人甲状旁腺激素N端肽中一个含有B细胞新表位的hPTH13-27片段的研究

目的研究并证明人甲状旁腺激素（hPTH）N端肽内的hPTH^13-27片段是一个新的B细胞抗原表位。方法计算机辅助分析预测hPTH^1-37和hPTH^1-84多肽片段内hPTH^13-27抗原表位的特征;制备大鼠抗hPTH^13-27-KLH多克隆抗体;酶联免疫测定（ELISA）研究hPTH^13-27抗原表位和抗体的免疫特征。结果蛋白质结构分析预测在hPTH^13-27区域含有B细胞抗原表位。hPTH^13-27单表位多克隆抗体制备成功。ELISA分析证明hPTH^13-27片段含有B细胞抗原表位,与抗体的结合效果依次为：抗hPTH^13-27抗体〉抗hPTH^1-84抗体〉抗hPTH^1-37抗体。hPTH^13-27片段与固相板结合对免疫原性影响不大。抗hPTH^13-27抗体与含hPTH^13-27片段的多肽结合良好;与不含hPTH^13-27片段的多肽无交叉反应。结论hPTH^13-27可能是制备hPTH N端肽抗体的核心表位片段。     

人内质网分子伴侣BiP抗原表位筛选及其在RA血清学诊断中的作用研究

目的:筛选鉴定人内质网分子伴侣BiP抗原表位多肽,探讨其在类风湿性关节炎(Rheumatoid arthritis,RA)血清学诊断中的价值.方法:运用计算机软件分析BiP蛋白的结构特点及抗原决定簇分布,根据结果设计合成系列多肽.比较合成多肽与RA患者血清(BiP抗体阳性)IgG的反应强弱,鉴定抗原抗体反应最强的抗原表位多肽.进一步以获得的BiP抗原表位多肽作为包被抗原,检测79例RA患者、34例系统性红斑狼疮(SLE)患者、66例干燥综合征(SS)患者和173例正常人血清中抗BiP抗原表位多肽IgG抗体水平.结果:筛选到一条与RA患者血清反应最强的多肽(N403),抗N403多肽IgG抗体在RA患者中的阳性率(67%),明显高于SS患者(29%)、SLE患者(0%)、和正常人(0%)(P＜0.01).抗N403多肽IgG抗体在RA血清学诊断中的敏感性为67%,特异性为93%.而且该抗体的检测对于RF、CCP、HRF、RA33、AKA、APF等抗体阴性的RA患者的诊断具有重要意义.结论:筛选获得的BiP抗原表位多肽N403,其检测抗体在RA临床血清诊断中具有很好的应用价值.     

毒蕈碱受体3抗原表位筛选及其在原发性干燥综合征诊断中的作用

运用计算机软件分析毒蕈碱受体3(M3R)蛋白的结构特点及抗原决定簇分布,根据结果设计合成3条多肽。比较合成多肽与抗M3受体抗体阳性干燥综合征(SS)患者血清IgG的反应强弱,获得一条抗原抗体反应最强的抗原表位多肽(N49)。进一步以多肽N49作为包被抗原,检测原发性SS(pSS)患者41例,继发SS(sSS)患者25例,系统性红斑狼疮(SLE)患者34例,类风湿关节炎(RA)患者80例和正常人174例血清中抗M3多肽IgG抗体,结果显示该多肽抗体在原发性SS患者血清中的阳性率为53.7%,明显高于sSS患者28%、SLE患者8.8%、RA患者25%和正常人12.1%(P<0.05)。抗N49多肽抗体在pSS诊断中的敏感性为53.7%,特异性为83.7%,而且该抗体的检测对于抗SSA抗体、抗SSB抗体或抗α-胞衬蛋白(-αfodrin)抗体阴性的pSS患者的诊断具有补充作用。表明,筛选获得的M3抗原表位多肽N49,其检测抗体在pSS临床血清诊断中具有很好的应用价值。     

噬菌体文库与计算机模拟筛选H1N1 流感病毒血凝素结合位点的比较

目的:对H1N1 流感病毒血凝素HA 的抗原表位初步筛选。方法:用噬菌体展示文库对2 株抗体的结合位点进行筛选,将筛选的抗原位点结合HA 序列进行多肽合成,免疫小鼠制备单克隆抗体,通过分子生物学手段对4 株抗体的轻链和重链可变区基因进行调取,通过计算机模拟抗体序列结合的抗原的结构域,推测抗体结合的氨基酸位点。结果:噬菌体文库筛选得出抗体的结合序列富含组氨酸和精氨酸,将针对多肽的单克隆抗体的序列和前期获得的2 株抗HA 单克隆抗体的序列比对分析,发现A1-8 和anti-14p 结合的HA 抗原序列相一致,H1-13 和anti-11p 结合的HA 抗原序列相一致。结论:通过将噬菌体展示文库技术和计算机模拟预测抗原抗体结合,可以有效筛选抗原表位,为流感病毒HA 抗原表位预测方法的完善提供理论基础。     

两种PTH载体蛋白连接法的特异抗体及载体抗体比较的初步探讨

人甲状旁腺激素(hPTH)是由84个氨基酸组成的一种重要的生物活性肽，对调节钙、磷代谢起着极为重要的作用，目前多使用针对限定抗原表位的抗体建立夹心法来测定人甲状旁腺激素(hPTH)分子。PTH结构简单，单独刺激机体不易产生抗体，只有与载体分子连接后才可得到较强的免疫应答效应。不同的连接方法对其特异及载体抗体的产生影响不同，     

重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用

目的：制备人自身抗原细胞色素P4502D6（CYP2D6）257-351位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗LKM-1抗体的敏感性和特异性。方法：以肝脏的cDNA混合文库为模板作PCR,将PCR产物与真核表达载体pEGH共同转化酿酒酵母Y258,碱裂解法进行质粒制备,PCR扩增鉴定。表达载体构建成功后,在半乳糖的诱导下表达产生重组融合蛋白,经GST亲和层析法进行纯化后,免疫印迹法鉴定抗原性,ELISA检测抗LKM-1抗体阳性血清及部分其他结缔组织病患者血清中的抗LKM-1抗体。结果：重组融合蛋白在宿主菌中获得表达,免疫印迹法鉴定表明其能与标准抗LKM-1抗体阳性血清反应,而与正常血清、其他抗血清无反应。在26份抗LKM-1抗体阳性血清中,6份抗HCV抗体阳性血清用重组多肽ELISA检测有5份呈阳性,其余20份血清用重组多肽ELISA检测均呈阳性；20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论：重组的257-351位氨基酸片段是CYP2D6抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗LKM-1抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。     

Trim22 B细胞表位预测及多克隆抗体制备与鉴定

目的:预测人Trim22的B细胞抗原表位,制备并鉴定其多克隆抗体。方法:利用生物信息学软件分析Trim22的氨基酸序列,确定抗原表位并人工合成多肽。将合成肽与牛血清白蛋白(BSA)偶联,免疫大白兔制备抗Trim22的抗体。经饱和硫酸铵沉淀法纯化抗体,并对抗体进行ELISA、Western blot鉴定。结果:获得抗Trim22的多克隆抗体,抗体效价为1∶16 000,该抗体能特异性地识别包含(382～397)区段的Trim22缺失突变体,同时该抗体不能识别Trim22旁系同源分子Trim5α、Trim6和Trim34α。结论:采用人工合成多肽作为半抗原制备出抗Trim22的多克隆抗体,对研究内源性Trim22与HIV-1衣壳蛋白的相互作用机制具有重要价值。     

BPI N端优势抗原表位的模拟合成及其抗血清的制备

目的通过生物信息学模拟合成杀菌/渗透增强蛋白氨基端(BPIN端)优势抗原表位肽,免疫动物获得相应抗血清。方法利用生物信息学分析BPIN端(1-199)氨基酸序列的抗原性、亲水性、可塑性、表面可及性和二级结构等理化特性,据此设计合成TA/IK两条多肽,将其与钥孔戚血蓝素(KLH)偶联后,免疫家兔获得相应抗血清;采用间接ELISA法鉴定多肽的抗原性、测定血清抗体效价,Westernblot鉴定抗血清特异性。结果人工合成BPIN端TA/IK两个B细胞表位肽;ELISA检测证实TA/IK抗原肽能与商品化兔抗人BPI55多克隆抗体结合;所获TA/IK抗血清效价分别为1∶51200和1∶25600;Westernblot证实TA/IK抗血清能与BPI55标准品特异性结合。结论模拟合成的TA/IK抗原肽确为BPIN端优势抗原表位,相应抗血清可用于BPIN端功能性片段的检测鉴定。     

核不均一性胞核核糖核蛋白Ⅰ(hnRNPI)抗原表位在系统性硬化症中临床意义的研究

目的探讨核不均一性胞核核糖核蛋白I(hnRNPI)抗原表位多肽在系统性硬化症(systemic sclerosis,SSc)中的临床意义,初步建立简便快捷的ELISA检测方法,为系统性硬化症的早期诊断寻找新的临床指标。方法根据已知的hnRNPI蛋白的氨基酸序列,应用不同的蛋白质抗原表位图谱分析软件对其进行表位分析,经比对筛选后,化学合成hnRNPI短肽序列2个,分别命名为I-1_(264-292)及I-2_(441-461),作为抗原对临床上包括硬皮病在内的多种结缔组织病患者血清相应抗体进行ELISA检测,包括SSc 42例、系统性红斑狼疮(SLE)102例、干燥综合征(SS)26例、混合型结缔组织病(MCTD)16例、未分化结缔组织病(UCTD)13例,其他结缔组织病(CTD)30例、类风湿关节炎(RA)26例及正常对照54例。结果抗hnRNPI-1及抗hnRNPI-2多肽抗体在SSc组中阳性率均明显高于其他疾病组(P<0.05),在SSc中敏感性分别为47.62%及38.1%,特异性分别为93.43%及91.08%,二者之间差异无统计学意义(P>0.05)。另外,除了与SSc患者病程相关外,该二抗体均与发病年龄、临床症状、器官受累、ESR、抗Scl-70抗体、抗着丝点抗体及抗核仁抗体之间未发现有统计学意义的相关性(P>0.05)。结论I-1及I-2分别是位于hnRNPI蛋白表面的抗原表位之一,具有抗原性,其抗体对SSc的临床诊断具有较高的敏感性及特异性,且在病程早期具有更高的阳性率,有助于SSc的早期诊断。     

抗幽门螺旋杆菌尿素酶B亚单位(UreB)单克隆抗体抗原识别表位的初步鉴定

目的 初步确定抗幽门螺杆菌尿素酶B亚单位(UreB)单克隆抗体(mAb)6E6识别的抗原表位.方法 采用截短法分段构建含UreB抗原的重组质粒,分别命名为U12,U13,U15,U16,U47.用IPTG诱导表达融合蛋白.对表达产物进行SDS-PAGE蛋白电泳及Western blot 分析.结果 6E6能够分别识别1～300位氨基酸的U12片段,1～260位氨基酸的U16片段,1～230位氨基酸的U15片段,而不能识别1～200位氨基酸的U13片段和251～389位氨基酸的U47片段.结论 mAb 6E6抗体识别的抗原表位位于200～230位氨基酸.     

Immunological evidences for the presence of small late carboxylterminal fragment(s) of human parathyroid hormone (PTH) in circulation in man

Two antisera, C-52 and C-97, raised against bovine (b)PTH(1-84) in guinea pigs, were evaluated with 125I-[tyr53] human (h)PTH(53-84) as tracer and intact hPTH(1-84) and synthetic hPTH(39-84), representative of large carboxylterminal ("C") fragments found in circulation, as standards. In both assays, hPTH(39-84) was 5-6 times more potent than hPTH(1-84) on a molar basis in displacing the tracer. With both antisera, progressive deletion at the aminoterminal end of large "C" fragments, as in hPTH(53-84) and hPTH(65-84), lead to decreased immunoreactivity, hPTH(69-84) being non-immunoreactive. The mid-carboxylterminal fragments, hPTH(44-68) and hPTH(39-68), did not react in either assay. Each antiserum measured known quantities of pure hPTH(1-84) or hPTH(39-84) standards similarly. Serum PTH values obtained with antiserum C-97 were about 3 times higher in renal failure, 1.75 times higher in normal individuals and those with primary hyperparathyroidism, while similar to values measured with antiserum C-52 in individuals with secondary hyperparathyroidism without renal failure or with pseudohypoparathyroidism. When circulating PTH taken from patients with these disorders was fractionated by gel chromatography, both antisera recognized similar peaks of intact hPTH(1-84) and of large "C" fragments while antiserum C-97 further recognized a peak of smaller "C" fragments. This explained the different clinical behavior of the latter antiserum. Our findings demonstrate the existence of small late "C" fragments in circulation. They further suggest an influence of serum calcium and of renal function on the quantity of these fragments.     

Human Parathyroid Hormone: Antibody Characterization

PTH antibodies were raised in two sheep (S 469 and S 478) by immunizing with porcine and bovine parathyroid extracts. Both antisera were characterized with various PTH preparations and fragments. Both antisera cross react with human, bovine and porcine PTH, one antiserum also binds rat PTH. Region specificity could be attributed to the mid region of the PTH molecule with particularly high affinities of both antisera for the fragment 44–68 hPTH. S 478 has smilarly high affinity for intact hormone (affinity constants 0.6 ± 10¹³ 1/mol), while S 469 has much higher affinity for the 44–68 fragment (affinity constant 0.84 ± 10¹³ 1/mol) than for intact hormone. The antibodies are useful not only for clinical radioimmunoassay, but also for experimental work. They have been distributed to many laboratories.     

Distribution and concentration of immunoreactive parathyroid hormone in brain and pituitary of sheep

Summary We have studied the presence of immunoreactive parathyroid hormone (PTH) in the central nervous system and pituitary of sheep. The PTH concentrations were measured radioimmunologically by two different region-specific antibodies. We could demonstrate PTH in various areas of the brain, whole pituitary, parathyroid glands and plasma of 21 sheep. Measurable concentrations of the two different parathyroid regions (35–84 and 44–68 amino acids fragments) were found in all samples.Abbreviations ACTH Adrenocorticotrophic hormone - CT Calcitonin - CSF Cerebrospinal fluid - CPM Counts per min - Leu-ENK Leucine-enkephalin - Met-ENK Methionine-enkephalin - PTH Immunoreactive parathyroid hormone - RIA Radioimmunoassay - TSH Thyroid stimulating hormone - T₄ Thyroxine - T₃ Triiodothyronine     

An Homologous and Sensitive Radioimmunoassay for the Synthetic Amino-Terminal (1-34) Fragment of Human Parathyroid Hormone: Application to the Clearance of This Peptide Administered in Vivo

The synthetic 1–34 amino-terminal fragment of human parathyroid hormone (hPTH 1–34) is undergoing multicentre clinical trials to assess its long term therapeutic potential in the treatment of osteoporosis. An homologous radioimunoassay (reagents prepared from the synthetic hPTH 1–34 peptide) has been developed to monitor the pharmacokinetics of hPTH 1–34 in man and in a dog model. The assay is rugged, sensitive (detection limit 1.75 ± 10^?11 moles/litre) and precise (coefficient of variation 6%). Three different ampouled preparations of the native intact hPTH 1–84, of different degrees of purity (approximately 3%-90% pure) gave complete log dose response curves parallel to that of the ampouled synthetic hPTH 1–34 peptide, and were equipotent on a molar basis. Native intact bovine PTH 1–84 showed an incomplete non-parallel displacement curve; there was no recognition of synthetic hPTH 44–68 and 53–84 peptides. Preliminary application of the assay to the determination of the plasma disappearance of hPTH 1–34 in man and dog gave half-times (t1/2) of 3–8 minutes for a first exponential component and 12–18 minutes for the second; in the dog, metabolic clearance rate was calculated to be 9ml/kg/minute and the distribution space 160ml/kg.     

Mesure de la parathormone : facteurs de variation et problèmes de standardisation

The parathyroid hormone (PTH) assay still involves some problems, considering the molecular heterogeneity and the different specificity of the available sandwich immunometric methods. A number of preanalytical variation factors can affect the PTH measurement: the intraindividual biological variability (pulsatile secretion, circadian rhythm) and the interindividual variability (sex, age, genetics factors), as well as the sample typology and way of storage, can be mentioned. Beside the molecular heterogeneity (C-terminal fragments, included the recently identified non-(1–84)) molecular forms which may interfere with "intact molecule" PTH methods), the factors affecting the analytical variability concern the antibody interference (anti-PTH autoandibodies and heterophilic antibodies) and particularly the calibration differences existing among the commercially available assays. In order to evaluate these differences, we performed recovery tests with both synthetic hPTH(1–84) from Bachem and recombinant hPTH(1–84) 95/646 from NIBSC. The results are similar to those recently obtained by UK-NEQAS and highlight remarkable between-method calibration differences, which are the main reason for the disagreements among PTH values obtained with different kits. On the contrary, no relationship apparently exists between the cross-reactivity with synthetic hPTH(7–84) fragment and the method bias (UK-NEQAS, 2001). Therefore the lack of an International Standard is the main obstacle to solving the problems related to PTH measurement?     

Comparison of Four Different Carboxylterminal Tracers in a Radioimmunoassay Specific to the 68–84 Region of Human Parathyroid Hormone

Two synthetic carboxylterminal fragments, {tyr⁵²}hPTH(52–84) and {tyr⁶³}hPTH(63–84), and purified bPTH(1–84) were iodinated with ¹²⁵Iodine to be compared as tracers in a late carboxylterminal radioimmunoassay. Tracer ¹²⁵I-bPTH(41–84) was generated in vitro by incubating ¹²⁵I-bPTH(1–84) with plasma membranes of rat kidney cortex. Region specificity was achieved by saturating the unwanted middle component of our multivalent antiserum with a molar excess of hPTH(44–68). A charcoal-dextran separation was worked out for each tracer. The titer of the antiserum giving ?30% specific binding of each tracer was used in all experiments. Displacement of each tracer with increasing molar concentration of hPTH(1–84), hPTH(53–841, hPTH(41–84) and of hPTH (64–84) was studied, hPTH(41–84) was also generated by incubating hPTH(1–84) with rat cortex kidney membranes and was calibrated against a commercial preparation of bPTH(37–84). A progressive increase in the titer of the antiserum was seen as the molecular weight of the tracers decreased from a titer of 1/20,000 with ¹²⁵I-bPTH(1–84) to a titer of 1/50,000 with the two synthetic tracers. Similarly the so-called damage seen during the charcoal-dextran separation in absence of antibody was reduced from 16.0±6.2% (mean ±SD) with ¹²⁵I-bPTH(1–84) to 1.3±.2 with the two synthetic tracers. 50% displacement of the ¹²⁵I-bPTH(1–84) tracer was achieved at 13.2±.8 fmol/tube for hPTH(1–84) and at 6.3±1.0 fmol/tube for hPTH(41–84), reflecting the greater reactivity of fragments in that system. With the two synthetic tracers, a concentration of 5.0±.4 fmol/tube of hPTH(1–84) or of 3.5±1.2 fmol/tube of hPTH(41–84) was necessary to achieve the same goal. With ¹²⁵I-bPTH(41–84) results were between the two extremes. These results indicated that an increase in antiserum titer, a decrease in assay damage, an improvement in assay sensitivity and in comparative molar reactivity of the various circulating forms of hPTH can be achieved by using synthetic carboxylterminal fragments as tracers in region specific radioimmunoassays of hPTH.     

Lack of serologically defined arthritogenic Shigella flexneri cell envelope antigens in post-dysenteric arthritis

Post-dysenteric or reactive arthritis (ReA) is closely associated with HLA-B27. This histocompatibility antigen is heterogeneous and consists of 2 serologically defined variants: B27M1+M2+ and B27M1+M2-. This paper gives a qualitative evaluation of the antibodies present in the sera of 62 patients with dysentery due to Shigella flexneri 2a, a known arthritogenic bacterium. The patients were classified in 4 groups: B27M1+M2+ReA+ (n = 5), B27M1+M2+ReA- (n = 7); B27M1+M2-ReA- (n = 1); B27-ReA- (n = 49). The isolated infectant possessed cell envelope antigens with B27M2-like epitopes (Mr 20,000). Analysis of the spectrum of antibodies directed against the separated cell envelope antigens of S. flexneri in the sera of these patients revealed 7 main patterns of reactivity. The detectable immunogens encompassed protein stainable antigens (Mr 98, 78, 68, 54, 50, 44, 41, 35, 14 and 13 kDa), lipopolysaccharides and peptidoglycan. None of the sera possessed detectable antibodies to the B27M2-like antigen. Consequently, this antigen is unlikely to be associated with ReA, and this applies equally to other antigens or patterns of antigens. The arthritogenicity of S. flexneri may therefore not be determined by the presence or absence of detectable antibody titers to certain cell envelope antigens. We hypothesize that other properties of these antigens could be of significance.     

Epitopes of the 44-68 human parathyroid hormone fragments: the importance of specific hydrophilic peptide sequences

Several pentapeptides included in the 44-68 sequence of human parathyroid hormone (hPTH) were synthesized simultaneously on benzhydrylamine and m-nitrobenzhydrylamine resins. The first polymer gave the free peptide and the second the peptidyl-resin complex. An ELISA test carried out with each peptidyl-resin complex showed that all the anti-44-68 hPTH antibodies raised in different animal species are directed against the same hPTH pentapeptidic sequence. This sequence is very hydrophilic and is specific to the hormone. This study demonstrates the importance of specific peptide chains in an epitope.     

Identification and localization of a limited number of predominant conformation-independent antibody epitopes of Theiler's murine encephalomyelitus virus.

Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease in mice is a well established animal model for human multiple sclerosis (MS). Identification of pathogenic epitopes may be helpful in understanding the pathogenesis of this immune-mediated disease. In order to analyze the viral epitopes, we have generated approx. 150 recombinant lambda gt11 clones expressing various capsid areas of TMEV. Six predominant areas, ranging from 13-26 amino acid residues, (3 in VP1, 2 in VP2 and 1 in VP3) are readily recognized by conformation-independent antibodies from virus-infected mice. These areas have been designated as A-1A (VP1 13-27th residues), A-1B (VP1 145-167), A-1C (VP1 251-276), A-2A (VP2 2-14), A-2B (VP2 165-179), and A-3A (tentatively VP3 24-43). Antibodies from TMEV-infected susceptible SJL/J mice strongly react with A-1B, A-2A and A-2B, in contrast to antibodies from resistant BALB/c mice which mainly recognize A-1A and A-2A. Interestingly, the reactivity pattern of antibodies from TMEV-infected mice are somewhat different from that of antibodies from TMEV-immunized mice. Although the majority of antibodies in TMEV-infected mice recognizes conformation-dependent epitopes, the differential recognition of the conformation-independent antibody epitopes by susceptible mice may play a role in TMEV-induced demyelination.