单克隆抗体免疫结合物~(125)Ⅰ—3A_1在裸鼠淋巴瘤模型体内示踪研究

本文报道利用~(125)I 标记的抗 CD_7抗原决定簇的单抗 3A_1,在人 T 淋巴瘤裸鼠模型及人鼻咽癌裸鼠模型体内进行生物学分布和显像研究。注射标记抗体26小时后,淋巴瘤裸鼠模型中出现肿瘤显像。96小时后处死裸鼠,测定肿瘤及各器官中的放射性分布,结果表明 McAb 3A_1在 CEM 荷瘤裸鼠的肿瘤组织中有特异性的浓集。~(125)I 标记的对照抗体,鼠抗乙型肝炎病毒核心抗原抗体,在 T 淋巴瘤裸鼠模型的肿瘤部位无明显浓集。     

单克隆抗体与基因工程抗体免疫结合物导向治疗肿瘤的进展

单克隆抗体治疗恶性肿瘤虽有些成功的报道，但其效果尚不甚理想，但它却是长期以来梦寐以求的治疗肿瘤以求的“魔弹”部件。因为抗肿瘤的单克隆抗体，在特异性与敏感性上均超过多克隆抗体并具有对肿瘤相关抗原的特异性，因此，它是药物、毒素、酶和放射性同位素的良好载体，如同导弹能够准确地攻击癌细胞。近年来由于基因工程抗体的问世，为肿瘤的免疫治疗开拓了新的前景。一、单克隆抗体免疫结合物1．单克隆抗体在核医学上的应用已获得很大进展，特别是对病人扫描技术的发展及肿瘤的定位。在原理上，抗肿瘤抗体可以携带放射活性物质，用于…     

抗荧光素单克隆抗体免疫磁珠的制备及初步应用

目的制备抗荧光素单克隆抗体免疫磁珠,并进行初步应用。方法采用B淋巴细胞杂交瘤技术制备抗荧光素单克隆抗体;制备腹水,经50%硫酸铵粗提后,再经阴离子交换树脂DE52进一步纯化单克隆抗体;采用氧化共沉淀法制备纳米磁性粒子;乳液聚合法制备羧基磁性微球;用碳二亚胺将抗荧光素单克隆抗体共价偶联于磁性微球表面,制备抗荧光素单克隆抗体免疫磁珠。用制备的免疫磁珠检测乙肝表面抗原,并与市售ELISA试剂盒的检测结果进行比较。结果共获得5株杂交瘤细胞株,其中1F12细胞株分泌的抗体效价、相对亲和力较高,特异性较好;纯化的1F12株单抗的纯度达95%,蛋白含量为2.4 mg/ml,ELISA效价为106,相对亲和力为0.2 mg/L,与FITC标记的BSA可特异性结合;纳米磁性粒子的平均粒径为150 nm,铁含量为71.63%;羧基磁性微球的平均粒径为210 nm,羧基含量约为2.15 mmol/g;每克羧基磁性微球可结合抗荧光素单克隆抗体约12 mg,制备的免疫磁珠可有效结合荧光素标记的蛋白。应用抗荧光素单克隆抗体免疫磁珠检测乙型肝炎表面抗原的灵敏度高于ELISA试剂,检测限达0.1 ng/ml。结论成功制备了抗荧光素单克隆抗体免疫磁珠,其具有应用于免疫检测分析的价值。     

多价肺炎链球菌荚膜多糖结合物对小鼠免疫应答的影响

目的探讨多价肺炎链球菌荚膜多糖结合物对小鼠免疫应答的影响。方法分别用单一载体(破伤风类毒素)和混合载体(破伤风类毒素和白喉类毒素)作为多糖结合物的载体蛋白,制备5～9价肺炎链球菌荚膜多糖结合物,免疫NIH小鼠,采用间接ELISA法测定免疫小鼠血清中抗荚膜多糖特异性IgG抗体水平。结果多价肺炎链球菌荚膜多糖结合物的免疫效果优于相应的单价结合物;随着结合物配制价数的增高,小鼠的免疫应答能力趋于下降;多价结合物中使用两种载体能缓解此现象,但载体含量过高时,依然会抑制免疫应答。结论多价肺炎链球菌荚膜多糖结合物免疫小鼠后,随着载体剂量的增高,会出现载体诱导的表位抑制现象,使小鼠的免疫应答反应减弱。两种载体蛋白同时使用能缓解此现象。     

抗旋毛虫硒蛋白T单克隆抗体的制备及其对裸鼠肝癌H7402细胞的抑制效果

目的制备抗旋毛虫与肝癌H7402细胞相关抗原硒蛋白T单克隆抗体,并观察其对裸鼠肝癌H7402细胞的抑制效果。方法用纯化的旋毛虫硒蛋白T重组蛋白常规免疫BALB/c小鼠,采用杂交瘤细胞技术制备其单克隆抗体;将肝癌H7402细胞经大腿内侧皮下接种BALB/c裸鼠,1×10^7个细胞/只,采用制备的高效价单抗,按20、50及100μg/只剂量接种裸鼠,隔日1次,共5次,评价单抗对裸鼠肝癌H7402细胞的抑制效果。结果成功获得了3株稳定分泌抗旋毛虫硒蛋白T的单抗的细胞株:4D4、4H2及4F1,效价均达1∶204800以上,其中4H2抗体效价最高;20、50及100μg/只剂量组的抑瘤率分别为51.6%、56.2%和80.3%。结论制备的抗旋毛虫与肝癌H7402细胞相关抗原硒蛋白T单克隆抗体对裸鼠体内肝癌H7402细胞具有明显的抑制效果。     

呋喃唑酮代谢物单克隆抗体及其新型免疫层析检测试纸条的制备

目的制备呋喃唑酮代谢物AOZ单克隆抗体及其胶体金与荧光淬灭免疫层析试纸条。方法将AOZ衍生成CPAOZ后,与载体蛋白BSA偶联,制备完全抗原CPAOZ-BSA,经皮下多点注射免疫BALB/c小鼠,共5次,取小鼠脾细胞与SP2/0细胞融合,获得稳定分泌特异性抗体的杂交瘤细胞株。利用获得的抗CPAOZ单克隆抗体制备胶体金及荧光淬灭免疫层析试纸条,并对试纸条的灵敏度及特异性进行验证。结果制备的完全抗原经紫外扫描鉴定偶联成功。共获得3株能稳定分泌抗CPAOZ单克隆抗体的杂交瘤细胞株,其中效价和特异性最好的1株2H11针对CPAOZ的50%抑制质量浓度(IC50)为0.88 ppb。以该抗体制备的胶体金及荧光淬灭免疫层析试纸条的最低检测限分别为7.5和0.062 5 ppb;两种试纸条与其他3种硝基呋喃类代谢物CPAMOZ、CPAHD、CPSEM均不存在交叉反应。结论成功制备了抗CPAOZ单抗及其胶体金免疫层析和荧光淬灭免疫层析试纸条,两种试纸条均具有较高的灵敏度和较强的特异性,其中荧光淬灭免疫层析试纸条的灵敏度较胶体金免疫层析试纸条高约100倍,具有良好的应用前景。     

治疗用抗人T淋巴细胞分化抗原单克隆抗体与人体组织反应性的体外研究

应用免疫组化酶染色法及免疫细胞荧光染色法体外检测了 WuT_3(抗 CD_3),WuT_4(抗CD_4),WuT_8(抗 CD_8),WuT_9(抗 CD_71 或 TfR),WuTac(抗 CD_(25)或 IL-2R)等5种临床治疗用小鼠抗人 T 淋巴细胞分化抗原单克隆抗体(简称单抗)与25种正常人体组织的反应性。结果显示,WuT_3与髓质胸腺细胞,淋巴结及扁桃体中 T 细胞区细胞,脾白髓中动脉周围淋巴鞘细胞及胃肠道粘膜层淋巴细胞呈阳性反应。WuT_4与皮质胸细胞及大多数髓质胸腺细胞呈阳性反应。WuT_8与皮质胸腺细胞及少数髓质胸腺细胞呈阳性反应。WuT_4 和 WuT_8阳性细胞在淋巴结、扁桃体、脾脏及胃肠道粘膜中的分布与 WuT_3 阳性细胞基本一致,但数量依次减少。淋巴结和扁桃体的生发中心以及脾脏的脾小结中散在分布有少数WuT_3,WuT4及 WuT_8阳性细胞。此外,睾丸中散在分布个别 WuT_8阳性淋巴细胞。WuT_9与26%的骨髓细胞及个别皮质胸腺细胞呈阳性反应。WuT_9 尚与小肠粘膜肠腺底部细胞呈较强染色。WuTac 仅与个别散在的髓质胸腺细胞呈阳性反应。上述5种单抗与其它非淋巴组织及细胞基本呈阴性反应。     

鼠抗人间变性淋巴瘤激酶单抗及其试剂盒的制备及鉴定

目的 制备并鉴定鼠抗人间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)单抗及其试剂盒.方法 以人ALK重组蛋白免疫BALB/c小鼠,常规杂交瘤技术制备人ALK单抗,ELISA及免疫细胞化学(immunocytochemical,ICC)法筛选稳定分泌ALK 单抗的杂交瘤细胞株;SDS-P...     

脱氧雪腐镰刀菌烯醇免疫分析核心试剂特异性单克隆抗体的研制与特性研究

采用1,1’-羰基二咪唑(CDI)活化法成功将脱氧雪腐镰刀菌烯醇分子上C-3和C-15位羟基与载体蛋白赖氨酸的氨基共价偶联得到免疫原DON-BSA和包被原DON-OVA,并用三硝基苯磺酸法对合成结果进行鉴定。以人工抗原免疫Balb/c小鼠,取小鼠脾细胞与SP2/O鼠骨髓瘤细胞融合,经筛选和多次亚克隆,得到了1株能稳定分泌脱氧雪腐镰刀菌烯醇抗体的单克隆细胞株1D5,并制备单克隆抗体腹水。经检测该抗体亚型为IgG1,亲和力常数Ka为8.33×107L/mol,交叉反应的试验结果显示该抗体与其他真菌毒素无交叉反应率,稳定性良好,为粮油食品中脱氧雪腐镰刀菌烯醇免疫快速检测技术建立及产品研发提供了关键试剂材料。     

人巨细胞病毒IgM抗体生物素-亲和素单克隆抗体捕获时间分辨荧光免疫法检测试剂盒的研制与评价

目的建立人巨细胞病毒IgM(human cytomegalovirus IgM,HCMV IgM)抗体生物素-亲和素单克隆抗体捕获时间分辨荧光免疫法(biotin-avidin monoclonal antibody capture time-resolved fluoroimmunoassay,BA Mac TrFIA)检测试剂盒,并对其性能进行评价。方法采用鼠抗IgMμ链单克隆抗体(monoclonal antibody,McAb)作为固相反应板的包被抗体,生物素(biotin)标记的HCMV基因重组抗原作为桥接抗原,链酶亲和素(SA)标记铕(Eu3+)作为示踪物,配制以β-萘甲酰三氟丙酮为主要成分的增强液,应用BA Mac TrFIA法进行HCMV IgM抗体的检测,对连续制备的3批试剂盒的最低检测限、重复性、阴性参考品符合率、阳性参考品符合率、热稳定性进行评价,并与巨细胞病毒IgM抗体诊断试剂盒同时对1 020份血清或血浆样本进行临床研究比对试验,采用Kappa检验进行一致性分析。结果选择4μg/ml作为鼠抗IgMμ链McAb最佳包被浓度,1∶10 000稀释作为HCMV-Bio最佳工作浓度,1∶1 500稀释作为SA-Eu3+最佳工作浓度,试剂盒参考值建议为阴性对照品检测荧光值(negative control counter,NCx)×2.1;连续制备的3批试剂盒最低检测限、重复性、阴性参考品符合率、阳性参考品符合率均达到国家检定标准;于37℃恒温箱放置6 d后,检测性能无明显改变;临床研究比对试验Kappa指数为1,一致程度达100%。结论成功制备了HCMV IgM BA Mac TrFIA法检测试剂盒,该试剂盒灵敏度高,精密性较好,与同类产品检测结果相关性好,为临床HCMV lgM抗体的检测提供科学、可靠的诊断依据。     

单抗导向药物3A1-Dextran-DM对白血病CEM细胞特异性杀伤效应的观察

抗T淋巴母细胞单克隆抗体与柔红霉素的结合物,在体外对T淋巴母细胞CEM显示出选择性杀伤作用,而对K562、Raji、SP2/0等非T淋巴细胞则无杀伤效应。由此证实单抗3Al-柔红霉素免疫结合物具有导向杀伤癌细胞的性能,为临床特异性治疗肿瘤带来了新的希望。     

流行性出血热病毒单克隆抗体的研究

应用流行性出血热病毒(EHFV)Z_(10)株制备的疫苗免疫BALB/c小鼠,取脾细胞与BALB/c小白鼠骨髓瘤SP2/0细胞融合,获得10株能分泌ENFV特异性单克隆抗体(McAb)的杂交瘤细胞,其McAbs小鼠腹水荧光抗体滴度达1:2000～1:51200。经免疫印迹试验及ELISA夹心法证明1 0株McAbs为抗EHFV核蛋白(NP)的McAb,均不具有中和抗体活性,有低滴度的血凝抑制活性;经间接免疫荧光试验(IFAT)证明,2株为组特异性的McAb,5株为亚组特异性的McAb,3株为型特异性的McAb。实验用冻存免疫脾细胞进行杂交获得成功,为杂交瘤细胞的研究提供了经验。     

抗急非淋M_2和M_5型白血病单抗对骨髓粒-单核细胞集落形成(CFU-GM)的影响

为了使单抗用于骨髓净化和导向药物的研究,应用分泌抗急非淋M_2和M_5型单抗1D_1和1A_1,观察对正常骨髓粒-单核细胞集落形成单位(CFU-GM)的影响。结果实验组与对照组比较无显著性差异,说明单抗对CFU-GM没有影响,不损伤正常骨髓造血干细胞,为M_2和M_5型单抗用于骨髓净化和导向药物的研究提供实验依据。     

Short-Term Effect of SARS-CoV-2 Spike Protein Receptor-Binding Domain-Specific Antibody Induction on Neutrophil-Mediated Immune Response in Mice

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2⁺CD101⁺ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.     

Combination of the Glutaminyl Cyclase Inhibitor PQ912 (Varoglutamstat) and the Murine Monoclonal Antibody PBD-C06 (m6) Shows Additive Effects on Brain Aβ Pathology in Transgenic Mice

Compelling evidence suggests that pyroglutamate-modified Aβ (pGlu3-Aβ; AβN3pG) peptides play a pivotal role in the development and progression of Alzheimer’s disease (AD). Approaches targeting pGlu3-Aβ by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Donanemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aβ-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16–41%) but statistically insignificant reduction of Aβ42 and pGlu-Aβ42 in mice brain, the combination of both treatments resulted in significant reductions of Aβ by 45–65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aβ in different compartments, the antibody is able to clear existing pGlu3-Aβ deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aβ-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect.     

家兔角膜结膜模型用于痢疾活菌苗免疫效果的研究

采用家兔角膜结膜模型对依链株口眼痢疾福氏2a 和福氏3a 菌苗进行了免疫效果的研究。结果表明这两型菌苗的完全保护率分别为28.5%和19.1%,部份保护率为47.6%和61.9%,病程下降率为55.0%和47.4%,经与对照组作 Ridit 分析,差异非常显著;综合保护率分别为80.5%和76.5%。试验证明,这种模型是适用的,而且较灵长类动物便宜,操作易于掌握,无早期感染;较豚鼠容易获得和饲养,便于操作和观察,值得研究和推广。     

Evidence of a Gastro-Duodenal Effect on Adipose Tissue and Brain Metabolism,Potentially Mediated by Gut–Liver Inflammation: A Study with Positron Emission Tomography and Oral 18FDG in Mice

Interventions affecting gastrointestinal (GI) physiology suggest that the GI tract plays an important role in modulating the uptake of ingested glucose by body tissues. We aimed at validating the use of positron emission tomography (PET) with oral ¹⁸FDG administration in mice, and to examine GI effects on glucose metabolism in adipose tissues, brain, heart, muscle, and liver, and interfering actions of oral lipid co-administration. We performed sequential whole-body PET studies in 3 groups of 10 mice, receiving i.p. glucose and ¹⁸FDG or oral glucose and ¹⁸FDG ± lipids, to measure tissue glucose uptake (GU) and GI transit, and compute the absorption lumped constant (LCa) as ratio of oral ¹⁸FDG-to-glucose incremental blood levels. GI and liver histology and circulating hormones were tested to generate explanatory hypothesis. Median LCa was 1.18, constant over time and not significantly affected by lipid co-ingestion. Compared to the i.p. route, the oral route (GI effect) resulted in lower GU rates in adipose tissues and brain, and a greater steatohepatitis score (+17%, p = 0.03). Lipid co-administration accelerated GI transit, in relation to the suppression in GIP, GLP1, glucagon, PP, and PYY (GI motility regulators), abolishing GI effects on subcutaneous fat GU. Duodenal crypt size, gastric wall ¹⁸FDG uptake, and macro-vesicular steatosis were inversely related to adipose tissue GU, and positively associated with liver GU. We conclude that ¹⁸FDG-PET is a suitable tool to examine the role of the GI tract on glucose transit, absorption, and bio-distribution. The GI effect consists in the suppression of glucose metabolism selectively in organs responsible for energy intake and storage, and is blunted by lipid ingestion. Modulation of gut and liver inflammation, as reflected by high GU, may be involved in the acute signalling of the energy status.     

Fourier Transform Infrared Microspectroscopy Combined with Principal Component Analysis and Artificial Neural Networks for the Study of the Effect of β-Hydroxy-β-Methylbutyrate (HMB) Supplementation on Articular Cartilage

The potential of Fourier Transform infrared microspectroscopy (FTIR microspectroscopy) and multivariate analyses were applied for the classification of the frequency ranges responsible for the distribution changes of the main components of articular cartilage (AC) that occur during dietary β-hydroxy-β-methyl butyrate (HMB) supplementation. The FTIR imaging analysis of histological AC sections originating from 35-day old male piglets showed the change in the collagen and proteoglycan contents of the HMB-supplemented group compared to the control. The relative amount of collagen content in the superficial zone increased by more than 23% and in the middle zone by about 17%, while no changes in the deep zone were observed compared to the control group. Considering proteoglycans content, a significant increase was registered in the middle and deep zones, respectively; 62% and 52% compared to the control. AFM nanoindentation measurements collected from animals administered with HMB displayed an increase in AC tissue stiffness by detecting a higher value of Young’s modulus in all investigated AC zones. We demonstrated that principal component analysis and artificial neural networks could be trained with spectral information to distinguish AC histological sections and the group under study accurately. This work may support the use and effectiveness of FTIR imaging combined with multivariate analyses as a quantitative alternative to traditional collagenous tissue-related histology.