用自制抗HRP－抗HBc双特异性单克隆抗体检测抗HBc的初步应用

用杂交瘤融合法建立了抗ＨＲＰ－抗ＨＢｃ双特异性单克隆抗体（ＢｓＡｂ）杂交－杂交瘤细胞系，它分泌的腹水抗体经ＦＰＬＣ－离子交换层析梯度盐洗脱呈现三个主峰，抗体活性检测表明依次为抗ＨＲＰ、ＢｓＡｂ及抗ＨＢｃ。试用ＢｓＡｂ－ＨＲＰ复合物检测乙肝病例血样，与市售试剂盒初步比较，效果满意。     

抗人IgG-抗HRP双特异性单克隆抗体细胞株的建立与鉴定

目的 制备人ＩｇＧ检测用双特异性单克隆抗体诊断试制。方法 用人ＩｇＧ免疫ＢＡＬＢ／ｃ小鼠脾细胞与抗ＨＲＰＭｃＡｂ杂交瘤细胞ＨＡＴ敏感株进行第２次融合。结果 获得５株能稳定分泌抗人Ｉｇ－抗ＨＲＰ的双特异性单克隆抗体的杂交－杂交瘤细胞,分泌的抗体亚类其中１株为ＩｇＧ２ａ／ＩｇＧ１,余为ＩｇＧ１／ＩｇＧ１。培养上清与腹水效价分别为２＾７、１０＾０４以上,经ＨＲＰ亲和层析有２个蛋白吸ＢｓＭｃＡｂ存在于第２     

腺病毒抗独特型单克隆抗体的制备及性质研究

采用了多种免疫方式及一种新的检测方法：用经固定的杂交瘤细胞（Ａｂ１）包被酶标板，加入待测上清孵育后加入只与哺乳类动物抗体Ｆｃ段结合的ＨＲＰ－ＰｒｏｔｅｉｎＡ。共得到了６株分泌抗独特型单抗的杂交瘤细胞。其中两株与Ａｂｌ的结合可被腺病毒抑制，且都能在Ｂａｌｂ／ｃ小鼠诱导出与腺病毒结合的Ａｂ３。因此为Ａｂ２β。     

抗HBeAg单克隆抗体的制备及初步鉴定

为获得能替代抗ＨＢｅＡｇ多克隆酶标记抗体做ＥＬＩＳＡ，我们用纯化的ＨＢｅＡｇ阳性血清，免疫Ｂａ１ｂ／ｃ小鼠，采用杂交瘤技术，获得８株能稳定分泌小鼠抗ＨＢｅＡｇｍＡｂ的杂交瘤细胞。对其特性进行了初步鉴定，并就这些单克隆抗体（ｍＡｂ）在ＥＬＩＳＡ反应中的特性进行了分析。１　材料和方法１－１　材料　ｅ抗原阳性的人血清经亲和层析纯化的ＨＢｅＡｇ，由本室收集并纯化。Ｂａ１ｂ／ｃ小鼠，购自上海西普尔－必凯实验动物有限公司。小鼠Ｓｐ２／０骨髓瘤细胞，由中国科学院上海细胞生物学研究所提供。１－２　杂交瘤细胞株的…     

抗HBs独特型抗体免疫复合物意义的初步探讨

抗ＨＢｓ独特型抗体免疫复合物意义的初步探讨周艺峰，王艾丽，武建国，丁庆（南京军区南京总医院临床免疫科，南京２１０００２）业已证明，乙型肝炎患者血清中存在抗ＨＢｓ独特型抗体（抗ＨＢｓ－Ａｂ＿２）及其抗ＨＢｓ独特型抗体特异复合物（抗ＨＢｓ－Ａｂ＿２－ＩＣ...     

斑点金免疫渗滤法测定人全血中抗HBc抗体

目的：建立一种适合斑点免疫渗滤法（Ｄｏｔ Ｉｍｍｕ ｎｏ ｇｏｌｄ Ｆｉｌｔｒａｔｉｏｎ Ａｓｓａｙ ＤＩＧＦＡ）特点的快速全血诊断方法,方法：选择可瞬间分离血中细胞和血清的膜,依据ＤＩＧＦＡ的原理对３３份ＨＢｃＡｂ阳性血样和４８份阴性血样进行了全血抗ＨＢｃＡｇ抗体的检测。结果：测定阳性标本率为９６．７％,阴性标本符合率为１００％。结论：提供了一种简便、快速、可行的全血检测抗ＨＢｃＡｇ抗体的方法。     

MCAb、PcAb不同标记ELISA技术对HBeAg／抗－HBe检测的评估

用单克隆抗体（ＭｃＡｂ）和多克隆抗体（ＰｃＡｂ）进行辣根过氧化物酶标记或生物素标记作酶联免疫吸附试验（ＥＬＩＳＡ），检测乙型肝炎病毒ＨＢｅＡｇ／抗－ＨＢｅ标志物。结果显示，ＭｃＡｂ的灵敏性高于ＰｃＡｂ，且标记用的抗体量较少，一孔一期法同时测ＨＢｅＡｇ／抗－ＨＢｅ只能使用ＭｃＡｂ。利用生物素与亲和素系统与ＭｃＡｂ建立的Ｍｃ－ＡＢＣ－ＥＬＩＳＡ技术，其灵敏性与放射免疫法（ＲＩＡ）相同，特异性好，稳定性优于ＲＩＡ。生物素标记抗体至少１年保持效价不变，是一种新的较理想的检测ｅ系统的方法之一。     

用人抗HBs在体内诱导的远期免疫应答

作者对三年前曾经过高价人抗ＨＢｓ的１１名健康志愿者复查血清内的ＨＢｓＡｇ，抗独特型抗体和抗－抗－独特型抗体水平，发现ＨＢｓＡｇ皆为阴性，Ａｂ３水平高于Ａｂ２，说明在这三年期间，无人感染乙肝病毒。     

HBsAg阳性,抗HBs阴性和抗HBc阳性者乙肝病毒DNA的检测

ＨＢｓＡｇ阳性、抗ＨＢｓ阴性和抗ＨＢｃ阳性者乙肝病毒ＤＮＡ的检测蒋挺英，陈兆军，张腊红，楼德利，及晓英病毒性肝炎血清标志的检测方法，随着免疫学技术的进展而取得迅速发展［１］，其测定原理主要依赖于抗原与抗体间的特异性反应，如ＲＩＡ和ＥＬＩＳＡ。随着分子...     

用荧光标记、流式细胞仪无茵分选法建立二次杂交瘤

双功能抗体（ＢｓＡｂ），即一个单抗分子具有两种不同抗原特异性结合能力，是近年来一种新型免疫治疗剂。ＢｓＡｂ的产生可通过化学交联技术、细胞融合技术或基因工程技术及二次杂交技术。二次杂交瘤可以是杂交瘤与脾细胞的融合（三源杂交源，ｔｒｉｄｏｍａｓ），也可以是两种杂交瘤细胞之间的融合（四源杂交源，ｔｅ－ｔｒａｄｏｍａｓ）。显然，应用两种已鉴定好的杂交瘤细胞作融合，在抗体的筛选与鉴定上优于杂交瘤与脾细胞的融合，因此，本文通过化学诱变产生的抗ＣＤ３ＨＡＴ敏感（ＨＧＲＴ－）细胞株标记荧光后与抗ＨＢｘＡｓ杂交瘤…     

Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies

Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.     

抗人膀胱癌/抗VEGF双功能基因抗体制备及体外效应研究

目的：研究制备抗人膀胱癌和抗血管内皮生长因子的双功能抗体，用以导向抑制肿瘤新生血管的形成。方法：在制备抗人膀胱癌和抗血管内皮细胞生长因子单克隆抗体的基础上制备双功能抗体。结果：双功能抗体的IC50为10^-9.5，抗血管内皮生长因子抗体的IC50为10^-8.9，抗人膀胱移行细胞癌单克隆抗体的IC50为10^-8.3。结论：双功能抗体的有效率明显高于单克隆抗体。     

The combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb elicited antitumor immunity by T-cell adoptive immunotherapy in lung cancer

Lung cancer remains a global challenge due to high morbidity and mortality rates and poor response to treatment, and there are still no effective strategies to solve it. The bispecific antibody (BsAb) is a novel antibody, which can target two different antigens and mediate specific killing effects by selectively redirecting effector cells to the target cells. In this study, we combined two BsAbs to achieve a dual-target therapy strategy of EpCAM⁺ and MUC-1⁺ with high affinity and specificity. The results showed that the combination of two BsAbs against EpCAM and MUC-1 could inhibit the growth of lung cancer more effectively in cell lines and primary tumors. The superior antitumor effect of two BsAbs could be attributable to enhanced CTL and increased production of type I IFNs. At the same time, the combination of EpCAM/CD3 BsAb and MUC-1/CD3 BsAb significantly regulated T population in the TDLNs. Therefore, we have found a potential immunotherapeutic strategy, which was the combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb for the treatment of non-small cell lung cancer.     

A novel pancreatic β-cell targeting bispecific-antibody (BsAb) can prevent the development of Type 1 diabetes in NOD mice

To prepare a novel Bispecific Antibody (BsAb) as a potential targeted therapy for T1D, we produced a “functionally inert” monoclonal antibody (mAb) against Glucose transporter-2 (GLUT-2) expressed on β-cells to serve as an anchoring antibody. The therapeutic arm is an agonistic mAb against Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), a negative regulator of T-cell activation expressed on activated CD4 + T-cells. A BsAb was prepared by chemically coupling an anti-GLUT2 mAb to an agonistic anti-CTLA-4 mAb. This BsAb was able to bind to GLUT2 and CTLA-4 in vitro, and to pancreatic islets, both in vitro and in vivo. We tested the safety and efficacy of this BsAb by treating Non-Obese Diabetes (NOD) mice and found that it could delay the onset of diabetes with no apparent undesirable side effects. Thus, engagement of CTLA-4 on activated T cells from target tissue can be an effective way to treat type-1 diabetes.     

新型抗IL-1β和IL-17A双特异抗体的构建及特性鉴定

目的 双特异性抗体(bispecific antibody,BsAb)具有双重的生物学功能.本实验旨在设计并原核表达抗人IL-1β和抗人IL-17A的双特异性抗体(BsAbl/17),获得具有生物活性的BsAb1/17,为深入研究和利用双特异性抗体奠定基础.方法 利用重叠PCR方法构建VH1VL17-CL和VL1VH17-CH1基因片段,并且在所用引物的5'和3'端附加Nco Ⅰ和BamH Ⅰ的酶切位点.将重叠PCR产物进行胶回收后用Nco Ⅰ/BamH Ⅰ进行双酶切,酶切产物再次胶回收,将其连接到用Nco I/BamH I消化的pET-27b载体上.将重组质粒pET-27b-VH1VL17-CL(petA)和pET-27b-VL1VH17-CH1(petB)转化到E. coli Rosetta中.SDS-PAGE和Western blot进行鉴定,用real-time PCR检测其阻断IL-1 β刺激人T细胞表达细胞因子IL-18的活性,人IL-6定量酶联检测试剂盒检测其阻断IL-17A刺激HeLa细胞表达人IL-6的活性.结果 DNA测序结果证明成功构建了pET-27b-VH1VL17-CL(petA)和pET-27b-VL1VH17-CH1(petB)表达质粒.petA、petB诱导产物主要以包涵体形式存在,成熟蛋白纯化产物纯度超过90%以上,经SDS-PAGE分析表明表达产物的相对分子质量约为38×103,与理论值相符.Western blot和ELISA结果证实双特异抗体BsAb1/17对IL-1 β和IL-17A均具有很好的亲和力.通过RT-PCR检测证明其具有阻断IL-1 β刺激人T细胞大量表达细胞因子IL-18的活性,并且具有阻断IL-17A刺激HeLa细胞产生IL-6的活性.结论 成功构建了同时抗IL-1β和IL-17A的双特异抗体,并利用大肠杆菌表达系统高效表达较高纯度的具有生物活性的双特异抗体.

Abstract：

Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.     

Designing of a bispecific antibody against SARS-CoV-2 spike glycoprotein targeting human entry receptors DPP4 and ACE2

COVID-19 originated in Wuhan city, China, in 2019 erupted a global pandemic that had put down nearly 3 million lives and hampered the socio-economic conditions of all nations. Despite the available treatments, this disease is not being controlled totally and spreading swiftly. The deadly virus commences infection by hACE2 receptor and its co-receptors (DPP4) engagement with the viral spike protein in the lung alveolar epithelial cells, indicating a primary therapeutic target. The current research attempts to design an in-silico Bispecific antibody (BsAb) against viral spike glycoprotein and DPP4 receptors. Regdanvimab and Begelomab were identified to block the D614G mutated spike glycoprotein of SARS-CoV-2 and host DPP4 receptor, respectively. The designed BsAb was modified by using KIH (Knobs into Holes) and CrossMAb techniques to prevent heavy chain and light chain mispairings. Following the modifications, the site-specific molecular docking studies were performed, revealing a relatively higher binding affinity of BsAb with spike glycoprotein and DPP4 co-receptor than control BsAb. Also, for blocking the primary entry receptor, hACE2, an anti-viral peptide was linked to the Fc region of BsAb that blocks the hACE2 receptor by linker cleavage inside the infected host. Thus, the designed BsAb and anti-viral peptide therapy could be a promising triumvirate way to obstruct the viral entry by blocking the receptor engagement.     

Di-diabody: a novel tetravalent bispecific antibody molecule by design

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.     

Release of Cytokines and Soluble Cell Surface Molecules by PBMC after Activation with the Bispecific Antibody CD3 × CD19

Bispecific antibodies (BsAb) consist of two different heavy and light chains and may bind to two different antigens present on different cell types. With their dual specificity BsAb may recognize effector cells (e.g. T cells) on one hand and tumour cells (e.g. malignant B cells) on the other hand. The authors analysed whether T cell activation and subsequent killing of malignant B cells mediated by the bispecific antibody CD3 × CD19 was reflected by the release of cytokines. In addition, the authors investigated whether the in vitro cytokine release was similar to that observed in vivo in the patients treated with BsAb. The in vitro release of cytokines into the supernatant of cell cultures of peripheral blood mononuclear cells (PBMC) and malignant B cells was measured after incubation with either the bispecific antibody CD3 × CD19 or the monospecific anti-CD3 (aCD3) antibody in the presence or absence of interleukin (IL)-2. Release of tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, IL-8, IL-10, soluble (s) CD4, sCD8 and sCD25 by PBMC was equal under both conditions and could be used as an indicator for T cell activation. However, the cytokine pattern and level did not correlate with the cytotoxic capacity, which was 4 logs higher with BsAb + IL-2 compared to aCD3 + IL-2. The in vitro pattern of cytokine release was similar to that observed in vivo in the serum of patients treated with BsAb and IL-2, indicating the possibility of predicting cytokine release in future patients with other therapeutic regimens.     

Bispecific anti-human red blood Rhesus-D antigen x anti Fc gamma RI targeted antibody-dependent cell-mediated cytotoxicity and phagocytosis by mononuclear leucocytes.

The Fc receptor mediated antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis induced by bispecific antibody (BsAb) to the high-affinity Fc receptor for IgG (Fc gamma RI) and to human red blood group antigen RhD were studied in vitro, using human mononuclear leucocytes as effector cells. The results were compared with those obtained by using a human monoclonal IgG1 anti-RhD used alone and a reference human polyclonal anti-RhD antibody. The effect of non-specific human IgG on FcR-mediated functions by mononuclear leucocytes was checked. The results demonstrate that BsAb presents a high resistance of Fc-mediated function to blockade by non-specific human IgG compared with that of both polyclonal and monoclonal anti-RhD antibodies. These results further encourage possible clinical application of bispecific antibody in passive immunotherapy.