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.

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.

BASIC STUDY AND PHASE I CLINIC TRIAL OF ANTI-IDIOTYPIC ANTIBODY MIMICKING NASOPHARYNGEAL CARCINOMA CELL ANTIGEN

An anti-idiotypic monoclonal antibody 2A9 (Ab2)was prepared, which mimicked the nasopharynegealcarcinoma (NPC) cell antigen defined by anti-NPC McAbFel. The abilities of 2A9's inducing humoral and cellularimmunity against NPC cell antigen were studied insyngeneic mice by inducing anti-Ab2 sera (Ab3) anddelayed-type hypersensitivity. Two periods of phasc Ⅰclinical trials were carried out, stage Ⅳ NPC patientsreceiving radiotherapy were chosen. Human anti-mouseantibodies (HAMA), anti-anti-idiotypic antibodies (Ab3),and anti-NPC cell antibodies (Ab1') were detected byELISA. TNF-α,IL-2, IFN-γ levels in sera were determinedby ELISA Kits. IL-2 mRNA expression in peripheralblood mononuclear cells (PBMC) were shown by in situhybridization. The results showed that 2A9 was safe inapplying on NPC patients and induced some humoraland/or cellular immunc responses.

广州市花地街道55岁及以上健康体检居民乙型病毒性肝炎流行状况分析

目的:了解广州市花地街道55岁及以上中老年健康体检人群乙型病毒性肝炎(简称乙肝)感染及疫苗接种情况。方法:对940名55岁及以上中老年体检居民采集静脉血,采用酶联免疫吸附试验进行血清学检测,描述其阳性率及分布特征。结果:共调查940人,乙肝病毒表面抗原(HBsAg)阳性率、乙肝病毒表面抗体(HBsAb)阳性率和乙肝病毒核心抗体(HBcAb)阳性率分别为8.30%,60.74%,30.11%。不同年龄、乙肝既往史比较,HBsAg阳性率差异有统计学意义(P<0.05);不同乙肝既往史、乙肝疫苗接种史比较,HBsAb阳性率差异有统计学意义(P<0.05)。有乙肝既往史的户籍、已婚人群为高危人群,无乙肝疫苗接种史的女性、高中/中专、户籍、已婚人群为高危人群;不同乙肝既往史比较,HBcAb阳性率差异有统计学意义(P<0.05)。乙肝病毒易感率为27.26%。不同户籍类型比较,乙肝病毒易感率差异有统计学意义(P<0.05)。结论:花地街道55岁及以上人群乙肝表面抗原阳性率水平较高,应对易感人群补种、加强或重新接种乙肝疫苗,提高人群免疫水平,降低乙肝病毒易感率。

Identification of colorectal cancer metastasis markers by an angiogenesis-related cytokine-antibody array

AIM:To investigate the angiogenesis-related protein expression profile characterizing metastatic colorectal cancer (mCRC) with the aim of identifying prognostic markers.METHODS:The expression of 44 angiogenesissecreted factors was measured by a novel cytokine antibody array methodology.The study evaluated vascular endothelial growth factor (VEGF) and its soluble vascular endothelial growth factor receptor (sVEGFR)-1 protein levels by enzyme immunoassay (EIA) in a panel of 16 CRC cell lines.mRNA VEGF and VEGF-A isoforms were quantified by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) and vascular endothelial growth factor receptor (VEGFR)-2 expression was analyzed by flow cytometry.RESULTS:Metastasis-derived CRC cell lines expressed a distinctive molecular profile as compared with those isolated from a primary tumor site.Metastatic CRC cell lines were characterized by higher expression of angiogenin-2 (Ang-2),macrophage chemoattractant proteins-3/4 (MCP-3/4),matrix metalloproteinase-1 (MMP-1),and the chemokines interferon γ inducible T cell α chemoattractant protein (I-TAC),monocyte chemoattractant protein I-309,and interleukins interleukin (IL)-2 and IL-1α,as compared to primary tumor cell lines.In contrast,primary CRC cell lines expressed higher levels of interferon γ (IFN-γ),insulin-like growth factor-1 (IGF-1),IL-6,leptin,epidermal growth factor (EGF),placental growth factor (PlGF),thrombopoietin,transforming growth factor β1 (TGF-β1) and VEGF-D,as compared with the metastatic cell lines.VEGF expression does not significantly differ according to the CRC cellular origin in normoxia.Severe hypoxia induced VEGF expression up-regulation but contrary to expectations,metastatic CRC cell lines did not respond as much as primary cell lines to the hypoxic stimulus.In CRC primary-derived cell lines,we observed a twofold increase in VEGF expression between normoxia and hypoxia as compared to metastatic cell lines.CRC cell lines express a similar pattern of VEGF isoforms (VEGF 121,VEGF 165 and VEGF 189) despite variability in VEGF expression,where the major transcript was VEGF 121.No relevant expression of VEGFR-2 was found in CRC cell lines,as compared to that of human umbilical vein endothelial cells and sVEGFR-1 expression did not depend on the CRC cellular origin.CONCLUSION:A distinct angiogenesis-related expression pattern characterizes metastatic CRC cell lines.Factors other than VEGF appear as prognostic markers and intervention targets in the metastatic CRC setting.

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.

Comprehensive Online Management Based on Cloud Platform for Breast Cancer Patients Using Dual-Targeted Therapy with Macromolecular Monoclonal Antibodies

Objective: The purpose of the study was to explore the application effects of the cloud platform-based comprehensive online management for breast cancer patients using dual-targeted therapy with macromolecular monoclonal antibodies. Methods: 120 breast cancer patients treated by dual-targeted therapy with macromolecular monoclonal antibodies were managed by a cloud platform from March to November 2019. Comprehensive online management included consultation about drugs and side effects and frequently asked questions in the dual-targeted therapy with macromolecular monoclonal antibodies. Results: In the consultation about drugs and side effects, there were five patients with fever, neutrophil, cough, and fatigue;24 with diarrhea;25 with nausea;11 with oral mucosal inflammation;10 with rashes and dry skin;8 with insomnia;and 1 with palpitation. Moreover, 110 patients with anxiety about the missed or delayed treatment were properly handled. Conclusion: The comprehensive online management of dual-targeted therapy with macromolecular monoclonal antibodies based on the cloud platform is helpful to satisfy the at-home breast cancer patients’ needs, ensure the continuity of dual-targeted therapy with macromolecular monoclonal antibodies for breast cancer patients, prevent misinformation, alleviate patients’ negative psychological emotions, and reduce patients’ economic losses. The online cloud platform integrated management model is crucial for managing patients with breast cancer treated by dual-targeted therapy.

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.

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.

单抗免疫酶斑点法检测GGT-Ⅱ在肝癌诊断中的价值

背景与目的:肝癌特异性γ-谷氨酰转肽酶同工酶Ⅱ(gamma-glutamyltransferaseisoenzymeⅡ,GGT-Ⅱ)被认为是甲胎蛋白(AFP)以外的最佳肝癌标志物,但目前尚无简便的检测方法。本研究目的是探讨单克隆抗体免疫酶斑点法检测GGT-Ⅱ对肝细胞癌的诊断价值。方法:纯化GGT-Ⅱ同工酶,免疫BALB/c小鼠,利用杂交瘤技术制备单克隆抗体。采用免疫酶斑点法与电泳法同步检测123例肝癌和164例不同良性肝病患者血清中GGT-Ⅱ。结果:单克隆抗体免疫酶斑点法检测GGT-Ⅱ对肝细胞癌的诊断阳性率(71.5%)与电泳法(76.4%)相比无明显差异。但免疫酶斑点法假阳性率(肝硬化23.7%,慢性肝炎27.1%)高于电泳法(肝硬化10.0%,慢性肝炎8.4%)。结论:单克隆抗体免疫酶斑点法检测GGT-Ⅱ对肝细胞肝癌诊断有一定价值,但诊断特异性有待提高。

YggG截短蛋白的表达及其抗体的制备

目的 :在大肠杆菌中表达截短的YggG蛋白 ,并制备兔抗YggG截短体抗体。方法 :从含有大肠杆菌yggg基因全长DNA的质粒中 ,用PCR扩增截短的yggg基因序列 ,克隆入非融合表达载体pDH2中 ,构建YggG截短体的高表达工程菌 ,并温敏诱导表达非毒性的YggG蛋白 ,薄层扫描检测目的蛋白含量。免疫家兔制备兔抗YggG突变体抗血清 ,并以Western blot检测抗体效价、鉴定抗体特异性。结果 :大肠杆菌中表达的YggG截短突变体蛋白占菌体总蛋白的 3 1.7%。抗血清效价约 1∶2 0 0 0 ,Westernblot分析显示抗血清具有较高的特异性。结论 :成功地获得了YggG截短体蛋白 ,并制备了兔抗YggG突变体抗血清。

卵巢癌抗独特型微抗体刺激树突细胞激活的T细胞对自体卵巢癌细胞杀伤作用的研究

背景与目的:以抗原肽、蛋白、肿瘤细胞冻融物致敏树突细胞的肿瘤免疫治疗已在多种肿瘤展开。6B11抗独特型微抗体能模拟卵巢癌相关抗原OC166-9,本研究用6B11抗独特型微抗体体外刺激树突细胞,以诱导出对卵巢癌患者自体肿瘤细胞抗原特异性的细胞毒作用。方法:取10例卵巢癌患者的外周血,分离并诱导培养树突细胞,在培养过程中用6B11抗独特型微抗体刺激,促成熟后在体外激活自体T细胞。3H-TdR掺入法测DCs对自体T细胞刺激增殖的作用;51Cr6h释放实验测激活的T细胞对自体肿瘤细胞特异性的杀伤。结果:9例中有4例(#4,#5,#7,#10)6B11微抗体负载组DC刺激自体T细胞增殖3H-TdR掺入的cpm值高于对照组;杀伤实验的10例患者中5例(#1,#2,#4,#7,#9)6B11微抗体负载组DC刺激自体T细胞(简称MINI-DC-T)对自体肿瘤细胞有特异性杀伤。在效靶比为20∶1时,各组不同的效应细胞对自体肿瘤细胞的杀伤率分别为:MINI-DC-T组25%～100%、F(ab)2-DC-T组18%～40%、unpulsed-DC-T组13%～43%、T细胞组9%～58%,MINI-DC-T组高于其它各组。在效靶比为20∶1时,4例(#1,#2,#4,#9)患者MINI-DC-T对自体肿瘤细胞及SKOV3、HLE、K562细胞的杀伤率分别为25%～100%、5%～51%、2%～38%和2%～25%,MINI-DC-T对自体肿瘤细胞的杀伤高于对照组靶细胞。

EB病毒VCA－IgA抗体阴性鼻咽癌病人的远期疗效

本文首次报告５０例ＥＢ病毒ＶＣＡ－ＩｇＡ抗体阴性鼻咽癌病人的临床特征及远期疗效。５０例病人中，颈淋巴阴性（Ｎｏ）病人占５８％（２９／５０）其中Ｔ１－２ＮｏＭｏ病人１９例。本组病人５年生存率６６％，１０年生存率５４％．较高的生存率与Ｎｏ病例多有关。随着Ｎ分期的升级，生存率下降。Ｔ３－４ＮｏＭｏ病人给予较高剂量照射，其１０年生存率为３３．３％；Ｔ分期较早病人给予低剂量照射，也获得较高的生存率，提示不同了分期的病人需要不同的放射剂量。本文还总结、讨论了此类病人的临床症状，抗体阴性可能原因及诊断方法。

临床实习学生SARS感染流行病学分析

目的 了解临床实习学生感染SARS的情况 ,为做好今后的防治工作提供依据。方法 采用整群抽样和流行病学个案调查 ,对中山大学临床医学各年级本科生进行回顾性调查 ;对患过SARS病的 9名学生以及与他们密切接触的 2 0名学生进行SARS病毒特异性IgG抗体检测。 结果 在 2 3 7名实习学生中 ,有 10人罹患SARS ,患病率为 4 2 2 %。参与“抗非典”一线医疗工作的有 2 0人 ,其中 6人感染SARS ,患病率为 3 0 0 % ;在医院实习或见习 ,但未参与“抗非典”一线医疗工作有 2 17名 ,其中 4人感染SARS ,患病率为 1 84%。结论 临床实习学生是SARS的高危人群 ,应积极做好防护工作 。

腰椎间盘突出症中自身体液免疫异常的实验研究

目的从动物实验的角度探讨腰椎间盘突出症中自身体液免疫异常。方法在SD大鼠身上建立非机械压迫性腰椎间盘突出症模型,运用透射比浊法和免疫组织化学染色针对大鼠外周血免疫球蛋白IgG、IgM和髓核中抗原抗体复合物沉积进行检测,并用HE染色观察大鼠神经根的变化。结果大鼠模型建立后,后肢机械缩爪阈值明显降低(P<0.01),其外周血IgG、IgM水平出现有统计学意义的升高(P<0.05),髓核中出现抗原抗体复合物阳性沉积(P<0.01),其相应节段神经根出现较明显的炎性反应。结论腰椎间盘突出症中存在自身体液免疫异常,这可能是其发病机制之一。

五种赤潮藻单克隆抗体的制备

目的:建立分泌抗海洋褐胞藻(Chattonellamarina)、卵圆褐胞藻(Chattonellaovata)、赤潮异弯藻(Heterosigmaakashiwo)、具齿原甲藻(Prorocentrumdentatum)、尖刺拟菱形藻(Pseudonitzschiapungens)等5种藻的高特异性单克隆抗体杂交瘤细胞株.方法:利用细胞融合技术制备杂交瘤细胞,并通过反相间接血凝、间接酶联免疫吸附测定(ELISA)等方法筛选克隆阳性杂交瘤细胞株.结果:获得15CA7、23BG2和23BG9等3株杂交瘤细胞分泌的单克隆抗体与5种藻具有一定的特异性,但抗体的亲和力较低,仅与海洋褐胞藻具有较高的亲和力;而获得的M18杂交瘤细胞株分泌的单克隆抗体可与以上除尖刺拟菱形藻外4种藻产生高效价的明显交叉反应.结论:获得了3株分泌对5种藻具有一定的特异性较低亲和力抗体;而M18细胞株分泌的单抗与除尖刺拟菱形藻外4种藻产生明显的高效价的交叉反应,显示此单抗可代替5种海洋藻的抗血清.

自身免疫性溶血性贫血输血前检查的研究

目的探讨自身免疫性溶血性贫血(AIHA)对输血前检查3项的影响,建立AIHA患者血液标本的研究性实验方法及交叉配血策略。方法对392份AIHA患者血液标本进行血型鉴定、抗体筛选和鉴定、DAT分型及交叉配血试验,结合患者的临床诊断、治疗及免疫史,分析试验结果并选择盐水介质直接离心法、LISS/Coombs卡、经典抗人球蛋白试验等不同方法进行抗体筛选、鉴定及交叉配血。结果 392份AIHA标本中338份(86.2%)为温自身抗体,17份(4.3%)为冷自身抗体,37份(9.4%)为混合型自身抗体(温+冷自身抗体),其中45份同时存在同种抗体;48份自身抗体具有类特异性,344份自身抗体无特异性(全凝集素);交叉配血方法选择LISS/Coombs卡相合21份,试管法LISS-IAT相合22份,经典抗人球蛋白法相合218份,配血均不相合153份。结论对于AIHA患者血液标本应建立完整的输血前检查试验方法,根据试验结果选择适合的交叉配血方法,保障临床输血的及时、安全和有效性。

自身免疫性感音神经性聋血清抗膜迷路蛋白抗体的检测

采用改良免疫转印法分析了拟诊为自身免疫性感音神经性听力减退患者体内抗膜迷路蛋白抗体，并将该法与常规法比较，发现用该法在患者血清中最多可检出５条强阳性带（含６８０００），阳性率为７２％（１８／２５），显著高于常规法（４０％）。还在部分患者体内检出了抗肺炎克雷白杆菌膜蛋白抗体（７／９）。

都江堰地区美沙酮门诊吸毒人群丙肝、梅毒抗体检测分析

目的探讨都江堰地区吸毒人员的社会学特征及其丙肝、梅毒的感染情况。方法对都江堰市精神卫生中心美沙酮门诊的120位吸毒人员发放调查问卷,并抽取静脉血检测丙肝抗体、梅毒抗体。结果本调查发现都江堰地区的吸毒人群存在几个显著特点:①吸食的毒品以海洛因为主;②低龄化吸毒现象较为突出:初次吸食毒品在18岁以下的占17.50%(21/120);③无业人员占74.17%(89/120),初中以下文化程度占75.00%(90/120);④情感、婚姻问题成为吸毒主要诱因,未婚、离异及丧偶共占62.50%(75/120);⑤调查的吸毒人员女性梅毒阳性率高于男性,差异有统计学意义(P<0.01);丙肝阳性率性别差异无统计学意义(P>0.05)。结论针对都江堰地区吸毒人员特点,重点应做好以下工作:①关注高危人群,进一步提高其对毒品及相关疾病的认识,以减少其对药物的依赖,并保持有效的控制体系;②倡导社会和家人帮助吸毒人员回归社会,重新树立对生活的希望;③从小加强对毒品的宣传教育,增强全民禁毒意识。

系统性红斑狼疮患者血清抗内皮细胞抗体和抗β_2糖蛋白I抗体水平及其对人脐静脉内皮细胞增殖的影响

目的:检测抗内皮细胞抗体(AECA)和抗β2GP-Ⅰ抗体在SLE患者血清中的水平及探讨AECA对人脐静脉内皮细胞(HUVEC)增殖的影响。方法:收集系统性红斑狼疮(SLE)患者90例和正常对照40例,留取血清,经酶联免疫吸附法(ELISA)检测AECA和抗β2GP-Ⅰ抗体水平。收集SLE患者部分临床指标,分析AECA和抗β2GP-Ⅰ抗体水平与临床指标间的关系。将不同浓度AECA的SLE患者血清加入HUVEC培养基,应用四唑盐比色试验(MTT)法检测各组OD值判断内皮细胞增殖状态。结果:SLE患者血清中AECA和抗β2GP-Ⅰ抗体水平显著高于正常对照组(P<0.01)。在90例SLE患者中33例抗β2GP-Ⅰ抗体阳性,该33例SLE患者对应的AE-CA、APTT水平和SLE活动评分(SLEDAI)积分均显著高于其余57例SLE患者(P<0.01,P<0.05,P<0.05)。同时该33例患者AECA和抗β2GP-Ⅰ抗体水平呈高度正相关(r=0.984,P<0.01),AECA和抗β2GP-Ⅰ抗体水平均与SLE-DAI呈正相关(P<0.05),抗β2GP-Ⅰ抗体水平与凝血酶原时间(PT)间存在正相关(r=0.349,P<0.05)。空白对照组、健康对照血清组和低浓度AECA血清组间OD值无差异,而高浓度AECA血清组OD值显著低于以上3组(P<0.05)。结论:AECA和抗β2GP-Ⅰ抗体与SLE疾病活动相关,AECA在体外对HUVEC增殖有抑制作用。