黄曲霉毒素B_1半抗原分子设计与抗原合成及抗体特性研究进展

本文综述了国内外半抗原分子设计方法、基本要求、应遵循原则、抗原合成方法等方面的研究进展,尤其是对黄曲霉毒素B_1(AFB_1)半抗原分子设计与抗原合成及抗体特性进行了详尽阐述,旨在为AFB_1制备AFB_1及其他小分子半抗原高质量的抗体提供新的方法和思路。

抗BVDV Erns蛋白单克隆抗体制备及生物学特性鉴定

为制备抗牛病毒性腹泻病毒(BVDV)Erns蛋白单克隆抗体,本试验用Erns重组蛋白免疫Balb/c鼠,刺激小鼠脾脏产生特异性免疫细胞;通过化学剂诱导剂PEG-4000诱导免疫细胞和骨髓瘤细胞融合,形成杂交瘤细胞;间接ELISA方法结合亚克隆方法筛选阳性细胞孔;鉴定为稳定分泌抗体的细胞株做扩大培养,细胞悬液计数并无菌注射KM小鼠腹腔,制备、收集腹水抗体;SDS-PAGE鉴定抗体大小、ELISA测定抗体效价、Western blot和IFA鉴定抗体特异性。本试验建立了3株稳定分泌抗体的细胞株;腹水抗体重链大小为55 kDa,轻链25 kDa,均为IgG1型,效价高于10^(4),抗体可特异性结合病毒抗原。结果证实,制备的单克隆抗体效价高、特异性好,为制备检测BVDV试剂盒奠定基础。

PAICA法检测ITP病人血小板特异抗体的临床应用

目的 检测特发性血小板减少性紫癜 (ITP)病人及非免疫性血小板减少症病人的抗血小板特异性抗体 ,并探讨其临床意义。②方法 采用血小板相关抗体特性检测技术 (PAICA法 ) ,检测 5 2例ITP病人、31例非免疫性血小板减少症病人抗血小板GPⅡb/Ⅲa ,GPⅠb/Ⅸ的特异性抗体 ,并与血小板相关抗体IgG(PAIgG)检测结果进行了比较。③结果 血小板特异抗体检测的可能性比值及阳性预测值较高 ,但阴性预测值较低 ,而PAIgG检测的可能性比值、阳性预测值及阴性预测值均较低。④结论 血小板特异抗体检测对肯定ITP诊断有重要的意义 。

抗人N端脑钠肽前体抗原表位分析及单克隆抗体的研制

目的:制备并鉴定特异性抗人N端脑钠肽前体(NT-proBNP)抗原表位的单克隆抗体。方法:通过Bepipred在线表位预测方法和Epitope Prediction System软件预测人NT-proBNP B细胞抗原表位,综合2种预测结果对比优化选取合适的2个表位肽段P1和P2,偶联KLH为免疫原,免疫BALB/c小鼠,选择血清效价较高的小鼠脾细胞与SP2/0细胞融合;经HAT筛选,间接ELISA法检测和有限稀释法进行亚克隆,得到阳性杂交瘤细胞株;采用体内诱生法制备腹水并用Protein G柱纯化抗体;经鉴定,对特异性最好的2株进行纯度、效价、亚型及亲和力的分析。结果:通过P2-KLH免疫获得的阳性杂交瘤细胞株1F9和P1-KLH免疫获得的阳性杂交瘤细胞株4H2能稳定分泌抗NT-proBNP单克隆抗体,纯度较高,能与NT-proBNP重组蛋白特异性结合,效价分别达到4×10^4、4×10^5,抗体亚型均为IgG1,相对亲和力常数分别为1.14×10^7和1.6×10^7 L/mol。结论:获得2株特性良好的单克隆抗体,可为下一步研制NT-proBNP检测试剂盒提供原材料。

基于噬菌体展示纳米抗体的绿色免疫PCR检测脱氧雪腐镰刀菌烯醇

目的:在获得脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)抗独特型纳米抗体的前期基础上,将纳米抗体作为酶标抗原的替代物,应用于荧光定量免疫聚合酶链式反应(polymerase chain reaction,PCR)体系,实现DON的高灵敏、绿色免疫分析。方法:将特异性结合DON抗体的噬菌体展示纳米抗体(P-28)作为竞争抗原,以编码P-28纳米抗体的DNA为靶标,设计特异性PCR扩增引物,优化荧光定量免疫PCR退火温度、抗DON抗体浓度、P-28投入量等参数,建立基于间接竞争模式的荧光定量免疫PCR检测DON的方法。结果:本研究建立的荧光定量免疫PCR方法线性检测范围为0.1～1 000 ng/mL,IC50值为(3.96±2.21)ng/mL,最低检出限为0.048 ng/mL,并与其他真菌毒素无交叉反应。结论:该方法直接使用噬菌体展示纳米抗体作为竞争抗原的替代物,应用于免疫PCR体系,避免了使用传统的化学合成酶标抗原所带来的环境污染、操作毒性等缺陷,并具有良好的特异性及灵敏度。

抗-hTSH抗独特型抗体的制备及生物活性的研究

本文根据独特性-抗独特性免疫网络学说的理论，探讨Graves病发病机理。本文用hTSH免疫家兔制备了抗TSH抗体（TSHAb1）并进行纯化。用纯化后的TSHAb1作为免疫源，免疫豚鼠获得了抗TSHAb1的抗独特型抗体（TSHAb2），并从免疫、生物活性方面进行鉴定，证明所制备出的抗体TSHA2有类似TSH样的功能。

糖皮质激素联合依诺肝素钠对复发性早期流产患者BA、LP、AIA、ADPN影响

目的:探讨糖皮质激素联合依诺肝素钠治疗复发性早期流产效果及对患者封闭抗体(BA)、瘦素(LP)、抗独特性抗体(AIA)、脂联素(ADPN)作用。方法:选择2018年3月-2020年3月在本院接受治疗的复发性早期流产患者115例,随机数表法分为联合组(n=58)和单药组(n=57)。单药组给予依诺肝素钠治疗,联合组给予糖皮质激素联合依诺肝素钠治疗。比较两组临床疗效、血清BA、LP、AIA、ADPN、孕酮(P)、雌二醇(E2)、绒毛膜促性腺激素(β-hCG)水平变化情况、妊娠结局及不良反应。结果:治疗后联合组总有效率(91.4%)高于单药组(71.9%),两组BA、AIA转阳率均提高且联合组(87.9%、79.3%)高于单药组(64.9%、54.4%)(均P<0.05);血清激素LP、ADPN、P、E2、β-hCG两组均高于治疗前且联合组高于单药组(P<0.05)。联合组成功分娩率(87.9%)高于单药组(66.7%),异位妊娠(1.7%)、再次流产率(0)低于单药组(15.8%、8.8%)(P<0.05);两组不良反应总发生率(3.5%、7.0%)无差异(P>0.05)。结论:复发性早期流产患者应用糖皮质激素联合依诺肝素钠治疗可提高疗效,可能与其可有效改善患者BA、LP、AIA、ADPN水平有关。

Development and Characterization of Monoclonal Antibody Specific to Nuclear Protein of Avian Influenza Virus Type A

Five monoclonal antibodies（Mabs） to nuclear protein of avain influenza virus（AIV） were developed by syncretizing SP 2/0 and the spleen cells from BALB of mice immuized with H9 subtype AIV. Specificity of these Mabs were identified by immunofluorescent assay（IFA） and enzyme linked immunosorbent assay （ELISA）. These five Mabs which were named as AIV-NP-2C3, AIV-NP-6A5, AIV-NP-3 H9, AIV-NP-7B4, AIV-NP-2H4 could react with all viruses of AIV-H9 strains in tests. The result of Western blotting showed that only the 60 ku protein antigen of AIV-H9 could be recognized by the Mabs but never recognized by New castle disease virus, REV and infectious bursa disease virus. The result of preliminary application showed that avian influenza viruses could be deetected bv Mabs in IFA and ELISA. All these Mabs will probably play important roles in preventing and monitoring avian influenza viruses.

Comparison of Multiplex Fluorescent PCR with Serum Type-specific Antibody Detection in Diagnosis of Genital Herpes

Objectives: To compare multiplex fluorescent PCRwith serum type-specific antibody detection in thediagnosis of herpes simplex virus (HSV) infection andto evaluate its significance in the diagnosis of genitalherpes.Methods: We detected HSV infection in 121 speci-mens collected from patients with genital herpesusing both multiplex fluorescent PCR and serum type-specific antibody detection. HSV viral isolation wasused as the standard control.Results: When compared with the viral isolation, thesensitivity and specificity for multiplex fluorescentPCR were 100% and 88.89%, respectively afterdiscrepant analysis. The sensitivity and specificity fortype-specific antibody detection was 77.68 % and77.78 %, respectively. However, the type-specificantibody detected HSV in two asymptomatic patientswhile the multiplex fluorescent PCR couldn’t detectany HSV DNA from those specimens.Conclusions: Multiplex fluorescent PCR is a verysensitive and specific method for detection and typingof HSV in the lesion of genital herpes, it failed todetect HSV DNA from the asymptomatic patients.Serum type-specific antibody detection was a lesssensitive and specific test but could detect the specificantibody from some asymptomatic patients. Thecombination of these two techniques would allow rapid,sensitive and accurate detection and typing of HSVand help clinical diagnosis and epidemiologic survey-ing of genital herpes.

Potential implications of Helicobacter pylori-related neutrophil-activating protein

Helicobacter pylori （H. pylori） virulence factors pro- mote the release of various chemoattractants/inflam- matory mediators, including mainly the neutrophil- attractant chemokine interleukin-8 and neutrophil- activating protein （NAP）, involved in H. pylor/-induced gastric pathologies. Co-administration of Chios mastic gum （CMG）, which inhibits H. pylor/NAP, with an H. pylori eradication regimen might add clinical benefits against H. pylori-related gastric pathologies, but pos- sibly not CMG as main therapy. Although H. pylori NAP and other H. pylori-related cytotoxins [i.e., vaculating cytotoxin （VacA）] appear to play a major role in gener- ating and maintaining the H. pylori-associated gastric inflammatory response and H. pylor/NAP is a promising vaccine candidate against H. pylori infection （H. pylori-1）, concerns regarding its potential drawbacks, particularly neurogenic ones, due to possible cross- mimicry, should be considered. Possible cross-mimicry between H. p, vlor/ NAP and/or bacterial aquaporin （AQP） and neural tissues may be associated with the anti-AQP-4 antibody-related neural damage in multiple sclerosis （MS）/neuromyelitis optica patients. Moreover, the sequence homology found between H. pylori VacA and human Na＋/K＋-ATPase A subunit suggests that antibodies to VacA involve ion channels in abaxonal Schwann cell plasmalemma resulting in demyelination in some patients. A series of factors have been im- plicated in inducing blood-brain barrier （BBB） disrup- tion, including inflammatory mediators （e.g., cytokines and chemokines induced by H. pylor/-I） and oxidative stress. BBB disruption permits access of AQP4-specific antibodies and T lymphocytes to the central nervous system, thereby playing a major role in multiple sclero- sis pathogenesis. Relative studies show a strong asso- ciation between H. pylori-I and MS. H. pylor/-I induces humoral and cellular immune responses that, owing to the sharing of homologous epitopes （molecular mim- icry）, cross-react with components of nerves, thereby contributing and perpetuating neural tissue damage. Finally, H. pylori NAP also plays a possible pathoge- netic role in both gastric and colon oncogenesis.

Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185^(c-erbB-2)

The c-erbB-2 proto-oncogene encodes a 185kDa protein p!85, which belongs to epidermal growth factor receptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis for certain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibits proliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. In order to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed the single-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichia coli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cell immunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain (ECD) of p!85. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion molecule with a homodimer form and the recombinant product was expressed in mammalian cells. In a series of subsequent analysis this fusion protein showed identical antigen binding site and activity with the parent antibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targeting drugs, with a view of application in the diagnosis and treatment of human breast cancer.

Affinity peptide developed by phage display selection for targeting gastric cancer

AIM:To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer.METHODS:A peptide screen was performed by biopanning the PhD-12 phage display library,clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues.Tumortargeted binding of selected peptides was confirmed by bound phage counts,enzyme-linked immunosorbent assay,competitive inhibition,fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues.RESULTS:Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal-appearing gastric mucosa.After the third round of positive screening,the peptide sequence AADNAKTKSFPV(AAD) appeared in 25%(12/48) of the analyzed phages.For the control peptide,these values were 6.8 ± 2.3,5.1 ± 1.7,3.5 ± 2.1,4.6 ± 1.9 and 1.1 ± 0.5,respectively.The values for AAD peptide were statistically signif icant(P < 0.01) for gastric cancer as compared with other histological classif ications and control peptide.CONCLUSION:A novel peptide is discovered to have a specific binding activity to gastric cancer,and can be used to distinguish neoplastic from normal gastric mucosa,demonstrating the potential for early cancer detection on endoscopy.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

化学发光微粒子免疫检测法在梅毒血清学检测中的应用

目的观察化学发光微粒子免疫检测法(CMIA)在梅毒螺旋体特性抗体(TP-Ab)血清学检测中的应用。方法采取前瞻性研究,选择2018年1月至2021年1月商丘市中心医院收治的60例高度疑似梅毒患者作为研究对象。所有患者均接受TPPA及CMIA检测,以TPPA检测结果为参比,分析CMIA在梅毒TP-Ab血清学检测中的应用价值。结果 60例高度疑似梅毒患者,TPPA检测阳性46例(76.67%),阴性14例(23.33%);与TPPA作为参比,CMIA对梅毒TP-Ab抗体的检出的敏感度为95.65%,特异度为92.86%,准确度为95.00%,一致性较好;TP-IgM对梅毒TP-Ab抗体的检出的敏感度为76.09%,特异度为71.43%,准确度为75.00%,一致性一般;TP-ELISA对梅毒TP-Ab抗体的检出的敏感度为78.26%,特异度为85.71%,准确度为80.00%,一致性一般;CMIA诊断TP-Ab敏感度、准确度高于TP-IgM、TP-ELISA,三组比较差异有统计学意义(P <0.05)。结论 CMIA在梅毒的诊断中相较于TP-IgM、TP-ELISA具有较好的诊断效能,临床可选择CMIA作为梅毒血清学检测方法。

Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer

AIM： To detect and evaluate the antibodies against Helicobacter pylori （H pylori） neutrophil-activating protein （HP-NAP） in patients with gastric cancer and other gastroduodenal diseases.METHODS： Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coll. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 （IL-8） and growth-related oncogene （GROα） cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected.RESULTS： The serum positivity and mean absorbancevalue of HP-NAP-specific antibodies in the gastriccancer group （97.7% and 1.01 ± 0.24） were significantly higher than those in the chronic gastritisgroup （85.7% and 0.89 ± 0.14, P 〈 0.005） and healthy control group （27.7% and 0.65 ± 0.18, P 〈 0.001）. The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly upregulate IL-8 production in gastric epithelial cell lines but had no effect on GROα production.CONCLUSION： Infection with virulent H py/ori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma, rHP-NAP- based ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

Immunogenicity and protective efficacy of Vibrio harveyi pcFlaA DNA vaccine in Epinephelus awoara

The FlaA gene from Vibrio harveyi marker, was cloned into the eukaryotic expression with a short nucleotide sequence encoding the Flag vector pcDNA3.1（＋） （designated as pcFlaA）. Ninety grouper （Epinephelus awoara） were separated into three equal size groups. An experimental group was immunized with pcFlaA, Control I group was immunized with the vector pcDNA3.1（＋）, and Control 1I group was immunized with PBS. The expression of pcFlaA mRNA and protein was examined using reverse transcription-polymerase chain reaction （RT-PCR） and immunohistochemistry. We also evaluated the immunogenicity and protective efficacy of pcFlaA against V. harveyi by measuring the lymphocyte proliferation response and serum levels of specific antibody and conducting a bacterial challenge test. We successfully transfected the fish muscle with pcFlaA. The pcFlaA mRNA and protein was expressed in the muscle cells for up to one month following injection. The proliferation response of lymphocytes in fish immunized with pcFlaA was significantly higher than in control group II. Furthermore, the immunized fish generated specific antibody. The vaccination also resulted in significantly higher survival during the bacterial challenge test.

ANALYSIS OF THE ANTIGEN-ANTIBODY SPECIFICITY IN THE SEMEN OF PATIENTS WITH NEISSERIA GONORRHOEAE

This study was done to define the human genital immune response to infection with Neisseria gonorrhoeae. The semen specimens were obtained from 15 patients with uncomplicated gonococcal infection in the acute and convalescent phases and 15 men with uninfected control- After precipitated with amoniasulfate,the semen was tested against the outer membrane protein of gonococcal isolates from the same patients to examine antigen-antibody interactions by use of the western blot technique. The antibodies in the semen reacted with more gonococcal antigens in the acute phase than in the convalescent phase. IgA in the semen reacted with more antigens than did IgG in the same specimens. The predominant reacted antigens were protein I, protein II, 46～ 48, kD, 14 ～16 kD and 88～ 90 kD protein.

Preparation of monoclonal antibody to P53 and its clinical application

Objective：The aim of this study was to prepare monoclonal antibody against P53, a kind of tumor suppressor protein,and use the antibody initial y in clinical immunoassay. Methods：Monoclonal antibody was prepared and identified via the classic protocol of monoclonal antibody preparation. Identified monoclonal antibodies were purified by af inity chro-matography. Antibody titer was determined by enzyme linked immunosorbent assay （ELISA）. The specific binding activity of antibody was detected by Western blotting and immunohistochemistry. Results：Three strains of monoclonal antibodies named 1P15, 2P37 and 3P40 were obtained and purified by af inity chromatography. The purity of antibodies was higher than 90%. The titers of antibodies were more than 1：6000. Western blot and immunohistochemistry assay showed that the specific antibody can combine with endogenous P53 protein in the tumor celllines and determine the expression of P53 in tumor tis-sue. Conclusion：Three strains of monoclonal antibodies with high af inity to P53 were successful y established, which can be used for detecting the expression of P53 in tumor cells or tissue.

A Woman with Psychogenic Non-epileptic Seizures and Pelvic Mass

MOST cases of encephalitis are caused by viruses but a few have an immunological basis, such as paraneoplastic encephalitis, with specific antibodies identified. One recently characterized encephalitis caused by antibodies is anti-N- methyl-D-aspartate （NMDA） receptor encephalitis. It is a form of paraneoplastic limbic encephalitis associated with ovarian teratoma and has recently been described.The NMDA receptor mediates excitatory neurotransmission. It is important for synaptic plasticity, and thus for higher function such as learning and memory. This disorder results in prominent psychiatric symptoms followed by a rapid decline of the level of consciousness, central hypoventilation, seizures, involuntary movements and dysautonomia.