B7-H3单抗交联作用对Mo-DC体外生物学功能的影响

探讨B7-H3分子对人外周血单核细胞来源树突状细胞(Mo-DC)体外成熟和生物学功能的影响。采用常规方法从健康人外周血单核细胞诱导DC,在诱导过程中,加入B7-H3单抗21D4共培养,经流式细胞术检测Mo-DC上B7-H3分子和其他共刺激分子的表达,ELISA试剂盒检测培养上清中细胞因子IL-10和IFN-γ的分泌量,并采用3H-TdR掺入法测定T细胞的增殖。结果:B7-H3分子在未成熟和成熟Mo-DC上均有高水平表达,抗人B7-H3单抗21D4能上调Mo-DC表面CD80、CD86和CD83的表达,提高Mo-DC的共刺激能力,促进T细胞的体外增殖,并能显著促进T细胞分泌IL-10。由此表明,B7-H3单抗21D4交联作用可以促进Mo-DC体外成熟,上调其共刺激T细胞的能力。     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Allergic conversion of protective mucosal immunity against nasal bacteria in patients with chronic rhinosinusitis with nasal polyposis

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HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the -helix of DRβ chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DRβ chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR β₁ and β₂ domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(β 1^*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(β 1^*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR16-DR7M and TAL 13. 1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the β₂ domain, β180 and β181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptidebinding groove contribute to polymorphic antibody-binding epitopes.     

纤溶酶-α2抗纤溶酶复合物单克隆抗体的制备及鉴定

目的 :制备纤溶酶 α2 抗纤溶酶复合物 (PAP)的单克隆抗体(mAb)。方法 :以从血浆中纯化的PAP免疫BALB/c小鼠。按常规方法融合 ,以固相等分子浓度的纤溶酶原、α2 抗纤溶酶(α2 AP)及PAP为抗原 ,建立间接ELISA筛选杂交瘤细胞培养上清 ,并对杂交瘤细胞分泌的mAb的特异性和亲和力进行鉴定。结果 :共获得 2 4株可稳定分泌特异性mAb的杂交瘤细胞。其中 ,针对PAP分子中纤溶酶结构的mAb 16株 ,针对α2 AP结构的mAb 1株 ,针对新抗原 (PAP分子中新出现的不同于前体分子纤溶酶原及α2 AP的抗原决定簇 )结构的mAb 7株。这些腹水中抗PAPmAb的滴度为 2× 10 -4～ 1× 10 -8,其中 4株mAb的亲和常数为 5 .6 2× 10 -9～ 3.5 8× 10 -11mol/L之间。结论 :成功地制备针对PAP新抗原的具有高亲和力的mAb ,为建立不受其前体分子干扰的PAP特异性检测方法 ,研究纤溶系统的激活状态提供了工具。     

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。     

CD3AK细胞过继免疫治疗的实验研究

目的: 分析抗CD3分子的单克隆抗体 (mAb)yCD3的免疫学特性和生物学活性, 观察其激活的免疫活性细胞CD3AK体外及动物体内的抑瘤作用。方法: 用流式细胞术(FCM)测定yCD3的特异性, 以及CD3AK细胞的免疫表型和产生细胞因子的情况。用 3H -TdR法测定yCD3对淋巴细胞转化的作用; 乳酸脱氢酶法 (LDH)测定CD3AK细胞的体外细胞毒活性。建立荷瘤小鼠模型, 观察静脉注射CD3AK细胞后肿瘤生长的情况、转移灶数量和小鼠存活天数。结果: yCD3与T细胞呈特异性反应, 5μgyCD3可竞争抑制 70%的标准抗CD3抗体与细胞表面CD3分子的结合。yCD3刺激外周血淋巴细胞增殖的有效浓度为 8μg/L, 并与IL- 2、抗CD28抗体有协同作用。活化的CD3AK细胞中CD3 、CD8 和CD25 细胞增多; 产生IL- 2和IFN γ的CD3 细胞均有不同程度的增加, 在抗CD28抗体协同刺激下分别增加 3. 29和 2. 47倍。当效靶细胞比为 80∶1时, CD3AK细胞对体外肿瘤细胞杀伤的百分率为 57. 54%。分组观察荷瘤动物, CD3AK细胞治疗组的抑瘤率为 33. 17%, 对小鼠肿瘤肺转移的抑制率为39. 70%, 与LAK细胞联合治疗的疗效更显著。结论: yCD3可活化T细胞, 诱导的CD3AK细胞在体外及动物体内显示抑制肿瘤的作用, 在临床抗肿瘤过继性免疫治疗中具有重要意义。     

抗青霉素单克隆抗体的制备及初步应用

目的:制备抗青霉素的单克隆抗体(mAb)并建立双抗体夹心ELISA检测方法,对临床上引起青霉素过敏反应的过敏原青霉噻唑蛋白进行研究。方法:将半抗原青霉素和载体蛋白偶联后免疫BALB/c小鼠,应用杂交瘤技术建立稳定分泌抗青霉素mAb的杂交瘤细胞株。常规制备腹水,用辛酸-硫酸铵法纯化,并对纯化的mAb进行特异性鉴定。通过对不同抗体组合的分析和条件的优化,建立检测过敏原的双抗体夹心ELISA方法。结果:经细胞融合、筛选及克隆化,共获得9株稳定分泌抗青霉素mAb的杂交瘤细胞株,其中5株亲和力较高。建立了双抗体夹心ELISA相对定量检测方法,该方法灵敏度达到870 U/L,平均回收率为107.81%,批内变异系数平均为6.7%,批间变异系数平均为9.3%,可用于A群链球菌制剂中青霉噻唑蛋白的检测。结论:成功地制备了抗青霉素的mAb,并建立了相对定量检测青霉噻唑蛋白的双抗体夹心ELISA法。