PRODUCTION OF PHAGE-DISPLAYED ANTI-IDIOTYPIC ANTIBODY SINGLE CHAIN VARIABLE FRAGMENTS TO MG7 MONOCLONAL ANTIBODY DIRECTED AGAINST GASTRIC CARCINOMA

Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Isolation and Characterization of Recombinant Variable Domain of Heavy Chain Anti-idiotypic Antibodies Specific to Aflatoxin B_1

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain （VHH）. The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F（ab＇）z fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic （anti-ld） responses. Antigen-specific elution method was used for panning private anti-ld VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay （ELISA）. Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.

PREPARATION OF ANTI-IDIOTYPIC ANTIBODIES SPECIFIC FOR ANTI-HEL AND ANALYSIS OF THEIR FUNCTIONAL MIMICRY

Objective. This study is to investigate the functional mimicry by using antiidiotypic antibodies of enzymes. Methods.Monoclonal antiidiotypic antibodies against antiHEL(hen eggwhite lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal antiHEL antibodies against HELs different antigenic epitopes. Then bacteriolysis of the antiidiotypic antibodies were observed. Results.Eight hybridomas strains secreting antiidiotypic antibodies were selected and characterized. It was shown that two of eight antiidiotypic antibodies secreted by two hybridomas(1A 10 C 9 and 2A 11 C 1B 3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed antiidiotypic antibodies of 1A 10 C 9 and 2A 11 C 1B 3 was stronger than that of one of them, but less than HEL. Conclusion. The results demonstrated that the antiidiotypic antibodies that could mimic enzyme activity existed in the idiotype network during antienzymatic immune response.

Enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.

ANTI-IDIOTYPIC MONOCLONAL ANTIBODIES AGAINST ANTI-OVARIAN CARCINOMA MONOCLONAL ANTIBODY COC166-9 GENERATION AND APPLICATION

Anri-idiotypic monoclonal antibody (Mab Ab2 ) by MAb COC166-9 against ovarian serous papillary adenocarcinoma was prepared. Hybridomas of Ab2 screened by sandwich ELISA and immunocompetitive inhibition tests were procured and named as 6B11 and 1H12. The number of their chromosomes were 93 and 91, and DNA analysis also proved the characteristics of hybridomas. These Ab2s could induce delayed type hypersensitivity (DTH), the cellular immune response. The results of the immune reaction of 6B11 with SKOV3 (ovarian carcinoma cell line) were similar to OC166-9 (Ag), the positive control, while 1H12 was weaker. Anti-and-idiotypic antibody (Ab3) was also raised by 6B11 and 1H12 respectively. They all showed positive immunohistochemical stainings with ovarian serous adenocarcinoma tissue sections and immunocytochemical stainings with SKOV3 cells as was shown by COC166-9. In the antibody dependent cell mediated cytotoxicity (ADCC) tests, they showed no differences against SKOV3 as compared with COC166-9. We anticipate that 6B11 and 1H12 may be used as vaccines against ovarian carcinoma and may provide a clue for its prevention and treatment.

Induction of Human Anti-Human Antibody Responses (Ab2) after Application of a Humanized Lewis Y Carbohydrate Specific Antibody (Ab1): Connection of Prolonged Disease Stabilization with Ab3 Induction?

Purpose: Detailed analysis of a patient with epithelial Lewis Y (LeY) positive cancer who received twice 50 mg of the humanized Lewis Y carbohydrate specific mAb IGN311 and developed a clinically significant human anti-human antibody (HAHA) response (Ab2). Results: Clinical stabilization of the disease was assigned to in this patient. The HAHA response consisted mainly of IgG1 and was found to be directed against the IGN311 binding site. Consistent with the induction of the HAHA response, CDC activity against Lewis Y positive target cells was completely abolished at day 8 and could not be restored by the second 50 mg infusion indicating complete neutralization of applied IGN311. The ADCC reactivity was also significantly reduced and anti-anti idiotype-specific antibodies (Ab3) were detectable at day 65. Conclusions: Induction of Ab3 antibodies should be considered as an additional factor influencing the efficacy of humanized antibodies. In this context, the potential threat of induced HAHA responses against therapeutic mAbs might have to be reconsidered because they might actually have also beneficial immunological long-term effects leading to an active immunization component induced by therapeutic antibodies.

Immunomodulation of Human Carcinogenesis by the Blood Serum Antibodies against Benzo[a]pyrene, Estradiol and Progesterone

It was supposed that lung and breast cancer risks significantly increased when the levels of serum immunoglobulins A antibodies against benzo[a]pyrene and estradiol increased together, but did not separately. However, the cancer risks dramatically decreased when the levels of immunoglobulins A against progesterone elevated separately or together with immunoglobulins A against benzo[a]pyrene and estradiol. So, immunoglobulins A against benzo[a]pyrene and immunoglobulins A against estradiol acted as co-initiator and co-promoter in developing cancer scenario, but immunoglobulins A against progesterone acted along or conjointly with immunoglobulins A against benzo[a]pyrene and estradiol as strongly inhibitor in human carcinogenesis. Also it was suggested the precise mechanism of carcinogenesis modulation using anti-idiotypic antibodies against estradiol and progesterone through their membrane steroid receptors.

Humoral immune responses induced by anti-idiotypic antibody fusion protein of 6B11scFv/hGM-CSF in BALB/c mice

Background We have previously developed and characterized a monoclonal anti-idiotype antibody, designated 6B 11, which mimics an ovarian carcinoma associated antigen OC166-9 and whose corresponding monoclonal antibody is COC166-9 （Abl）. In this study, we evaluate the humoral immune responses induced by the fusion protein 6B11 single-chain variable fragment （scFv）/human granulocyte macrophage colony-stimulating factor （hGM-CSF） and 6B 1 lscFv in BALB/c mice. Methods The fusion protein 6B 11 scFv/hGM-CSF was constructed by fusing a recombinant single-chain variable fragment of 6B11scFv to GM-CSE BALB/c mice were administrated by 6B11scFv/hGM-CSF and 6B11scFv, respectively. Results The fusion protein 6B11scFv/hGM-CSF retained binding to the anti-mouse F（ab）2＇ and was also biologically active as measured by proliferation of human GM-CSF dependent cell TF1 in vitro. After immunization with the 6B11scFv/hGM-CSF and 6BllScFv, BALB/c mice showed significantly enhanced Ab3 antibody responses to 6B11 scFv/hGM-CSF compared with the 6B11 scFv alone. The level of Ab3 was the highest after the first week and maintained for five weeks after the last immunization. Another booster was given when the Ab3 titer descended, and it would reach to the high level in a week. Conclusion The fusion protein 6B11scFv/hGM-CSF can induce humoral immunity against ovarian carcinoma in vivo. We also provide the theoretical foundation for the application of the fusion protein 6B11 scFv/hGM-CSF for active immunotherapy of ovarian cancer.

Preparation of Anti-Idiotypic Antibody against Avian Influenza Virus Subtype H9

To generate monoclonal anti-idiotypic antibodies(mAb2)against avian influenza virus subtype H9(H9 AⅣ), BALB/c mice were immunized with purified chicken anti-H9-AⅣ IgG and the splenocytes of immunized mice were fused with myeloma cells NS-1.Hybridoma cells were screened by indirect enzyme-linked immunosorbent assays with both chicken and rabbit anti-H9-AⅣ IgG as coating antigens.One hybridoma cell clone secreting monoclonal antibody against idiotypes shared by both chicken and rabbit anti-H9-AⅣ IgG was established.Experiments demonstrated the mAb2 was able to inhibit the binding of hemagglutinin to anti-H9-AⅣ IgG and to induce chickens to generate hemagglutination inhibition antibodies,indicating this anti-species-sharing-idiotypic antibody bore the internal image of hemagglutinin on avian influenza virus.Cellular & Molecular Immunology.2005;2(2): 155-157.

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis

Background The development of new adjuvants for human use has been the focus of attention. This study’s aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms. Methods Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG,IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-γ and IL-4 in supernatant of splenocytes were determined via ELISA. Results Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG,IgG1 and IgG2a antibodies against NP30 increased remarkably,as compared with those of the group immunized with NP30 alone. The concentration of IFN-γ in supernatant of splenocyte was drastically elevated [the groups immunized with CA-NP30 and NP30 alone were (493.80±400.74) pg/ml and (39.03±39.58) pg/ml,respectively],but the concentration of IL-4 showed no significant difference from that of NP30 alone [(27.94±9.84) pg/ml vs (27.28±14.44) pg/ml]. Conclusions Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.

蝶蛹金小蜂卵黄蛋白单克隆抗体的制备及其应用方法的建立

为深入研究寄生蜂卵黄发生及其内分泌调控，特采用杂交瘤细胞技术，制备4株能稳定分泌抗蝶蛹金小蜂Pteromalus puparum卵黄蛋白（vitellin，Vt）的单克隆抗体（mAb），即PpVtmAb1，PpVtmAb2，PpVtmAb3和PpVtmAb4。这4株单克隆抗体的重链和轻链的亚类均分别为IgG1和κ类型，不仅特异性识别Vt，而且识别雌蜂血淋巴中卵黄原蛋白（viteUogenin，Vg），但与雄蜂体液无反应。通过比较4种不同的ELISA方法，确定了微量检测蝶蛹金小蜂体内Vg／Vt的最适ELISA法，即双夹心ELISA法。该方法可用于单头雌蜂体内Vg／Vt的检测，其检测灵敏度为20ng／mL。用Western免疫印迹的方法证实了该蜂Vg的合成始于刚羽化的成虫，并在羽化后12～36h内含量达到高峰。

异色瓢虫卵黄蛋白单克隆抗体的制备及鉴定

【目的】为了能准确地追踪异色瓢虫Harmonia axyridis(Pallas)卵黄原蛋白(vitellogenin,Vg)的合成、转运途径和吸收方式,以及卵黄蛋白(vitellin,Vn)在卵母细胞内的积累及分布情况,本研究对异色瓢虫的Vn进行了单克隆抗体(monoclonal antibody,Mc Ab)的制备。【方法】以异色瓢虫Vn免疫BLAB/C小鼠,应用杂交瘤技术,经过3次亚克隆筛选,制备能稳定分泌抗Vn的单克隆抗体。【结果】实验获得4株能够稳定分泌抗异色瓢虫Vn的单克隆抗体,即5E2,5E11,1E9和5H8。其中1E9,5E11和5E2亚型均为Ig G1,5H8亚型为Ig M。Western blot免疫印迹分析显示,4株单克隆抗体可以特异性地识别Vn,而与雄虫血淋巴无反应。其中,5E2和1E9可以与异色瓢虫抗原的4个亚基发生较强的免疫反应,结合腹水制备前上清效价检测结果最终选取5E2制备单克隆抗体。5E2单克隆抗体的效价为1∶81 000,SDS-PAGE分析显示5E2重链和轻链的分子量分别为50和27 k D。【结论】本实验成功制备出一株能够稳定分泌抗异色瓢虫Vn的单克隆抗体,为建立酶联免疫吸附试验(ELISA)方法测定其动态变化奠定了基础。

ELISA测定德国小蠊卵黄蛋白含量方法的建立

本文采用蒸馏水沉淀法提取了德国小蠊卵黄蛋白，免疫家兔，并将所得抗血清经雄虫血淋巴吸收，获得抗卵黄蛋白特异抗体，用ＤＥＡＥ－纤维素批次法纯化抗体ＩｇＧ，并用辣根过氧化物酶标记，建立ＥＬＩＳＡ直接法测定卵黄蛋白含量的方法。

A clinical trial of active immunotherapy with anti-idiotypic vaccine in nasopharyngeal carcinoma patients

OBJECTIVE: To investigate the effect of active immunotherapy with anti-idiotypic vaccine in patients with nasopharyngeal carcinoma (NPC). METHODS: Anti-idiotypic antibodies (2H4/5D3) bearing the internal image of the NPC antigen were used in active immunotherapy in NPC patients receiving radiotherapy. Antibodies and cytokine levels in patient sera were determined using ELISA before and after active immunotherapy. IL-2 mRNA expression in the peripheral blood mononuclear cells (PBMC) was measured by in situ hybridization. RESULTS: Nineteen patients with NPC at stage IV were treated with alum-precipitated 2H4 or 5D3. Neither hypersensitivity nor adverse side effects were observed. The levels of anti-anti-idiotypic antibodies (Ab3) and anti-NPC antibodies (Ab1') were increased. Human anti-mouse antibodies (HAMA) were seen in 19 patients of the experimental group; the levels of Ab1' did not increase in the control group. Serum IL-2, IFN-gamma and TNF-alpha levels were increased in most patients in the experimental group, while no differences were observed in Ab1' and cytokine levels between pre- and post-therapy in the control group. In addition, IL-2 mRNA expression in PBMCs from NPC patients was closely related to serum IL-2 (r = + 0.8829) levels by in situ hybridization. CONCLUSIONS: Anti-idiotype vaccine is safe for clinical active immunotherapy. Anti-idiotypic vaccine might be able to enhance humoral and/or cellular immunity in NPC patients receiving radiotherapy.

鸭病毒性肝炎高免卵黄抗体的研制

用DVH弱毒疫苗和DVH油乳剂苗免疫健康高产蛋鸡制备高免卵黄抗体,研究了高免卵黄抗体治疗雏鸭DVH的疗效。结果表明,高免卵黄抗体治疗DVH的疗效取决于卵黄抗体的中和效价、治疗时间和免疫途径。卵黄抗体的中和效价应达28.5以上,并且在感染DVH 24 h以内对雏鸭进行肌肉注射,免疫效果最好。

鸭病毒性肝炎高免卵黄抗体的研制

用DVH弱毒疫苗和DVH油乳剂苗免疫健康高产蛋鸡,可以制备高免卵黄抗体。高免卵黄抗体治疗雏鸭病毒性肝炎疗效的试验表明,用卵黄抗体治疗DVH的疗效取决于卵黄抗体的中和效价及治疗的时间。卵黄抗体的中和效价应达28.5以上,并且在感染DVH24 h以内对雏鸭进行肌肉注射,免疫效果为最适宜。

长角血蜱卵黄蛋白单克隆抗体的制备及鉴定

为研究蜱类的卵黄发生及其机理,用长角血蜱Haemaphysalis longicornis卵黄蛋白(vitellin,Vn)免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0进行融合,经3次克隆化筛选,获得6株能稳定分泌抗Vn的单克隆抗体(McAb),即1B5,2A7,2B8,2F2,3A1和3G1。1B5,2B8和2F2亚型为IgGA;2A7亚型为IgG1;3A1和3G1亚型为IgG2a。6株McAb均具有高度特异性,效价在1∶105以上。选取效价和特异性最好的1株抗体1B5进行SDS-PAGE分析和亲和力测定,测得1B5重链和轻链的分子量分别为58kD和21kD,亲和常数为2.8993×10-6。Western免疫印迹分析发现6株单抗均与Vn的8个亚基发生免疫反应。本研究成功制备了6株抗长角血蜱Vn的单克隆抗体,为深入阐明长角血蜱卵黄发生的过程与调控机理提供了重要的工具。

Construction and Expression of a Single Chain Antibody Mimicing Human Ovarian Cancer Antigen CA125

One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody W J02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02, could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.

Localization of Abscisic Acid Binding Proteins in Maize Root Tip Using Immunogold-Silver Staining Method

Antisera against abseisic-acid-binding proteins(Ab Ⅰ)and anti-idiotypic antibodies to ABAmonoclonal antibody(Ab Ⅱ)have been developed and tested to comparatively localize abacisic-acid-bindingproteins(ABBPs)in maize root tips using immunocytochemistry technique.Based on the staining pattern ob-served along the root tip,an apicobasal gradient of ABBP has been revealed with either Ab Ⅰ or Ab Ⅱ.Thelabels are mainly concentrated in,the root cap and apical meristem.In the elongation zone and root hair zone,only cells in pericycle and epidermis are obviously labeled.The silver labels are distributed evenly in thewhole cell of the apical meristem,but in the cells of the root cap and elongation zone,the positive stainingdegree in the cytoplasm is reduced even to completely negative staining;nevertheless,the nuclei and plasmamembranes are strongly labeled.As in the labeled cells of the root hair zone,only some immunoactive sitescan be seen in the nucleus compacted to the edge of the cells.The staining pattern in the whole root tip alsoshows obvious dependence of the ABBPs localization on the activity of the nucleus.The significance ofABBPs localization is discussed.

Immunohistochemical localization of dopamine and norepinephrine receptors in human early placental villi

Evidence so far accumulated suggests that the human early placenta can be considered as a complicated neuroendocrine organt. Our previous reports have demonstrated that the human early placenta presents not only a variety of neuropeptides but also neurotransmitters such as 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE). It has been shown that these neurotransmitters in human placenta may play an important role in the process of blastocyst implantation and maintenance of normal pregnancy. As far as we are aware, no information concerning the DA and/or NE receptors localized in