多粘菌素B琼脂糖层析柱清除体液中内毒素

用多粘菌素B琼脂糖亲和层析法清除内毒素,结果表明:5ml多粘菌素B层析柱的总吸附内毒素能力为450 μg,用此法可完全清除体液或各种液体中的内毒素,对血清及腹水中的内毒素也有明显的吸附作用,而其他各种主要成分(除内毒素外)经过处理后无明显改变。去氧胆酸是一种强有力的去污剂,可使已饱和的柱子复活,复活率达85％左右。该方法有简便,可靠,吸附能力大,柱子的复活率高等优点。本方法的建立为内毒素血症的治疗展示了新的前景。     

凝集素受体在膀胱癌中的分布

目的：探讨膀胱癌中凝集素受体分布与其分化程度和浸润深度的关系。方法：应用生物素标记的花生凝集素（ＰＮＡ）、麦胚凝集素（ＷＧＡ）及刀豆凝集素（ＣｏｎＡ）等３种凝集素对５２例人体膀胱癌、１０例正常人体膀胱粘膜，进行亲合组织化学法研究。结果：发现正常膀胱粘膜ＰＮＡ、ＷＧＡ受体阴性，ＰＮＡ受体阳性率随膀胱癌病理分级的上升而递增，差异具有显著性意义（Ｐ＜０．０５）。ＰＮＡ、ＷＧＡ受体阳性率在浸润性肿瘤中明显高于浅表性肿瘤（Ｐ＜０．０５）。结论：提示ＰＮＡ、ＷＧＡ受体阳性率与膀胱癌分化程度和浸润深度有关。     

Response of V beta 8.1+ T cell clones to self Mls-1a: implications for the origin of autoreactive T cells.

Clonal deletion and anergy are two major mechanisms of self-tolerance. However, the molecular mechanisms underlying clonal deletion and anergy, as well as the threshold of TCR affinity/avidity required for these processes, are not known. Expression of the V beta 8.1 TCR correlates with the reactivity of the T cells to the minor lymphocyte stimulating locus-1a (Mls-1a) and T cells expressing this TCR are deleted in the thymus of Mls-1a mice. Similarly, in TCR V beta 8.1 transgenic mice, the number of CD4+CD8-T cells is reduced in Mls-1a mice. However, small numbers of CD4+CD8-T cells remain in the periphery of adult Mls-1a transgenic mice. We have generated T cell clones from TCR V beta 8.1 transgenic mice by stimulation of lymph node T cells with C57BL/6 alloantigens. Interestingly, CD4+CD8-V beta 8.1+ clones isolated from the transgenic mice of Mls-1a background responded to the self-antigen Mls-1a, to which they did not respond in primary assay. Reactive patterns of the clones were compared with clones derived from Mls-1b mice. Proliferation and cytokine production of the clones from Mls-1a mice to the self-antigen Mls-1a were generally reduced when compared with clones from Mls-1b mice. More importantly, T cell clones from Mls-1a mice required more Mls-1a antigen for their activation, and were more susceptible to the inhibitory effects of anti-CD4 antibody on the proliferative responses to Mls-1a than those from Mls-1b mice. These results suggest that the T cell receptor on clones derived from Mls-1a mice have functional but reduced affinity/avidity for self-antigen Mls-1a.     

兔乳酸脱氢酶C4的分离纯化及动力学研究

建立兔乳酸脱氢酶Ｃ４（ＬＤＨ－Ｃ４）分离纯化的新方法，研究该酶的动力学特性。采用Ｂｌｕｅ Ｄｅｘｔｅｒａｎ和Ｓｅｐｈａｒｏｓｅ ４Ｂ交联制成Ｂｌｕｅ Ｄｅｘｔｒａｎ－Ｓｅｐｈａｑｒｏｓｅ ４Ｂ染料配体亲和层析柱，结合ＱＡＥ－Ｓｅｐｈａｄｅｘ Ａ４５０离子交换层析分离兔ＬＤＨ－Ｃ４，以作图法测定该酶动力学数据。     

生物工程技术制备人源抗-HBs Fab 片段

目的：用生物工程技术制备人源性抗-HBsFab。方法：将从抗体文库中筛选出的人源抗-HBsFab基因克隆入pBAD/gⅢA载体，进而转化Tpo10大肠杆菌，对重组质粒菌发酵表达后，利用Ni-NTA-Agarose螯合层析柱纯化周质腔可溶性Fab蛋白。对所得包涵体依次变性，溶解，纯化后，利用透析进行复性，用Western blot检测Fab蛋白的特异性，Dot blot测定其生物学活性。结果：经Ni-NTA-Agarose柱纯化的周质腔可溶性Fab蛋白，有较好的生物学活性，并且总量达到80mg/L。对所获包涵体进行透析复性后，也可得到少量有活性的蛋白，但比例很小。结论；用pBAD/gⅢA-Top10表达系统表达人源抗-HBsFab片段，发酵培养后，经有效纯化可得到生物学活性较好的可溶性蛋白。为人源抗-HBsFab片段的大量制备提供了有效手段。     

Isolation of equine infectious anemia virus glycoproteins. Lectin affinity chromatography procedures for high avidity glycoproteins

Lectin affinity chromatography procedures were evaluated for the isolation of enveloped virus glycoproteins. The major glycoprotein of equine infectious anemia virus (E1AV) bound to concanavalin A (Con A)-Sepharose through interactions which could not be reversed by α-methylglucoside, but elution could be accomplished with buffers containing guanidine hydrochloride or sodium dodecyl sulfate. These denaturants, however, also released about one-half of the Con A protein from the Sepharose matrix. This degradation does not appear to have been recognized previously, as denaturants are frequently employed for the isolation of virus glycoproteins from Con A-Sepharose. In contrast, the virus glycoprotein bound equally well to Sepharose-bound Lens culinaris (lentil) lectin affinity columns and was effectively eluted with buffer containing 0.2 M α-methylglucoside. The lentil lectin-Sepharose procedure described is rapid, inexpensive and results in the efficient separation and recovery of EIAV glycoproteins. Thus, lentil lectin-Sepharose can provide a useful alternative to Con A-Sepharose for isolating other high avidity glycoproteins from viral envelopes or cell membranes.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

抗慢性B淋巴细胞白血病患者IgM单抗相对亲和力的分析

本文采用间接ELISA法检测了11株抗B-CLL患者IgM腹水和杂交瘤上清McAb的相对亲和力。结果提示,各株McAb结合抗原的相对亲和力不同。根据50％最大结合浓度分为两组。其中4株杂交瘤上清McAb与纯化腹水McAb测得结果基本吻合。实验结果为合理地选用这些单抗提供了重要依据。     

抗Ig融合蛋白Fc段单克隆抗体的制备、鉴定与应用研究

目的：制备并鉴定抗Ig融合蛋白Fc段的单克隆抗体(mAb)，建立用于检测Ig融合蛋白的夹心ELISA法和纯化Fc融合蛋白的亲和层析法。方法：以hBCMA—Ig融合蛋白为抗原免疫BALB／c小鼠，通过细胞融合制备抗Fc段mAb，用ELISA等方法鉴定mAb的Ig亚类、表位以及种属特异性，建立用于检测Ig融合蛋白的夹心ELISA法;Western blot检测mAb对变性Ig融合蛋白的反应性。将mAb与Sepharose4B交联，制备亲和层析柱，对LAIR1-Ig融合蛋白进行纯化。结果：获得7株稳定分泌抗Fc段mAb的杂交瘤(FMUFcl-FMUFc7)。利用FMUFc4作为包被mAb，FMUFc5作为酶标记mAb，成功地建立了检测Ig融合蛋白的ELISA法，敏感度达到2μg／L；在7株mAb中，FMUFc6可用于Ig融合蛋白的western blot检测。用FMUFc 6mAb制备的亲和层析柱，可有效地纯化LAIRl—Ig融合蛋白。结论：成功地制备了抗Ig融合蛋白Fc段的mAb，建立了可用于Ig融合蛋白检测和纯化的方法，为Ig融合蛋白的应用提供了有力手段。