大鼠抗小鼠组织相容性抗原抗体的制备与纯化

为进一步了解异种移植间免疫排斥反应的抗体特性，采用ＮＩＨ小鼠脾细胞免疫ＳＤ大鼠，制备大鼠抗小鼠组织相容性抗原抗血清，经辛酸沉淀、羟基磷灰石层析技术纯化抗体。结果：制备的大鼠抗小鼠组织相容性抗原抗血清具有较高效价（〉１：６４０），辛酸、羟基磷灰石二步法纯化的抗体具有纯度好、收获率高、能保留免疫原性、条件温和等特点。表明此法是制备各种抗组织相容性抗原抗体的有效方法。     

抗人膀胱癌抗独特型抗体的制备和鉴定

目的：制备抗人膀胱癌抗独特型抗体并进行体外实验鉴定。方法：提取膀胱癌抗原（Ag)。应用膀胱癌抗原通过杂交瘤技术制备抗人膀胱癌单克隆抗体（Ab1),Ab1经胃蛋白酶水解制备Ab1的F（ab)2片段，以F（ab)2片段免疫新西兰白兔，其血清经凝胶纯化后获抗人膀胱癌抗独特型抗体（Ab2)。再应用ELISA等方法进行鉴别。结果：Ab2与Ab1呈阳性反应，与BALB/C小鼠r球蛋白呈阴性反应；Ab2与Ag竞争结合Ab1；Ab2抑制Ab1与Ag的结合。结论：Ab1是针对膀胱移行细胞癌的单克隆抗体，Ab2是具有膀胱癌抗原内影像的抗独特型抗体。     

抗人肾癌G250单克隆抗体的制备及其功能鉴定

目的 大量制备、纯化抗人G250单克隆抗体(G250-MeAb),并鉴定其与G250合成多肽及细胞天然抗原的结合特异性.方法 将抗人G250杂交瘤细胞接种BALB/C小鼠腹腔,大量制备G250-McAb.采用正辛酸-硫酸铵沉淀法获得粗制抗体,然后经蛋白A亲和层析柱纯化抗体.采用荧光激活细胞分类术及免疫组织化学法鉴定G250-McAb与抗原结合特异性.结果 成功制备抗人G250-MeAb,腹水经纯化后,获得了高纯度抗人G250单克隆抗体,蛋白浓度达4.78 g/L.荧光激活细胞分类术及免疫组织化学结果 显示G250-McAb与G250表达阳性的Ketr-3细胞特异性结合,而不与G250表达阴性的ACHN细胞结合.结论 成功制备并纯化G250-McAb,为进一步开展G250-McAb介导的肾癌诊断、治疗奠定了基础.     

单克隆抗体OX7的制备及大鼠抗Thy1系膜增生性肾炎模型的建立

目的：制备单克隆抗体OX7（monoclonal antibody OX7，mAbOX7）7i建立大鼠抗Tbyl系膜增生性肾炎模型。方法：弗氏不完全佐剂预免疫Balb／c小鼠，腹腔注射OX7细胞，ProteinA亲和层析法纯化腹水，用SDS—PAGE鉴定纯化后抗体的纯度。将所得抗体尾静脉注射Wistar大鼠，PAS染色观察正常对照组注射前及注射后第3天、第7天大鼠肾脏病理组织学改变。结果：SDS—PAGE电泳显示，纯化的抗体有IgG的轻链和重链两条带，无其他杂带，抗体浓度为2．06mg／ml。PAS染色显示正常对照组肾小球结构正常，毛细血管袢开放良好；模型组第3天开始出现大量系膜细胞破坏，系膜基质溶解，肾小球毛细血管扩张；第7天系膜细胞重度增生。结论：通过将0X7细胞注入小鼠腹腔并用ProteinA亲和层析法纯化腹水，可成功制备高效价、高纯度及特异性强的mAbOX7，该抗体可成功建立抗Thyl系膜增生性肾炎动物模型。     

猪骨型羟基磷灰石的临床应用

本文介绍了猪骨型羟基磷灰石的制备及保存，并临床应用12例，经3月至～2年4月的随访，无全身排异反应，具有良好的组织相容性及支撑强度。     

纳米羟基磷灰石-40%二氧化锆生物陶瓷材料组织相容性评价

[目的]评估纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性.[方法]根据ISO10993-1标准,采用细胞毒性试验、急性毒性试验、溶血试验和体内植入(90 d)试验对纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性进行评估.[结果]纳米羟基磷灰石-二氧化锆生物陶瓷材料组织相容性的细胞毒性评分小于I级,细胞生长无明显抑制现象,无急性毒性反应,无溶血反应,体内植入符合植入材料生物学评价要求.[结论]纳米羟基磷灰石-二氧化锆生物陶瓷材料具有良好的组织相容性,作为骨组织工程中生物支架材料具有广阔临床应用前景.     

抗人精浆磷脂酶A2单克隆抗体细胞株的建立与鉴定

目的：制备并鉴定人精浆磷脂酶A2（PLA2）的单克隆抗体（McAb)，方法：通过PEG沉淀，Sephacryl S-300柱层析，DEAE－Sephadex A-25柱层析及羟基磷灰石（HA）柱层析分离纯化人精浆中的PLA2，并以SDS－PAGE鉴定其分子量及蛋白浓度，免疫BALB／C小鼠制备抗PLA2，McAb，通过ELISA技术，Western-Blot方法鉴定其敏感性及特异性。结果：从精浆中提纯的PLA2的相对分子质量（Mr)约为34900，纯化倍数约为245倍，制备的McAb 经鼠Ig亚类分析为IgM Kappa型，敏感性可达1：56-1:5^8，Western-Blot证明其特异结合的抗原即为PLA2蛋白酶，结论：Mr为34900的PLA2可能为一种新型PLA2，该PLA2的McAb 的制备将为研究人精浆中PLA2与男性生 殖能力的关系提供广阔的前景。     

狼疮性肾炎发病机制研究进展

系统性红斑狼疮是一种原因不明的多器官炎症的自身免疫病，其重要特征是Ｔ和Ｂ淋巴细胞对自身核抗原的耐受性丧失，出现多种抗核抗原成分的自身抗体，特别是ＩｇＧ 型抗－ ＤＮＡ 抗体，在狼疮性肾炎发病机制中有重要的地位。近年来，动物模型及人类系统性红斑狼疮研究表明，本病具有复杂的多基因遗传特性，主要组织相容性复合物和非组织相容性复合物基因决定了本病的易感性，尤其是自身抗体的产生。凋亡为自身抗体的产生提供了可能的抗原。阳离子抗－ ＤＮＡ抗体在介导狼疮性肾炎中发挥了重要的作用。     

抗人类癌抗原单克隆抗体的制备与肝组织中的鉴定

目的运用杂交瘤技术制备人类癌抗原(Human carcinoma antigen,HCA)的IgG单克隆抗体。方法用现有的HCA-IgM抗体HAE3分别从PC3、PCaT及LCaT总蛋白中分离纯化得到三组糖蛋白复合物组分作为抗原分别免疫Balb/c小鼠,获得鼠抗人HCA抗体.并对这些抗体进行ELISA检测效价、western-blot以及免疫组化等分析,筛选合适的抗体进行下一部分研究。结果共制备出62株高亲和力、高特异性并且针对不同抗原结合位点的鼠抗人HCA单克隆抗体。通过在肝组织上的鉴定,发现这些抗体无论效价、western-blot以及免疫组化都适合进行进一步研究。结论我们利用天然HCA抗原制备了高亲和力,高特异性并且针对不同抗原结合位点的单克隆抗体,为我们进一步研究其结构及功能提供了有力的特异的工具。     

珊瑚羟基磷灰石的细胞相容性实验研究

目的探讨珊瑚羟基磷灰石(CoralHydroxyapatite,CHAP)与骨髓基质细胞(BoneMarrowStro-macell,BMSc)的生物相容性,为用组织工程方法修复骨缺损提供依据。方法将骨髓基质细胞与珊瑚羟基磷灰石复合体外培养,进行形态学、细胞增殖、蛋白含量、碱性磷酸酶测定。结果骨髓基质细胞能粘附在珊瑚羟基磷灰石上,增殖、生长不受珊瑚羟基磷灰石的影响。结论珊瑚羟基磷灰石具有良好的细胞相容性,可作为骨组织工程的材料应用于骨缺损修复。     

Unimpaired first-set and second-set skin graft rejection in agammaglobulinemic mice

B cell and antibody-deprived B10.BR chronically suppressed by rabbit antimouse IgM serum rejected first-set allogeneic skin grafts as rapidly as control mice. We confirmed this finding using BALB/c mice born from B cell-deprived mothers and chronically treated with anti-IgM antibodies. Such mice had previously been shown to be agammaglobulinemic except for the suppressing monoclonal rat antimouse IgM antibody in their serum. These results indicate that in mice neither first nor accelerated second-set skin graft rejection reactions depend upon preexisting natural or specifically induced antibodies.     

Neutralization of immunosuppression by antibodies against variable as well as constant regions of monoclonal anti-Thy-1 xenoantibodies and their ability to be suppressed by initial T cell depletion

Timing, magnitude, and effect of the murine antibody response to rat pan-T-cell antibodies were studied in a bone marrow (C57BL/6-to-CBA mice) transplantation model. Prospective C57BL/6 marrow donor mice were sensitized against pan-T-cell (Thy-1, Thy-1.2, Lyt-1) monoclonal antibodies of various rat isotypes or against polyclonal rat antimouse-thymocytes (rat ATG) antibodies. Three days prior to transfer of spleen and bone marrow cells, the sensitized C57BL/6 donors received a dose of anti-Thy-1 mAb (RmT1) known to abolish graft-versus-host reactivity of unsensitized donors. The injected mAb provoked anti-antibodies reacting with RmT1. The anti-antibodies inhibited immunosuppression of the rat mAb RmT1 even if they bound only to nonidiotypic epitopes on RmT1. Avoiding cell-binding of the sensitizing rat anti-Thy-1.2 mAb by its injection into Thy-1.1 mice induced only low-titer and delayed anti-antibodies. This indicated the enhanced immunization potential of anti-Thy-1 when bound to cells. Finally, sensitization leading to the mouse antirat anti-antibodies and reversion of immunosuppression was prevented or reduced considerably by T cell depletion with a mouse IgG2a anti-Thy-1.2 mAb or high-dose cyclophosphamide or by rabbit ATG, provided it was initiated before starting the sensitizing injections of the rat antimouse T cell antibodies.     

Antimouse laminin antibodies in IgA nephropathy and various glomerular diseases.

IgG, IgA and IgM class antibodies to mouse laminin and human fibronectin in sera from patients with various glomerular diseases (50 cases of IgA nephropathy, 5 cases of minimal-change nephrotic syndrome; 6 cases of membranous nephropathy, 5 cases of systemic lupus erythematosus, 2 cases of Henoch-Sch?nlein purpura, 3 cases of poststreptococcal nephritis and 4 cases of preeclampsia) and from 30 normal controls were tested using a solid-phase enzyme-linked immunosorbent assay method. IgA antimouse laminin antibody titers in sera from IgA nephropathy patients were significantly higher (p less than 0.05) than in controls. There were no statistical differences in IgA antimouse laminin antibody titers between patients with other glomerular diseases and normal controls. IgM antimouse laminin antibody was significantly raised (p less than 0.01) in sera from patients with preeclampsia. The reaction of mouse laminin with the IgA nephropathy and preeclampsia sera on each of the IgA and IgM assay systems was inhibited by the antigen at up to 5 micrograms/ml. However, it was not inhibited by anti-C3d, anti-C1q, anti-J chain and antisecretory component sera or saccharides. The reaction of mouse laminin with an exceptionally high-titer IgA antimouse laminin antibody serum from a normal control on the IgA assay system was clearly inhibited by 1 mM of melibiose, which contains alpha-galactosyl residues. The same concentration of melibiose, however, did not inhibit the reaction of mouse laminin with IgA nephropathy sera on the same assay system. Treatment of mouse laminin with alpha-galactosidase did not alter any binding from IgA nephropathy sera but binding was lost from an exceptionally high-titer normal control serum. There were no correlations between serum IgA level and IgA antimouse laminin antibody titer in sera from IgA nephropathy patients. Immunoblot techniques revealed the presence of antibody in sera from IgA nephropathy patients reacting with both subunits A and B of laminin, somewhat stronger with laminin A. None of the sera tested contained antifibronectin antibodies. These results indicate that the IgA antimouse laminin antibody is a specific antibody in IgA nephropathy and might play a role in the pathogenesis of the nephritis since mouse laminin and human mesangial laminin present a common epitope.     

Partial purification of a major type of rat prostatic growth factor: characterization as an epidermal growth factor-related mitogen

The dorsolateral prostate of rats contains a mitogen that shares several properties with epidermal growth factor (EGF), which was designated as prostatic EGF-related mitogen (PEM). PEM was purified about 2,100-fold using molecular-sieve and ion-exchange chromatography. Final preparation stimulated DNA synthesis in BALB/c 3T3 cells at a concentration as low as 1.5 ng/ml and competed with 125I-EGF for binding to cell surface receptors. PEM had a molecular weight of about 14,000 and an isoelectric point of about 4.5, being heat- and acid-stable but inactivated by dithiothreitol. The primary cultured rat dorsolateral prostate epithelial cells required EGF for maximum growth. Partially purified PEM fully substituted for EGF in the primary culture system at a concentration as low as 90 ng/ml. However, the activity of PEM was hardly suppressed by antimouse EGF antiserum. These findings suggest that PEM is a member of the EGF family but has a higher molecular weight (high molecular weight EGF).     

Therapeutic potential of monoclonal antibodies to the leukocyte-common antigen. Synergy and interference in complement-mediated lysis

A series of rat monoclonal antibodies against the human leukocyte-common antigen were isolated and, by means of competitive binding assays with purified antigen, two distinct groups were defined that recognize different epitopes of the molecule. None of these antibodies were lytic with human complement, but when antibodies against each of these two epitopes were used in combination, synergistic lysis with human complement could be obtained. Synergistic lysis was only seen when each antibody of the pair was of the IgG2b subclass. IgG2a antibodies could not synergize, and in fact could interfere with lysis obtained by the pair of IgG2b antibodies. Although synergy has so far only been studied for rat monoclonal antibodies we also show that it is possible with mouse antibodies or with combinations of mouse and rat antibodies. The information about the principles of synergy and its interference should provide a rationale for using well-planned cocktails of monoclonal antibodies for therapy rather than a shotgun polyclonal antiserum.     

In vivo administration of lymphocyte-specific monoclonal antibodies in nonhuman primates. V. Evidence that humoral immune response to monoclonal antibodies and immunotoxin conjugates abrogates their cytotoxic activity

Monoclonal antibodies, either alone or conjugated to toxins, hold promise as important therapeutic agents. However, the immune response to these foreign protein agents may markedly limit their therapeutic utility in vivo. We have administered both an interleukin-2 receptor-specific monoclonal antibody (anti-IL-2R) and a CD2-specific monoclonal antibody linked to the ribosome-inactivating protein gelonin to macaque monkeys. The monkeys developed high-titer antibody responses to mouse Ig and, when immunotoxin was administered, to the toxin gelonin. Their antimouse Ig antibody responses were broadly reactive with mouse Ig of differing idiotypes and isotypes. Furthermore, sera from these monkeys blocked the in vitro cytotoxic effect of anti-IL-2R or immunotoxin. This blocking was mediated by both the antimouse Ig and the antigelonin antibodies. Serum from a monkey infused with one CD2-specific monoclonal antibody blocked the in vitro cytotoxicity of two other isotypically different CD2-specific monoclonal antibody conjugates. In addition, this serum blocked the in vitro cytotoxicity of a gelonin-monoclonal antibody conjugate of an unrelated specificity. These data indicate that the immune response to some monoclonal antibodies and toxins might preclude the later use of this class of substances in an individual. Therefore, strategies for the parental therapeutic use of monoclonal antibodies and immunotoxins must take into consideration the possible limiting effects of the humoral immune response to these agents.     

Long-term survival of cardiac xenografts in fully xenogeneic (mouse --> rat) bone marrow chimeras

BACKGROUND: The shortage of human hearts remains a major barrier to the efficacy of heart transplantation for the treatment of end-stage heart disease. One potential solution to the supply problem would be the use of hearts from nonhuman donors (xenografts). We have established a model of mouse to rat xenogeneic bone marrow chimerism, and in this study we have hypothesized that such chimeric rats will accept both donor and recipient specific heart grafts while rejecting third-party mouse and rat grafts. We also investigated humoral responses in naive and chimeric rats with and without donor murine cardiac grafts. METHODS: Recipient Lewis rats (n = 22) were given 1100 cGy lethal total body irradiation and the same day received 300 x 10(6) donor B10.BR mouse bone marrow cells intravenously. Peripheral blood of surviving rats (n = 18) was typed at 4 weeks and then monthly thereafter. Donor and recipient specific and third-party heterotopic heart transplantations were performed at 6 to 8 weeks after reconstitution with bone marrow. RESULTS: Multilineage bone marrow chimerism was produced in all experimental animals with complete replacement of recipient marrow by donor cells. Murine donor and rat recipient strain hearts transplanted in chimeric rats survived indefinitely. Third-party rat and mouse hearts were rejected, though at a slower rate than bone marrow matched naive controls. High levels of antimouse antibodies were detected in rats with rejected hearts. These antibodies were absent in chimeric animals with long-term surviving heart grafts. CONCLUSIONS: Long-term multilineage bone marrow chimerism can be produced in a mouse --> rat bone marrow transplant model. Long-term survival of donor specific and recipient specific vascularized cardiac grafts can be produced in these chimeric animals. These animals are clinically normal but show signs of subclinical immunosuppression regimen as they reject third-party hearts later than naive animals. Our results suggest that antibodies also play a significant role in concordant xenograft rejection, and induction of bone marrow chimerism can overcome this barrier.     

兔抗大鼠LM23蛋白合成肽多克隆抗体的制备与初步应用

目的制备大鼠精子发生相关蛋白LM23的合成肽多克隆抗体,对其特性进行初步鉴定,并对LM23多克隆抗体进行初步应用。方法利用生物信息学方法分析选择LM23蛋白的多肽序列。应用Fmoc法化学合成大鼠LM23蛋白N端第1～20和第274-291个氨基酸多肽（接近C端）,经C18的RP—HPLC纯化后,通过高碘酸钠法,将纯化的LM23蛋白的多肽与KLH交联,免疫新西兰大耳白兔,获得LM23蛋白的多克隆抗体。用ELISA方法鉴定多克隆抗体效价。通过Westernblot方法鉴定LM23多克隆抗体的特异性并研究LM23在大鼠睾丸中的表达;通过免疫组化方法研究LM23在大鼠睾丸组织和细胞中的定位。结果化学Fmoc法分别合成大鼠LM23蛋白N端第1～20,第274-291个氨基酸多肽,纯化后两条多肽纯度都达到90％以上,符合免疫用抗原标准。将多肽与KLH交联,用于免疫动物。经ELISA鉴定,两种多克隆抗体的效价分别达到1：64000和1：128000。应用大鼠睾丸组织切片进行LM23—18肽多克隆抗体的免疫组织化学研究,发现精母细胞有阳性颗粒反应,且LM23主要表达于细胞核。Westernblot研究证实,LM23—18肽多克隆抗体可特异识别大鼠睾丸组织中相对分子萤（Mr）约为36kD的LM23蛋白。结论所制备的LM23合成肽多克隆抗体可用于ELISA、Westernblot及免疫组化研究。LM23合成肽多克隆抗体的制备为进一步研究LM23的功能和作用机制提供了有力支持。     

Effect of IgG subclasses on in vivo bioavailability and metabolic fate of immune-complexed insulin in Lewis rats

The bioavailability, distribution, and metabolic fate of 125I-labeled insulin complexed to antibodies in guinea pig antiserum, purified guinea pig IgG1, IgG2, a mixture of IgG1 and IgG2, and homologous Lou/m rat antiserum were studied in inbred Lewis rats. 125I-insulin complexed to purified guinea pig IgG2 antibodies was rapidly cleared from the blood and sequestered in increasing amounts with time in the liver. Large amounts of the 125I-insulin complexed to guinea pig IgG1 antibodies remained in the blood for at least 30 min. The bioavailability of 125I-insulin bound to IgG1 and IgG2 antibodies was inhibited for at least 30 min because significantly less was available for rapid binding to insulin receptors on hepatocytes and renal tubular cells and its subsequent rapid degradation. The bioavailability of 125I-insulin was further decreased when bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies compared with the 125I-insulin complexed to either purified IgG1 or IgG2 antibodies alone. The 125I-insulin bound to antibodies in native guinea pig antiserum or a mixture of IgG1 and IgG2 antibodies was distributed in vivo in a manner reflecting the relative concentrations of the IgG1 and IgG2 antibodies present. The bioavailability, distribution, and metabolic fate of 125I-insulin in immune complexes prepared with homologous Lou/m rat insulin antiserum was qualitatively similar to that observed with immune complexes prepared with guinea pig insulin antiserum. It appears that the Lewis rat can be used as an in vivo model to study the bioavailability,distribution,and metabolic fate of insulin bound to xenogenic or homologous insulin antibodies.     

Immunoregulation via adhesion molecules in allogenic and xenogenic hepatocyte transplantation to nagase's analbuminemic rats

We investigated the effects of monoclonal antibodies (mAbs) against lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) on intrasplenic allogenic and xenogenic hepatocyte transplantation (HCTx) to analbuminemic rats. Ten to 12-wk-old male Nagase's analbuminemic rats (RT1¹) were used as recipients, Wistar/Shi rats (RT1^k) were used as donors for allografts and BALB/C mice were used as donors for xenografts. The experimental groups were as follows: group 1, allo-HCTx (n = 7); group 2, allo-HCTx + antirat ICAM-1/antirat LFA-1 mAbs (1.0 mg/kg/day, for 7 days, respectively) (n = 6); group 3, xeno-HCTx (n = 5); group 4, xeno-HCTx + mAbs (antimouse LFA-1/antirat ICAM-1) (n = 5). group 5, xeno-HCTx + mAbs (antirat LFA-1/antimouse ICAM-1) (n = 5). Serum rat albumin levels were measured in groups 1 and 2, and serum mouse albumin levels were measured in groups 3, 4, and 5, as indicators of the function of grafted hepatocytes. In allotransplantation groups, the serum rat albumin levels in the mAbs-treated group were significantly higher than those in the control group for 6 wk after transplantation. In xenotransplantation groups, no increase in the serum mouse albumin levels was detected in any group.