小鼠腹水效价分别为1×10~(-4)和8×10~(-3);ELISA和IFA结果显示,2株单抗仅与Asial型口蹄疫病毒反应,不与O型口蹄疫病毒反应,表明它们均为抗Asial型口蹄疫病毒的型特异性单克隆抗体。westem blot结果显示,2株单克隆抗体均不与全病毒抗原反应,表明它们所针对的抗原表位均为构象表位。相加ELISA试验表明,两株单抗识别不同的抗原表位。经硫氰酸盐洗脱法测定,3H6和5G3的相对亲和力指数分别为1.0 mol/L和1.5 mol/L。这2株单抗的获得为建立口蹄疫病毒检测方法提供了强有力的工具。     

The anti VEGF monoclonal antibody conjugated with nanometer particle of 5-FU

AIM:The characteristics of nanometer particles, which were prepared by the conjugation of anti-VEGF monoclonal antibodies and 5-fluorouracil-loaded polylactic acid nanometer particles (5-FU-NPs), were investigated for improving the anticancer activity of 5-FU. METHODS:The method of couple linkage of chemical bonds was used to prepare the 5-FU-NPs with VEGF antibody, then the appearance, distribution of particle diameter, releasing in vitro and the immunological activity were detected. RESULTS:The 5-FU-Ab-NPs appeared as regular globular, the average particle diameter was (202±23)nm. The 5-FU-Ab-NPs possessed the similar delayed release character of 5-FUs. More than 80% of the immmunogical activity were detected in conjugates-retained antibody by the immunological methods and electron microscopy. CONCLUSION:The 5-FU-Ab-NPs possess the similar delayed released activity to 5-FU-NPs and have double activity of immune targeting and delayed releasing, which may increase the local concentration of 5-FU.     

Analyses of monoclonal antibodies reacting with porcine wCD6: Results from the Second International Swine CD workshop

Among the 57 monoclonal antibodies analyzed within the T-cell group of the Second International Swine CD Workshop, one mAb fell within cluster T14a that included the CD6 standard a38b2 (No. 175). The new mAb MIL8 (No. 082) and a38b2 both precipitated from activated T-cells a 150 kDa monomeric protein. Staining patterns on the various cell types were similar. There was no inhibition of binding of either mAb to peripheral blood T-cells with the opposite mAb. The new mAb, MIL8, reacts with a separate epitope on porcine wCD6.     

Monoclonal antibodies putatively identifying porcine B cells

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.     

Reactivity of workshop monoclonal antibodies on paraformaldehyde-fixed porcine blood mononuclear cells

One hundred sixty-four monoclonal antibodies (mAbs) of the second international swine CD workshop were tested for their reactivity with porcine blood mononuclear cells before and after fixing the cells with varying concentrations of paraformaldehyde (PFA) (1, 5 and 10 g l⁻¹). A total of 38 (out of 134) positive reacting mAbs were significantly affected in their binding behavior on fixed cells. Modulation was seen as reduction in binding (staining intensity and/or % positive cells, n=18) or in elevated values (n=20). Modified mAb binding occurred after fixing cells with 5 to 10 g l⁻¹ PFA.     

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.     

大肠杆菌内毒素多克隆抗体的制备与纯化

为制备大肠杆菌内毒素多克隆抗体,应用大肠杆菌内毒素作为抗原免疫5周龄家兔,辛酸-硫酸铵法和葡聚糖凝胶层析法分离提纯抗血清,十二烷基磺酸钠-聚丙烯酰胺凝胶(SDS-PAGE)电泳方法鉴定多抗纯度,紫外分光光度计测定抗体蛋白含量,ELISA法检测抗血清效价与交叉反应;大肠杆菌内毒素抗体含量和效价分别为6.95mg/ml和1:12800;成功制备出抗大肠杆菌内毒素多克隆抗体,为大肠杆菌内毒素病诊断试剂盒的研制奠定基础。     

Identification of B-cell epitopes on the betanodavirus capsid protein

The pepscan procedure was used to identify betanodavirus B-cell epitopes recognized by neutralizing mouse monoclonal antibodies (MAbs) and serum samples obtained from sea bass, Dicentrarchus labrax, naturally infected with betanodavirus. Pepscan was performed with a panel of thirty-four 12-mer synthetic peptides that mimicked the entire betanodavirus capsid protein. Sea bass serum samples reacted strongly with three regions of the capsid protein comprising amino acid residues 1-32, 91-162 and 181-212. The latter region was also recognized by neutralizing MAbs and coincided with a region of high antigenic propensity identified by an antigen prediction algorithm. These data suggest that a region of the betanodavirus capsid protein spanning amino acid residues 181-212 may represent a neutralization domain that could potentially be used to inform the development of nodavirus vaccines and immunodiagnostic reagents.     

Role of outer membrane proteins in immunity against murine salmonellosis--1. Antibody response to crude outer membrane proteins of Salmonella typhimurium

The humoral immune response to crude outer membrane proteins (comp) of S. typhimurium in mice has been characterized. Maximal and quicker antibody response was observed when 50 micrograms of comp was injected intraperitoneally. The comp of smooth C5 strain of S. typhimurium evoked antibody response to both lipopolysaccharide (LPS) and proteins. Absorption of these sera with LPS coated erythrocytes eliminated the antibodies to LPS completely, while the antibody level to protein was left unaltered. The comp from rough mutant (lacking O-specific chain of LPS) of S. typhimurium elicited antibodies to proteins but not to LPS. These results indicate the concomitant production of antibodies to Salmonella outer membrane proteins also. The significance of such antibodies in protection and diagnosis has been discussed.