甲胺磷人工抗原的合成和鉴定

目的：合成和鉴定甲胺磷人工抗原。方法：用磷酸化方法偶联氧，硫二甲基硫代磷酰氯到牛血清蛋白上，然后做一系列的实验，如：测定磷，紫外，31P核磁共振光谱和免疫小鼠等。结果：人工抗原的偶联比是16，人工抗原的紫外光谱图与牛血清蛋白质相比发生改变，人工抗原和标准甲胺磷的31P核磁共振光谱具有相同的化学位移峰，被免疫的小鼠产生了抗甲胺磷的抗体，结论：甲胺磷人工抗原合成成功，为其免疫测定方法的建立提供了条件。     

大黄素-BSA包被膜片人工抗原制备及其免疫原性与特异性检测

目的:通过大黄素-BSA包被膜片的免疫原性及特异性研究,探讨半抗原-载体包被膜片人工抗原制备法的可行性。方法:先将BSA包被于PVDF膜片,再与大黄素-偶联剂衍生物偶联,制备大黄素-BSA包被膜片,经膜片皮下包埋法免疫大鼠,用大黄素或大黄酚、大黄素甲醚包被CA膜片免疫分析法检测免疫原性和特异性。结果:大黄素-BSA包被膜片的免疫原性高于或等于液相抗原;其抗血清对三种蒽醌类化合物反应的特异性基本一致(P0.05)。结论:大黄素-BSA包被膜片抗原,具有良好免疫原性和特异性,提示用半抗原-载体包被膜片法制备人工抗原可行。     

大黄素-BSA不同表位构型的免疫原性和特异性研究

目的:研究不同偶联法制备大黄素-BSA的大黄素表位构型及其免疫原性和特异性。方法:用重氮化对氨基苯甲酸偶联法和琥珀酸酐偶联法制备两种不同表位构型的大黄素-BSA1和大黄素-BSA2抗原,免疫小鼠制备抗血清,用醋酸纤维素膜双向免疫扩散法检测免疫原性及其抗体特异性。结果:大黄素-BSA1和大黄素-BSA2抗血清与大黄素反应的抗体效价分别为1∶144 0±357.771和1∶440±219.089,二者有极显著性差异(P=0.006)。分别与大黄素等五种蒽醌类化合物反应,前者对大黄素有较高特异性,抗原结合效价可达1∶1 843.2±457.947。后者对大黄素的特异性较低,结合效价仅为1∶8.8±4.382。结论:两种偶联法制备大黄素-BSA的表位构型不同,其免疫原性和特异性存在着显著差异。重氮化对氨基苯甲酸偶联法制备大黄素-BSA的免疫原性较高、大黄素特异性较强。     

甲氰菊酯人工抗原的合成和鉴定

目的 合成与鉴定甲氰菊酯的人工抗原.方法 通过水解、酰化、酯化等反应合成了甲氰菊酯的半抗原2,2,3,3.四甲基环丙烷羧酸-a-(N-丁酸基)-甲酰氨-3-苯氧基苄酯(Ⅳ)和2,2,3,3-四甲基环丙烷羧酸-a-羧基-3-苯氧基苄酯(Ⅲ).通过碳二亚胺法将半抗原Ⅳ与牛血清蛋白(BSA)偶联制备免疫抗原,通过混合酸酐法将半抗原Ⅲ与卵清蛋白(OVA)偶联制备包被抗原.结果 用质谱和核磁共振对Ⅲ和Ⅳ进行结构表征,合成产物为目标物.紫外光谱法鉴定结果表明,免疫抗原和包被抗原都发生了有效偶联,偶联比38:1和15:1,免疫抗原通过免疫新西兰大白兔得到的抗体效价为5.12×105.结论成功的合成了甲氰菊酯的人工抗原,为其免疫方法的建立奠定了基础.     

抗甘草酸二铵抗血清的制备

目的通过人工抗原的构建,制备抗甘草酸二铵（DG）的特异性抗血清。方法采用碳二亚胺法将DG与牛血清蛋白（BSA）和鸡卵清蛋白（OVA）共价偶联,分别合成免疫抗原DG-BSA和包被抗原DG-OVA。用合成的人工抗原免疫家兔,制得抗甘草酸二铵的特异性抗血清,并通过酶联免疫方法（ELISA）对抗血清进行鉴定。结果制备了抗甘草酸二铵的多克隆抗体,获知理想的包被抗原的浓度为200ng／mL,抗血清的工作浓度为1：10000,雌、雄家兔抗血清的效价分别为1：51200和1：25600。结论采用化学反应构建人工抗原的方法,可以制备出针对甘草酸二铵的特异性抗血清,从而为甘草酸二铵的鉴定及体内代谢的免疫学分析研究奠定了基础。     

25-羟基维生素D3人工完全抗原的合成与鉴定

目的:合成25-羟基维生素D3人工完全抗原,并制备抗25-羟基维生素D3的特异性抗体。方法:将25-羟基维生素D3进行化学修饰加入羧基活性基团,合成具有半抗原结构特征的25-羟基维生素D3-半琥珀酸酯。采用碳二亚胺法,将25-羟基维生素D3-半琥珀酸酯,分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联,合成人工完全抗原25-羟基维生素D3-半琥珀酸酯-BSA和25-羟基维生素D3-半琥珀酸酯-OVA。通过紫外吸收光谱,SDS-PAGE和MALDI-TOF进行偶联鉴定。用25-羟基维生素D3-半琥珀酸酯-BSA免疫小鼠,获得抗25-羟基维生素D3抗体免疫血清。结果:25-羟基维生素D3-半琥珀酸酯与BSA的偶联比为(12±0.16)∶1,免疫小鼠后获得高效价(效价为6.25×10-4)的抗体,且标准品浓度在37.5～600 ng/mL范围具有显著的竞争性线性关系,检测的灵敏度为37.5 ng/mL。结论:成功合成了25-羟基维生素D3人工完全抗原,制备出25-羟基维生素D3的抗体且其线性关系显著,灵敏度较高,为进一步研制检测25-羟基维生素D3的试剂盒奠定了基础。     

[1,6-双(L-α,β-二氨基丙酸)]催产素的合成

通过7＋2片段组合方式，采用液相法合成了全新的含非蛋白质氨基酸L—α，β-二氨基丙酸（L—Dap）的催产素的类似物。合成中所需的保护氨基酸的α-氨基均采用苄氧羰基（Z）保护，L-Dap的侧链氨基采用叔丁氧羰基（Boc）保护。先用逐步增长方式采用对硝基苯酚酯法缩合得到重要中间体七肽片段．再用叠氮法缩合得到保护九肽。Z-保护用催化氢化法脱除，Boc-保护用CF3COOH脱除。共合成了含L—Dap的新化合物8个，所有寡肽都通过了氨基酸分析以及质谱确证。     

影响紧张型头痛患者就医行为的多因素分析

目的探讨影响紧张型头痛（TTH）住院患者就医行为的心理因素。方法将35例诊断为TTH的住院军人作为研究对象，分别用生活事件量表（LES）、防御方式问卷（DSQ）、应付方式问卷、内外在心理控制源量表、症状自评量表（SCL-90）及史森克人格问卷（EPQ）对他们进行心理特征评定。并以人口统计学资料、疾病归因方式及上述各量表的因子分为自变量，以TTH患者的就医行为（包括4项指标，本次住院的平均住院时间、出院后1年内因TTH而服药的天数、门诊就诊次数及因TTH而再次住院的次数）为应变量，进行多元逐步回归分析。结果抑郁、神经质、负性生活事件对平均住院时间有正向预测作用，而解决问题因子对其有负向预测作用。焦虑、神经质、躯体化对因TTH而服药的天数有正向预测作用。抑郁、负性生活事件对过去1年中因TTH而门诊就诊次数有正向预测作用，而对TTH的心理归因对其有负向预测作用。焦虑及负性生活事件对因TTH而再次住院的次数有正向预测作用。结论不同的心理因素对TTH患者就医行为不同指标的影响不同。     

有机磷半抗原诱导的抗神经性毒剂单克隆抗体的制备及鉴定

目的：制备新化合物1-羟基—1—对胺基苯基甲磷酸单频哪基醇酯(P4)的单克隆抗体(mAb)。方法：以P4为半抗原，与血蓝蛋白(LPH)化学偶联制备人工抗原(P4—LPH)，并以其免疫BALB／c小鼠，制备抗P4的mAb。用竞争抑制酶免疫分析法，测定mAb对梭曼等7种配体的结合活性，结果以mAb与P4-BSA的结合被抑制50％时的配体浓度(IC50)，表示mAb的交叉反应性。结果：建立了9株对P4-BSA阳性反应的mAb，它们分属3种Ig亚类；6株(2B2、3A10、3D5、4F1、6C9和6H4)为IgGl，1株(3B9)为IgG2b，2株(1B3和6H5)为IgM。9株mAb中，8株具有与半抗原P4的结合的特异性。4株(1B3、2B2、3D5和4F1)具有梭曼结合活性，其IC50值分别为10^-3、10^-3、10^-5和10^-6mol/L；1株(4F1)具有与对氧磷结合的特异性，其IC50值为10^-5mol/L。4株腹水的mAb鼠滴度高，分别达10^-6(1B3和2B2)和10^-3(3D5和4F1)。结论：用新的半抗原P4成功地制得抗梭曼和抗对氧磷的mAb，可用于梭曼抗体酶、梭曼的免疫抗毒和免疫检测研究。     

用肾综合征出血热病毒结构蛋白体外免疫正常人外周血淋巴细胞制备单克隆抗体

用亲和层析提取的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５０ｋＤ和６７ｋＤ）为抗原，在体外免疫正常人外周血淋巴细胞（ＰＢＬ）。ＰＢＬ经亮氨酸甲基酯处理后，在含有ｓＰＷＭ－Ｔ、ＩＬ－２及不同浓度抗原的培养基中培养６ｄ，计数存活ＰＢＬ数量。结果表明，在上述体外免疫条件下存活ＰＢＬ的数量与抗原剂量呈正相关。将此体外免疫的ＰＢＬ与人－鼠杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，获得了３株分泌抗ＨＦＲＳＶ人单抗的杂交瘤细胞，表明在体外免疫条件下，正常人ＰＢＬ能够对ＨＦＲＳＶ结构蛋白发生特异性免疫应答。     

Paralysis to serum albumins in T and B lymphocytes in mice. Dose dependence, specificity and kinetics of escape

Paralysis to bovine serum albumin (BSA) was induced in mice at three concentrations of antigen. The animals were kept for 10 weeks at 10^?8 M BSA (low zone), 10^?5 M BSA (high zone) or 10^?4 M BSA. The latter concentration approaches that of autologous serum albumin. In order to determine paralysis in helper T cells and antibody-forming cell precursors (AFCP), the animals were immunized with sheep serum albumin (SSA) that cross-reacts with BSA at the levels of both helper cell and AFCP receptors. Helper cell paralysis was quantitated by determining splenic helper activity for BSA in a cooperating cell transfer system. Paralysis in the AFCP was determined by measuring the cross-reactivity of BSA and SSA in the anti-SSA serum raised in paralyzed and control animals. The following results were obtained. (1) Helper activity was almost totally suppressed in both low zone abd high zone paralysis, and at the helper cell level, paralysis could not be “broken” by immunization with a cross-reacting antigen. (2) Recovery in the helper cell compartment was not detectable before 4–5 months after the last paralyzing injection. (3) In the AFCP, that are not affected in low zone paralysis, only a small fraction of the cells, namely, those expressing receptors with a high affinity for the antigen, is inactivated in high zone paralysis. This is also true for animals kept at 10^?4 M antigen over a 10-week period. (4) Recovery in the (peripheral) AFCP was rapid and complete 2–3 months after the last paralyzing injection.     

Quaternized chitosan/alginate nanoparticles for protein delivery

Quaternized chitosan (QCS)/alginate (AL) nanoparticles (QCS/AL) were successfully prepared in neutral condition for the oral delivery of protein. The physicochemical structure of the QCS/AL nanoparticles was characterized by IR spectroscopy and transmission electron microscopy. The diameter of the nanoparticles with a positive surface charge was about 200 nm. The load of bovine serum albumin (BSA) was affected by the concentration and the molecular parameters, i.e. degree of substitution (DS) and weight-average molecular weight (Mw) of QCS, as well as the concentration of BSA. The release of BSA from nanoparticles was pH-dependent. Quick release occurred in 0.1M phosphate buffer solution (PBS, pH=7.4), while the release was slow in 0.1M HCl (pH=1.2). The DS and Mw of QCS play important roles in the release of BSA in vitro. QCS with high Mw accelerated the release of BSA in acid, while high DS retarded the release of BSA in both 0.1M HCl and 0.1M PBS.     

Immunological Characterization of α Antigen of Mycobacterium kansasii: B-Cell Epitope Mapping

Mapping of B-cell epitopes on α antigen of Mycobacterium kansasii (K-α) was carried out by using recombinant truncated K-α fusion peptides. We observed that two immunodominant B-cell epitopes (amino acids 222–268 and 267–306) and one minor epitope (amino acid 249–286) were located in the C-terminal region of K-α. The other three minor B-cell epitopes were mapped in N-terminal (amino acids 80–98 and 99–166) and central (amino acid 174–204) regions of K-α. All defined epitopes were common to Mycobacterium tuberculosis and M. kansasii . Besides these common epitopes, a region in K-α (amino acid 290–319) revealed different reactivities between antibodies against K-α and α antigen of M. tuberculosis . These findings may provide a basis for development of serodiagnosis that can distinguish between M. kansasii and M. tuberculosis infections.     

Molecular cloning and characterisation of a carp (Cyprinus carpio) cytokine-like cDNA that shares sequence similarity with IL-6 subfamily cytokines CNTF,OSM and LIF

In the course of suppression subtractive hybridisation between sodium alginate-induced peritoneal cells (SA-PC) and normal head kidney cDNAs in common carp (Cyprinus carpio), a cytokine-like cDNA clone was found. The clone, named M17, contains a 1600bp nucleotide sequence that encodes a 215 amino acid putative protein that would have a pI of 9.01 and would include a 33 amino acid signal peptide. The 3' untranslated region has seven ATTTA mRNA destabilising motifs that are common in cytokines and oncogenes. In a BLASTP search, M17 was most similar to chicken ciliary neurotrophic factor (CNTF) with 25% amino acid identity, followed by mammalian CNTF, cardiotrophin-1 and leukemia inhibitory factor (LIF) all of which belong to the IL-6 subfamily. However, M17 has some differences with CNTF in that CNTF has no signal sequence, the gene organisation of M17 is three exons and two introns, whereas that of CNTF is two exons and one intron, M17 has seven cysteines while CNTF has one cysteine, and M17 mRNA is detected in peripheral blood leukocytes as well as brain, whereas CNTF is expressed only in the nervous system. Compared to other members in the IL-6 subfamily cytokines, M17's cysteine positions and gene organisation are similar to those of oncostatin M and LIF, although amino acid identities are only 15-17%. Southern hybridisation suggested that M17 is a single copy gene. SA-PC showed significantly higher M17 mRNA levels than normal head kidney cells, which are considered to be a source of the SA-PC, indicating that M17 is inducible by inflammatory stimulation.     

A monoclonal anti-glycophorin A antibody recognizing the blood group M determinant: studies on the subspecificity

A mouse monoclonal antibody (425/2B, IgM) was obtained which shows specificity for blood group M determinant of glycophorin A. The antibody is pH-dependent. At pH 6-7 it reacted strongly with blood group M antigen, but also cross-reacted distinctly with N antigen. At pH 8.3 the antibody showed moderately decreased reactivity with M antigen, but no interaction with N antigen was detectable by hemagglutination, immunoblotting, or microplate ELISA. The direct binding studies and inhibition of 425/B antibody by untreated or modified blood group M and N glycoproteins or tryptic glycopeptides showed that the binding to the antigens was not affected by acetylation of their amino groups, or removal of amino-terminal amino acid residue. Desialylation of the antigens decreased their reactivity with the antibody and this effect was distinctly stronger at pH 7 than 8.3. The antibody reacted strongly at pH 7 and 8.3 with glycophorin B of Henshaw phenotype, whereas its reactivity with normal glycophorin B was weak or undetectable at these pH values, respectively. The results obtained indicated that anti-M specificity of 425/2B antibody is related to the 5th amino acid residue of glycophorin A (anti-Mgly specificity) and that pH shift from 7 to 8.3 changes the fine specificity of the antibody. At pH 8.3 the reactivity of the antibody is more dependent on glycine residue (higher anti-M specificity) and less dependent on sialic acid residues in the antigen.     

阿莫西林人工抗原的合成及其单克隆抗体的制备

目的:合成阿莫西林(AMX)完全抗原,获得稳定、高效分泌抗AMX单克隆抗体(mAb)的杂交瘤细胞株。方法:采用N-羟基琥珀酰亚胺活性酯(NHS)法制备AMX-OVA(免疫原)AMX-BSA(检测抗原),紫外光谱分析检测偶联物克分子比,以AMX-OVA免疫BALB/c小鼠,用细胞融合技术筛选分泌抗AMX mAb的杂交瘤细胞,体内诱生法制备腹水,辛酸-硫酸胺法从腹水中纯化mAb,ELISA法测定mAb亚类。结果:紫外光谱显示偶联物表现出小分子与蛋白载体叠加的特征,具有良好的免疫原性和反应原性。获得了5株可稳定分泌特异性抗AMX mAb的杂交瘤细胞株,亚类鉴定均为IgG1。mAb对AMX最小检测极限(limit of detection,LOD)为0.25 ng/mL,但与同属β-内酰胺类抗生素的氨苄青霉素有一定程度的交叉反应。结论:该细胞株可满足建立免疫学检测方法的需要和开发应用的要求。     

Identification and characterization of epitopes shared between the mycobacterial 65-kilodalton heat shock protein and the actively secreted antigen 85 complex: their in situ expression on the cell wall surface of Mycobacterium leprae.

Both mycobacterial hsp65 and the actively secreted antigen 85 complex of 30-kDa region proteins are considered to be major immune targets in mycobacterial diseases. In this study, by using a novel series of monoclonal antibodies (MAbs) directed to these antigens, we identified and partially characterized three unique epitopes (Rb2, Pe12, and A2h11) that are shared between mycobacterial hsp65 and the individual components of the antigen 85 complex. Dot blot assays with native purified proteins revealed that all three MAbs are strongly bound to hsp65 and antigens 85A (MPT44) and 85B (MPT59), while a weak reaction or no reaction was found with antigen 85C (MPT45). Immunoblotting showed that MAb Rb2 reacted strongly with both hsp65 and the antigen 85 complex proteins, whereas MAbs Pe12 and A2h11 reacted strongly with the former but weakly with the latter. Moreover, these MAbs did not react with other closely related MPT51 and MPT64 secreted proteins. Further characterization of these epitopes was performed by using recombinant fusion and truncated proteins of Mycobacterium bovis BCG hsp65 (MbaA) and the M. leprae 30- and 31-kDa antigen 85 complex fusion proteins. In hsp65, Rb2-Pe12- and A2h11-reactive epitopes were found to reside in the C-terminal region of amino acid residues 479 to 540 and 303 to 424, respectively. In the M. leprae 30- and 31-kDa antigen 85 complex, all three epitopes were located in an N-terminal region of amino acid residues 55 to 266, one of the known fibronectin-binding sites of the M. leprae antigen 85 complex. Comparison of these MAb-reactive amino acid sequence regions between mycobacterial hsp65 and the components of the antigen 85 complex revealed that these regions show certain amino acid sequence identities. Furthermore, by immunoperoxidase and immunogold ultracytochemistry, we demonstrated that Rb2-, Pe12-, and A2h11-reactive epitopes are expressed both on the cell wall surface and in the cytosol of M. leprae bacilli within the lesions of lepromatous leprosy patients and in M. leprae-infected armadillo liver tissue.     

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.     

Removal of bovine serum albumin from cow's milk using chicken egg‐yolk antibodies immobilized on chitosan gel

Polyclonal chicken antibodies raised against bovine serum albumin (BSA) were immobilized on chitosan gel for the immunoaffinity isolation of BSA from cow's milk. Antibodies (IgY) against BSA were isolated from egg‐yolk, purified and antibody reactivity to antigen was measured. IgY developed against BSA was reduced by 2‐mercaptoethylamine. The reactivities of reduced and whole IgY against BSA were not significantly different. The reduced IgY was covalently linked to chitosan gel through stable covalent thioether linkages using sulfo‐succinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐l‐carboxylate (sulfo‐SMCC) as a cross‐linker. The density of antibody IgY immobilized on chitosan gel was approximately 3–5 mg per ml of chitosan gel. The ligand‐binding capacity of immobilized IgY towards BSA was 0.35–0.44 mg BSA per ml of chitosan gel. A single pass of skimmed milk through the column allowed the removal of BSA from the milk sample. The milk sample was analyzed, before and after immunoaffinity separation, by SDS‐PAGE. BSA was desorbed with 0.5 M‐glycine‐HCl buffer at pH 2.8 but the reusability of the column was limited to three cycles. Alternatively, BSA was desorbed with 0.5 M‐glycine‐HCl buffer containing 2 M‐NaCl at pH 4.6 after longer incubation times at a slower flow rate. The low ligand‐binding capacity was not an impedement to reuse of the column. The column was reused more than 20 times with minimal loss of binding capacity.