乙酰胆碱酯酶抗独特型抗体的研制

目的：研制乙酰胆碱酯酶（Acetylcholinesterase，AchE）抗独特型抗体并测定酶活性。方法：AchE单克隆抗体是一种IgG1抗体，先用木瓜蛋白酶酶切，然后采用AchE-Sepharose-4B和SPA亲和层析柱进行提取纯化，获得纯化的单克隆抗体Fab段。以Fab作为抗原，免疫小鼠，制备抗Fab独特型抗体，采用酶催化ELISA法研究该单克隆抗体的乙酰胆碱酯酶活性。结果：制备出高效价抗乙酰胆碱酯酶抗体Fab段的独特型抗体，该抗体具有乙酰胆碱酯酶活性。结论：AchE单克隆抗体的独特型抗体具有AchE活性。     

一种利用HCIC快速纯化抗rhTF_(243)单克隆抗体方法的研究

目的:建立从小鼠腹水中快速纯化抗人重组组织因子243(recombine human tissue factor243,rhTF243)单克隆抗体(monoclonal antibody,mAb)的方法。方法:含抗rhTF243单克隆抗体的腹水经离心、过滤及缓冲溶液变换等预处理后,先经疏水电荷诱导层析纯化处理,去除大部分杂蛋白,再经Protein A-Sepharose CL-4B亲和层析柱进一步纯化。结果:经两步纯化后获得抗rhTF243单克隆抗体,其生物学活性高、纯度大于95%,抗体效价保持良好,抗体总回收率达73%,且具有明显的抗促凝作用。结论:建立的单克隆抗体纯化方法简便、高效,所得mAb的纯度高、生物学活性好。     

抗人Toll样受体4单克隆抗体的制备及其生物活性研究

目的运用单克隆抗体技术制备抗人Toll样受体4(TLR4)单克隆抗体(mhTLR4抗体),并鉴定其生物学活性。方法以重组人TLR4(rhTLR4)作为抗原,腹腔注射免疫Balb/c小鼠。分离小鼠脾脏B淋巴细胞与骨髓瘤细胞融合并培养,挑选阳性杂交瘤细胞,扩大培养,制备和鉴定mhTLR4其生物学效应。结果经3次免疫后的小鼠血清中抗rhTLR4抗体的效价具有较高水平的表达。1∶500的小鼠免疫血清可明显抑制人PBMC的TNF-α表达。分离小鼠脾细胞与骨髓瘤细胞进行融合,获得3株阳性单克隆杂交瘤细胞。其中2株经过增殖培养、纯化浓缩制备出较高产量的鼠抗人TLR4单克隆抗体。生物学活性测定表明,产生的抗rhTLR4单克隆抗体,能够明显地阻断脂多糖(LPS)诱导的人PBMC细胞对TNF-α的表达。结论本文制备的鼠抗人TLR4单克隆抗体,经鉴定具有较高的阻断内毒素信号传导的效应。为进一步研制人源化抗TLR4抗体、研制新一代抗内毒素靶向药物奠定了基础。     

抗早孕因子单克隆抗体的制备与鉴定

目的建立分泌抗EPF(Early pregnancy factor，早孕因子)单克隆抗体的杂交瘤细胞株，纯化单抗并鉴定。方法用本实验室已纯化的早孕和肿瘤源性：EPF作为抗原刺激Balb／c小鼠，用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合，经4次克隆化，获得可稳定分泌抗EPF单克隆抗体的细胞株，注入Balb／c小鼠腹腔制备腹水型单抗，Proteirr-A亲和层析纯化，SDS电泳和Western-blot等方法分析纯化结果。结果融合后获得一株稳定分泌抗EPF抗体的细胞株(C3D11)，克隆化后，获得稳定分泌抗EPF单克隆抗体的细胞株，将增殖后的细胞注射Balb／c小鼠腹腔获得腹水型单抗，以亲和层析法纯化，SDS-PAGE分析显示纯化后去掉了大部分杂蛋白，免疫印迹分析抗体纯度较高，与抗原匹配性良好。结论本研究制备的EPF单克隆抗体为特异性抗EPF抗体。     

抗生物素单克隆抗体的制备及其在核酸检测中的应用

生物素与亲和素有相当强的亲和力 (Ｋｄ10 15Ｍ ) ,已被广泛用于细胞化学、免疫组织化学、免疫测定以及核酸原位分子杂交。但是生物素 亲和素系统中 ,亲和素是一种高碱性 (ｐＨ0 5 )糖蛋白 ,易引起较强的非特异性结合。最近Ｂａｇｅｒ报道抗生物素抗体有模拟亲和素功能作用 ,并显示较好的特异性[1] 。本文报道建立二株抗生物素单克隆抗体 ,并初步用于乙型肝炎ＤＮＡ的检测。1　材料和方法1 1　材料　生物素、Ｎ 羟基琥珀酰亚胺、二环已基碳二亚胺、ＢＳＡ、ＰＥＧ、ＨＡＴ、ＤＡＢ (3,3 ｄｉａｍｉｎｏｂｅｎｚｉｄｉｎｅ )、羊抗小鼠Ｈ…     

人淋巴结的内源性抗生物素结合活性

目的 论证人淋巴结为具有内源性抗生物素结合活性(EABA)的器官。方法 以LSAB法、EnVision法、实时生物分子相互作用分析技术及椭偏成像生物显微技术，通过肝癌单克隆抗体Hepama-1对人淋巴结的反应，进行实验论证。结果 LSAB法曾对人淋巴结呈强阳性，但EnVision法、实时生物分子相互作用分析技术及椭偏成像生物显微技术对人淋巴结呈阴性。结论 人淋巴结具有内源性抗生物素结合活性。     

抗SARS病毒N蛋白单克隆抗体的制备和初步应用

目的 制备抗SARS病毒N蛋白的单克隆抗体并研究其初步应用。方法 用基因重组N蛋白免疫小鼠，取免疫后的鼠脾细胞与骨髓瘤细胞融合，筛选分泌抗SAPS病毒N蛋白单克隆抗体细胞株。将阳性细胞株接种小鼠腹腔制备单克隆抗体腹水并对抗体进行纯化，分析纯化抗体的相对亲和力。选择亲和力较高的抗体制备检测SARS病毒抗原的酶联免疫诊断试剂，并对其敏感性和特异性进行分析。结果 共获得11株单克隆抗体细胞株，其中3株单抗与N蛋白具有较高的亲和力，4株纯化单抗与N蛋白反应很弱，其余4株单抗介于两者之间。用亲和力较高的单抗制备检测SARS病毒抗原的诊断试剂，其敏感性可达31PFU／ml，而且与其他呼吸道病毒无交叉反应。结论 该试剂特异性较好，可用于SARS病毒抗原的检测，其敏感性仍需用临床急性期样品进行评价。     

由人免疫球蛋白转基因小鼠制备人抗乙酰胆碱受体单克隆抗体

目的：从人免疫球蛋白（Ig）转基因小鼠制备人源性抗乙酰胆碱受体（AChR）单克隆抗体。方法：以电鳐（Torpedo）AChR（tAChR）作为基础免疫原，分别以tAChR或人AChR（hAChR）α亚单位1～210位氨基酸（Hα1-210）与thioredoxin（Trx）融合制备的融合蛋白Trx-Hα1-210作为加强免疫原，注射人Ig转基因小鼠。应用ELISA检查由该小鼠制备的杂交瘤细胞培养上清液中人源性抗tAChR或抗Trz-Hα1-210单克隆抗体的分泌，应用放射免疫测定法（RIA）测定抗tAChR或抗Trx-Hα1-210单克隆抗体与hAChR的交叉反应性。结果：从tAChR作为加强免疫原组小鼠得到433株分泌人源性抗tAChR单克隆抗体的细胞株，从抗Trx-Hα1-210作为加强免疫原组小鼠得到20株分泌人源性抗Trx-Hα1-210单克隆抗体的细胞株。但这些人源性抗体均不能与hAChR起交叉反应。结论：由人Ig转基因小鼠制备人源性抗AChR单克隆抗体是可行的。     

阴离子交换层析法纯化gp130单克隆抗体B-S12

目的：建立一种从小鼠腹水中获得高纯度、活性好、纯化过程易于放大的抗gp130单克隆抗体B-S12的纯化方法。方法：经硫酸铵沉淀等预处理后的腹水样品，在阴离子交换层析柱上进行分离纯化，用MTT法检测纯化后抗体的生物学活性。结果：腹水样品经硫酸铵沉淀、PBS复溶，用pH 7．0、20mmol／L Tris—HCl缓冲溶液稀释10倍后上强阴离子交换层析柱，NaCl梯度洗脱，可获得纯度大于95％的B-S12抗体，回收率达73％，在体外对XG-2细胞有明显的促增殖作用。结论：建立了快速高效从小鼠腹水中纯化B-S12的方法，为该抗体的进一步应用提供了必要的实验基础。     

抗人大肠癌单克隆抗体的制备及应用

目的制备和纯化抗人大肠癌单克隆抗体(ND-1),分析其在大肠癌诊断中的应用价值。方法常规免疫小鼠制备腹水,腹水经离心和过滤后,应用G蛋白亲和层析法进行纯化。采用SDS-PAGE、间接免疫荧光(IFA)、间接ELISA检测纯化后抗体的纯度、活性、效价和特异性。并用免疫组织化学SP法检测在大肠癌组织中的表达。结果经SDS-PAGE分离可见两条相对分子质量(Mr)分别为55 000和25 000明显的两条蛋白条带。间接ELISA检测抗体效价为1∶1 000 000,ND-1抗体对表达相应抗原的大肠癌细胞株具有特异结合活性。人大肠癌组织切片经ND-1抗体免疫组织化学染色阳性率为82.9%,其中高分化腺癌阳性率为100%。结论应用G蛋白亲和层析法可以很好分离纯化鼠腹水中的IgG1亚型的单克隆抗体,抗体的纯度高,特异性强,生物学活性好,可望成为有效的肿瘤诊断和治疗的手段。     

重组人可溶性gp130的纯化及生物学活性鉴定

采用转瓶培养可溶性gp130基因转染细胞。运用免疫亲和层析法,将抗人可溶性gp130的单克隆抗体偶联于CNBr活化的Sepharose 4B。收获基因转染细胞的培养上清,经抗gp130单抗/Sepharose 4B亲和层析柱吸附,用pH 2.8、0.1 mol/L甘氨酸溶液洗脱,获取可溶性gp130重组蛋白纯品。基因转染细胞培养上清中可溶性gp130的表达量为100~120μg/ml,纯化后的回收率为70%~75%,SDS-PAGE电泳后出现一条蛋白曲带。经Western blot分析,纯化的可溶性gp130能与特异性抗体结合。将可溶性gp130(终浓度为5μg/ml)与IL-6依赖性生长细胞株XG2共同培养,细胞的生长与增殖出现抑制。提示经免疫亲和层析法纯化的可溶性gp130具有良好的生物学活性。     

体外急性心肌梗死早期诊断方法的研究

从人心肌中提取心肌肌凝蛋白轻链(CMLC),免疫Balb/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞做常规融合,制备单克隆抗体.通过抗人心肌肌凝蛋白轻链单克隆抗体的相加指数、亲和常数测定和抗体夹心实验,筛选到配对较好的两对单抗,在此基础上建立起轻链夹心ELISA方法,为临床急性心肌梗死早期诊断方法研究提供条件.     

The role of interleukin-5 in protective immunity to Strongyloides venezuelensis infection in mice.

We depleted or neutralized interleukin-5 (IL-5) and IL-5 receptor of C57BL/6 mice, using rat anti-murine IL-5 monoclonal antibody (NC17) and anti-murine IL-5 receptor monoclonal antibody (H7). Mice treated with these monoclonal antibodies were infected with Strongyloides venezuelensis larvae. The time-course of faecal egg output and peripheral eosinophilia were monitored. In a primary infection, anti-IL-5 treatment did not affect faecal egg output, although the eosinophil count in peripheral blood was markedly reduced. There was no difference in intestinal worm burden or faecal egg output between anti-IL-5 treated and non-treated mice. In a secondary infection, worms were expelled from the small intestine of anti-IL-5-treated mice as well as from non-treated mice. Worm recovery from the lungs of mice treated with either anti-IL-5 or anti-IL-5 receptor monoclonal antibody was the same as that of normal controls. However, a marked reduction in worm recovery was observed in re-infected mice that had not been treated with monoclonal antibodies. Treatment with anti-IL-5 or anti-IL-5 receptor monoclonal antibody suppressed blood and tissue eosinophilia. Thus the results suggested that the host's protective immunity against tissue-migrating larvae was IL-5-dependent but intestinal immunity was not.     

人OX40胞外区基因的克隆、原核表达及单克隆抗体的制备

目的制备抗人OX40单克隆抗体,为进一步研究OX40/OX40L通路以及针对该通路进行疾病干预、疾病治疗和药物筛选奠定基础。方法 PCR扩增人OX40胞外区的基因序列,将其构建到原核表达载体pET-32a(+)中,利用大肠杆菌表达系统表达OX40胞外区蛋白,经Ni柱纯化后获得目的蛋白。以纯化的OX40胞外区蛋白为免疫原免疫小鼠,采用常规方法进行细胞融合,通过ELISA和多次克隆化培养,筛选出分泌特异性鼠抗人OX40单克隆抗体的杂交瘤细胞株;通过ELISA、Western blot和免疫荧光检测抗体的效价及特异性。结果成功构建人OX40原核表达质粒pET-32a(+)-OX40,并在BL21(DE3)中诱导表达,经SDS-PAGE和Western blot鉴定、Ni柱纯化、透析后获得OX40胞外区蛋白;以此纯化蛋白为免疫原,成功制备具有较强亲和力的单克隆抗体。结论克隆表达并纯化了人OX40蛋白,成功制备针对该蛋白的高亲和力单克隆抗体,为进一步研究OX40/OX40L的生物学功能奠定了实验基础。     

自制NSE单抗的鉴定及其双抗夹心ELISA方法的建立

目的:制备并鉴定NSE(Neuron-specific enolase)单克隆抗体,建立可检测NSE蛋白的双抗夹心ELISA方法。方法:用本实验室已表达纯化的NSE融合蛋白免疫BALB/c小鼠,采用杂交瘤技术制备单克隆抗体。采用WB、IP、IF、IHC等方法对获得的NSE单抗进行鉴定及亚型检测。利用辣根过氧化物酶标记纯化后的NSE单抗,建立一个可检测NSE蛋白的双抗夹心ELISA法。结果:通过分析和鉴定,选定2株可稳定分泌抗NSE抗体的杂交瘤细胞株,效价达4.2×107～6.5×107,亚型为IgG2b。免疫印迹结果显示,该抗体不仅能识别细胞内源NSE蛋白,还能识别分泌到细胞培养上清液中的NSE蛋白,此外还可用于免疫荧光及免疫组化检测。文中所建立的双抗夹心ELISA法,最低检测极限为8.85 ng/ml。结论:成功获得了效价高、灵敏度好及特异性强的NSE单抗,建立了一个双抗体夹心ELISA检测系统,具有良好的临床应用前景。     

Monoclonal opsonic mouse antibodies specific for streptococcal IgG Fc-receptor

Spleen cells from mice immunised with group-A type-M15 streptococci and boosted with purified IgG Fc-receptor (FcR) from this type were fused with Sp 2/0 mouse myeloma cells. The resulting hybridomas were screened by ELISA for antibody production. Two IgM-secreting cell lines were selected. The monoclonal antibodies and ascites fluids inhibited the binding of 125I-labelled human IgG and IgG Fc-fragments to group-A type-M15 streptococci. The monoclonal antibodies also displaced purified FcR towards the anode in electrophoresis. They opsonised group-A type M-15 streptococci for phagocytosis by human granulocytes in the presence of fresh human serum. It was concluded that FcR is important for group-A streptococcal virulence.     

A single-step method for the purification of anti-FITC antibodies by use of a coumarin immunosorbent

Monoclonal anti-fluorescein isothiocyanate (FITC) antibody cross-reacts with 7-hydroxy coumarin derivatives conjugated to BSA. This property permitted the affinity purification of monoclonal anti-FITC antibodies from ascitic fluid using an-immunosorbent consisting of a 7-hydroxy coumarin derivative linked to Sepharose 4B. Ascitic fluid was applied to the immunosorbent column and, after washing, the bound antibody was eluted under extremely mild conditions using 3 M MgCl2. Antibody eluted in this manner was greater than 96% pure as assessed by SDS-PAGE. A polyclonal sheep anti-FITC antibody was also purified from serum on the same immunosorbent to greater than 94% purity. This simple and rapid method for the purification of anti-FITC antibodies will find applications in both immunodiagnostic procedures and in studies of hapten-antibody interactions. The affinity constant of the purified monoclonal anti-FITC antibody conjugated to horseradish peroxidase was assessed by ELISA and was found to be 1.5 x 19(9) M-1.     

Production, characterisation and use of monoclonal antibodies to human interleukin-5 in an enzyme-linked immunosorbent assay

Two mouse monoclonal antibodies (Mabs) against recombinant human interleukin-5(rhIL-5) have been produced, characterised and purified. Both are IgG1 antibodies and neutralised the activity of rhIL-5 in the B13 assay. Neither Mab cross-reacted with mouse IL-5. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was developed with different combinations of the mouse Mabs and also a rat anti-mouse IL-5 Mab, TRFK5, which also has activity against rhIL-5. The most sensitive assay, with a lower detection limit of 0.5 ng/ml IL-5, used TRFK5 as the capture antibody and the mouse anti-human IL-5 Mab as second antibody. The sensitivity of this assay was increased by an enhanced chemiluminescent reagent and resulted in a lower limit of detection around 40 pg/ml IL-5.     

Development of polyclonal and monoclonal antibodies for immunoassay and neutralization of human interleukin-4

A rabbit antiserum to partly purified recombinant E. coli-expressed human interleukin-4 (IL-4) has been produced which neutralizes the T cell growth factor, B cell growth factor, and Fc epsilon R2/CD23 inducing activities of IL-4. The antiserum demonstrated sufficient avidity to immunoprecipitate labelled COS7-expressed recombinant human IL-4. In contrast, rabbits immunized with conjugates of various synthetic IL-4 oligopeptides produced antisera which recognized IL-4 in both enzyme-linked immunosorbent assay (ELISA) and Western blotting formats, but failed to immunoprecipitate IL-4 from solution, or to neutralize bioactivity. Two rat monoclonal antibodies, 11B4, 22C10 were produced from a rat immunized with purified COS7 cell-expressed IL-4. These IgG2a antibodies recognized both E. coli-expressed and mammalian cell-expressed (COS7 and L cell) recombinant human IL-4 in solution (immunoprecipitation), as well as on solid phase (indirect ELISA and dot-blotting). The 11B4 antibody inhibited IL-4 bioactivity at an IC50 which was 25-50-fold in molar excess of factor. Both antibodies also recognized IL-4 bound to an immobilized rabbit IgG fraction of anti-IL-4. The 11B4 antibody was used to develop an immunoenzymetric assay capable of detecting less than 100 pg of analyte/ml. Supernatants from PBL, activated under varying conditions were tested for IL-4 levels. PHA and ConA were found to induce a relatively low degree of IL-4 production by these PBL. An approximately ten-fold greater level of IL-4 production was observed when they were stimulated with A23187 in combination with PMA. Various patient sera and cell line supernatants were also tested. These IL-4 immunoreagents are important tools for further studies of IL-4 immunobiology.