Construction of human monoclonal antibodies against rabies NS-protein antigen by Epstein-Barr virus transformation

A human monoclonal antibody against Rabies NS-protein was developed using peripheral blood lymphocytes. A special immunization protocol was used in order to achieve maximal cell numbers of active secreting B cells. These B cells were subjected to Epstein-Barr virus transformation and the transformed cells were cloned stepwise by limiting dilution using supernatant of stimulated macrophages. Active anti-Rabies-IgG secreting cell clones were adapted to serum-free conditions producing stably for more than one year antibodies of the subtype IgG1 at a rate of 10 micrograms/ml/10(6) cells/24 h.     

Molecular characterization of a neutralizing murine monoclonal antibody against Tityus serrulatus scorpion venom.

Monoclonal antibodies (mAbs) against Tityus serrulatus venom were obtained by the fusion of SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with a toxic fraction (TstFG50) of the Tityus venom (this G50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. From the initial screening of over 200 hybridoma fusion wells, a panel of 9 anti-TstFG50 secreting hybridomas was established. The capacity of mAbs to neutralize the TstFG50 toxic fraction toxic was determined by in vitro neutralization assays and by inhibition of the binding of 125I-TsVII to its site on rat brain synaptosomes. Only mAbTs1 neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII at 10(-7) M. To map the epitope recognized by the protective mAbTs1, we prepared a comprehensive series of overlapping 15-mer synthetic peptides covering the amino acid sequences of the four Tityus proteins. MAbTs1 reacted with peptide 26 of TsIV (KKSKDKKADSGYSYW), peptide 30 of TsVII (KKGSSGYSAWPASYS) and peptide 31 of TsNTxP (KKGSSGYSAWPASYS). MAbTs1 was not reactive with any peptide from TsII. The N-terminal lysine residue from the epitope was found to be critical for mAbTs1 binding. The epitope was positioned on the available three-dimensional structure of TsVII together with the recently identified residues from the pharmacophore of beta-scorpion toxins. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.     

Role of neutralizing antibodies in protective immunity against HIV

HIV continues to be a major health problem world wide, however the situation is particularly serious in Asian and Sub-Saharan countries. Therefore, development of an effective HIV vaccine could help to reduce the severity of the disease and prevent infection. Over the last two decades significant efforts have been made towards inducing potent humoral and cellular immune responses by vaccination, however antibodies and CTL responses alone are likely not sufficient for inducing sterilizing immunity or long-term control of viral replication. Therefore, it is generally believed that both humoral and cellular responses will be needed for an effective HIV vaccine. In support of humoral immunity, monoclonal antibodies that recognize critical neutralizing epitopes have shown to be effective at passive transfer experiments in conferring protection against challenge infection. However, antibodies to similar epitope specificities are difficult to induce by vaccination. Therefore, optimization of Env structure is needed for exposing appropriate neutralizing epitopes and masking non-neutralizing epitopes. Since the crystal structure of the core of Env glycoprotein has been solved, efforts are in progress to design novel Env immunogens that may induce effective neutralizing responses. Furthermore, there are HIV-1 strains that are resistant to neutralization by monoclonal antibodies, yet neutralized by pooled sera from HIV-1 patients. Therefore, efforts should be made to identify these novel epitopes and to design strategies to incorporate them in potential vaccines. To facilitate comparative evaluation of vaccine immunogens for their ability to induce cross clade neutralizing antibodies, efforts should be made to use standardized neutralization assays and standard virus panels. Once potent HIV Env structure have been identified, their effectiveness may be enhanced through the use of adjuvants, delivery systems and prime and boost strategies to improve the quality and magnitude of neutralizing responses.     

广谱全人源化甲型流感病毒中和抗体在流感预防与治疗中的应用

针对多个亚型甲型流感病毒的广谱中和性单克隆抗体（monoclonal antibody,mAb）是极具潜力的新型而有效的流感防治手段.抗流感病毒血凝素（hemagglutinin,HA）的保护性mAb,无论通过主动免疫还是被动免疫,均具有很好的效果.迄今,已有不少甲型流感病毒广谱中和性mAb被开发出来.尽管这些mAb具备临床应用潜能以及作为研制新型疫苗（抗独特型抗体疫苗）的可能,但仅有几种HA mAb对人类流感防治真正有效.此文主要对能高效识别多个亚型HA的人源化甲型流感病毒mAb做一综述.     

抗肿瘤单克隆抗体药物研究进展

抗肿瘤细胞表面抗原的单克隆抗体(单抗)可以有效治疗肿瘤.这些单抗药物可通过直接作用于肿瘤细胞、改变机体免疫应答、运送药物和重新激发机体免疫等机制来抗击肿瘤.此文就单抗治疗肿瘤的作用靶点、机制、代表性药物和新策略进行综述.     

Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

Aim:

To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.

Methods:

The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F'' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting, indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.

Results:

A recombinant vector was constructed. The Fab was expressed in soluble form in E coli Top10F''. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL. The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).

Conclusion:

The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.     

氯霉素单克隆抗体的制备与纯化

目的制备氯霉素(CAP)单克隆抗体。方法用人工合成抗原氯霉素-牛血清白蛋白(CAP-BSA)免疫BALB/c小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0按5∶1的比例融合,间接竞争ELISA法和有限稀释法进行单克隆杂交瘤细胞的筛选;制备腹水抗体;采用HiTrap rProtein A FF亲和色谱柱纯化抗体,用间接竞争ELISA法和间接ELISA测定抗体特异性。结果得到两株能稳定分泌单克隆抗体的杂交瘤细胞株,细胞培养上清中抗体效价达10-4以上,腹水抗体效价达10-7以上,纯化后的单克隆抗体纯度达98%,回收率达80%,抗体活性好并且与BSA、甲砜霉素、磺二甲基嘧啶等无交叉反应。结论成功获得两株能稳定分泌单克隆抗体的杂交瘤细胞,对动物性食品中CAP的检测具有较大的价值。     

抗金霉素单克隆抗体的制备与鉴定

目的制备抗金霉素(CTC)的单克隆抗体(mAb)。方法采用甲醛作为连接基,将CTC与牛血清白蛋白偶联制备免疫原,免疫BALB/c小鼠,取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞融合,经筛选克隆,以ELISA法对杂交瘤细胞的稳定性和mAb的特性进行鉴定。结果获得1株可稳定分泌mAb的杂交瘤细胞1H4-C4-C10,间接ELISA方法测定,细胞上清抗体效价为1∶1000,腹水效价为1∶5×105,为IgG1,与四环素交叉反应率为44.4%,与土霉素的交叉反应为8.2%。体外传代培养和冻存复苏后抗体分泌稳定。结论成功制备了针对CTC的mAb,为进一步研制检测CTC的ELISA试剂盒奠定了基础。     

Preparation and characterization of monoclonal antibodies against pseudexin

Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A affinity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins. Three of the antibodies neutralized pseudexin A and B, but none of the 10 other phospholipase A2 toxins tested. These same three antibodies inhibited the enzymatic activity of pseudexin A and B and also that of notexin. After each antibody was labeled with biotin, competition experiments were carried out to determine the binding relationships among the antibodies and the pseudexins. Competitions were frequently observed, with a low of zero to a high of eight out of the 14 possibilities. Competition experiments were also carried out with biotin-labeled rabbit IgG against the pseudexins. Some of the monoclonal antibodies had no effect on rabbit IgG binding to pseudexin, while others blocked up to 50% of the binding.     

A non-radioactive method for detecting neutralizing antibodies against therapeutic proteins in serum

The presence of neutralizing antibodies against protein therapeutics continues to cause concern in the biomedical field. These antibodies not only reduce the efficacy of the protein therapeutics, but may also block the normal function of their endogenous counterparts, which can result in serious health risks to the patient. To date, a limited number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. However, many of the existing assays involve the use of radioactive materials. We have established a novel and non-radioactive bioassay system for detecting neutralizing antibodies in patient serum samples. Our assay measures the cell metabolic activities that are closely associated with cell proliferation and apoptosis. The biologic effect of the therapeutic protein and the capability of the antibodies to neutralize the therapeutics are reflected by changes of the cellular metabolic activities triggered by the administration of the therapeutics or presence of the anti-therapeutic protein antibodies. Compared with existing assays, this new assay is equally or more sensitive, and completely eliminates the use of radioactive materials.     

抗新型冠状病毒中和抗体研究进展

自首次报道以来,新型冠状病毒（2019 novel coronavirus,2019-nCoV）引起的新型冠状病毒肺炎已迅速在全球传播。2019-nCoV感染可引起严重肺炎,危及患者生命,目前尚无特效药物。2019-nCoV刺突蛋白及其受体血管紧张素转化酶2的结构已获得解析,为药物的开发提供了基础。通过人外周血单B细胞抗体筛选技术及其他抗体筛选技术,已获得多种针对2019-nCoV的中和抗体。抗体药物的开发为新型冠状病毒肺炎的预防和治疗提供了新的选择。此文简要综述抗2019-nCoV抗体类药物的研究开发。     

The interaction of monoclonal antibodies directed against envelope glycoprotein E1 of Sindbis virus with virus-infected cells

Two monoclonal antibodies specific for the Sindbis virus envelope glycoprotein E₁ were evaluated for their ability to maintain long-term infection when present in the medium of virus-infected cells. One of them, previously shown to have neutralizing activity and to inhibit haemagglutination, caused suppression of both virus expression at the cell surface and prolonged intracellular virus presence. The other monoclonal antibody which lacked neutralizing activity but inhibited virus-specific haemolysis caused redistribution of viral antigens on the cell surface but only slightly prolonged cell survival. Both epitopes were located on the surface of the virus. By electron microscopy it was demonstrated that the determinant associated with haemolytic activity resided near the virus membrane while the haemagglutination inhibition antibody attached near the apex of the virus spikes.     

小鼠Metrnl单克隆抗体的制备及鉴定

目的 制备抗小鼠Metrnl单克隆抗体,并进行初步筛选鉴定。方法 分别制备小鼠Metrnl多肽片段和全长蛋白作为免疫小鼠抗原,取免疫后小鼠脾细胞与SP2/0骨髓瘤细胞融合,筛选阳性杂交瘤细胞,并亚克隆获得稳定细胞株,制备腹水。用ELISA方法检测腹水抗体效价;用Western blot方法鉴定抗体。结果 由14种多肽抗原制备的56株单克隆抗体中,未筛选出可用于Western blot识别Metrnl的抗体;由全长蛋白制备的25株抗体中,有12株可识别Metrnl蛋白。结论 本实验成功制备了12株单抗,可用于识别检测小鼠Metrnl蛋白。     

抗百日咳毒素单克隆抗体的纯化及应用研究

目的纯化抗百日咳毒素(PT)的单克隆抗体(M cAb),并建立特异、准确的PT定量检测方法。方法采用辛酸-硫酸铵沉淀法和A蛋白亲和层析法纯化杂交瘤细胞腹水,并经阻断抑制试验筛选识别不同表位的M cAb,用于建立定量检测PT的ELISA方法。结果经SDS-PAGE分析,纯化M cAb的纯度均在90%以上,选择二株识别不同抗原位点M cAbs,应用于检测PT的双抗夹心ELISA方法,灵敏度为2.14μg/L,批内变异系数5.85%,批间变异系数9.27%,平均回收率为108.12%,应用该法测定了国内几大生产厂家送检的无细胞百日咳疫苗原液中PT含量。结论获得了纯度高的抗PT M cAb,建立了一种特异、准确的定量检测PT的ELISA方法,并用于无细胞百日咳疫苗原液中PT含量的检测,为无细胞百日咳疫苗的质量控制提供了有力的手段。     

In vivo destruction of canine lymphoma mediated by murine monoclonal antibodies

The effect of murine anti-canine lymphoma monoclonal antibodies (MAbs) on tumor cell lysis by thioglycolate activated murine macrophages in vitro and tumor growth inhibition in athymic mice was studied. All IgG1 and IgG2a MAbs tested were able to promote specific destruction of canine lymphoma 17-71 cell line by activated macrophages. A correlation between higher ADCC activity and MAb isotype was not clearly evident. In vivo IgG2a and IgG1 MAbs inhibited the growth of canine lymphoma. These results suggest that MAbs of IgG type have potential in immunotherapy of dogs with lymphoma since they have high tumoricidal activity in vivo.     

Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor II.

AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain III of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain III (KDR-III) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-III were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells. RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-III protein. Ycom1D3 bound specifically to both the soluble KDR-III and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

The therapeutic potential of monoclonal antibodies against respiratory syncytial virus

Attempts to develop a vaccine against respiratory syncytial virus (RSV), the major cause of lower respiratory tract disease in infants and young children, have been unsuccessful. Passive immunisation with antibody to RSV has been found to be an effective alternative method for prophylaxis. The product currently in use for RSV passive immunisation, a preparation of purified human IgG containing virus-neutralising activity, requires monthly iv. infusions. Monoclonal antibodies (mAbs) are currently under development as an alternative means of treatment that would require lower doses. The first such mAb was recently approved for RSV prophylaxis in the USA. The mucosal delivery of antibodies is also effective and a mAb nose drop treatment for immunoprophylaxis is under development. The potential of passive immunisation for the treatment of existing RSV infections is not clear. Antibody treatment following infection clearly suppresses viral replication but it may not reduce disease once inflammatory processes have been initiated.     

Inhibition kinetics of monoclonal antibodies against cytochromes P450.

Monoclonal antibodies (MAbs) inhibitory to individual cytochromes P450 (P450s) are of tremendous utility in identification of P450s responsible for the metabolism of a given drug or drug candidate in pharmaceuticals. In the present study, two inhibitory MAbs against CYP2D6 (MAb(2D6-50,) IgG(2b) and MAb(2D6-184), IgG(2a)) were developed by hybridoma technology to exhibit their high specificity and potency. The MAbs were further employed to assess the quantitative role (47-93%) of CYP2D6 to the metabolism of bufuralol in human liver microsomes from seven donors. Together with the MAb inhibitory to CYP3A4 as previously reported (Mei et al., 1999), the MAbs were used to study the inhibition kinetics of dextromethorphan O-demethylation (CYP2D6), testosterone 6beta-hydroxylation (CYP3A4) and aflatoxin B1 3-hydroxylation (CYP3A4), respectively, with an adequate size of sample measurement. A kinetic model was proposed to fit the experimental observations with three-dimensional nonlinear regression, thereby resulting in a solution of kinetic parameters, i.e., K(I), K(S), V(max), alpha, and beta (changes in K(I) or K(S) and V(max) in the presence of the MAb). As a result, dissociation constants (K(I)) of the MAbs for the enzymes and the maximal inhibition (beta) values for the P450-catalyzed reactions were predicted to have 0.04 to 0.25 microM and > or =94%, respectively. The results have demonstrated that the model can accurately predict the kinetic parameters and provide some insights into the understanding of the mechanism of MAb interaction with P450 enzyme in nature and the applications of the MAbs in qualitative and quantitative identification of P450s involved in drug metabolism.     

Production and characterization of monoclonal antibodies against Staphylococcus aureus alpha-toxin

Murine monoclonal antibodies against staphylococcal alpha-toxin were produced using a well-characterized alpha-toxin fragment preparation as immunizing agent. Three monoclonal antibodies were selected for anti-alpha-toxin activity in an ELISA using alpha-toxin as antigen. The monoclonal antibodies (MAbs) belonged to different immunoglobulin classes/subclasses and showed different abilities to neutralize the hemolytic, cell-membrane-damaging, dermonecrotizing and lethal action of alpha-toxin. One MAb was superior to mouse polyclonal antiserum in all test systems except for hemolysis, whereas another MAb neutralized essentially as the polyclonal serum. The third MAb did not neutralize the hemolytic or dermonecrotic effect but still inhibited the lethal and membrane-damaging effect of alpha-toxin. These results indicate that the three MAbs recognize different epitopes on the toxin molecule and that different biological activities might correspond to these epitopes.