犬C-反应蛋白荧光定量免疫层析检测方法的建立与应用

试验开展了犬C-反应蛋白(C-CRP)荧光微球免疫层析定量方法的研究,旨在研制一种操作简易、灵敏度高,用于快速定量检测C-CRP荧光免疫层析试纸条。采用双抗体夹心法和荧光免疫层析技术,以羧基荧光微球标记的抗C-CRP单克隆抗体及羊抗鸡IgY为标记抗体,抗C-CRP单克隆抗体和鸡IgY分别作为检测线和质控线制备荧光免疫层析试纸条。结果显示,所制备C-CRP检测试纸条的检测范围为0.5～250mg/L,检测限为0.5mg/L,批内和批间变异系数(CV)分别为0.68%～6.94%和0.89%～8.79%,平均回收率为98.15%～101.19%,试剂与9种干扰物质无交叉反应。加速破坏稳定性试验表明试纸条可以常温放置24个月。临床样本测试发现,其与I-CHROMA试剂盒检测结果相关性良好(R2=0.995)。综上所述,该荧光免疫层析试纸条灵敏度、特异度较高,且操作简单、快速,可用于宠物犬临床检测。     

A dominant antigenic epitope on SARS-CoV spike protein identified by an avian single-chain variable fragment (scFv)-expressing phage

Severe acute respiratory syndrome (SARS) is a newly emergent human disease, which requires rapid diagnosis and effective therapy. Among antibody sources, immunoglobulin Y (IgY) is the major antibody found in chicken eggs and can be used as an alternative to mammalian antibodies normally used in research and immunotherapy. In this study, phage-expressing chicken monoclonal scFv antibody was chosen and characterized with phage display antibody technology. Truncated fragments of SARS-CoV spike protein were cloned in pET-21 vector and expressed in BL-21 Escherichia coli (E. coli) cells. After purification, the purity of these recombinant spike proteins was examined on SDS-PAGE and their identity verified with Western blot analysis using anti-his antibodies and sera from convalescent stage SARS-CoV-infected patients. Using these bacteria-derived proteins to immunize chickens, it was found that polyclonal IgY antibodies in the egg yolk and sera were highly reactive to the immunogens, as shown by Western blot and immunocytochemical staining analysis. A phage displaying scFv library was also established from spleen B cells of immunized chicken with 5 x 10(7) clones. After four panning cycles, the eluted phage titer showed a 10-fold increase. In sequence analysis with chicken germline gene, five phage clones reacted, with large dissimilarities of between 31 and 62%, in the complementarity-determining regions, one dominant phage 4S1 had strong binding to fragment Se-e, located between amino acid residues 456-650 of the spike protein and this particular phage had significantly strong binding to SARS-CoV-infected Vero E6 cells. Based on the results, we conclude that generating specific scFv-expressing phage binders with the phage display system can be successfully achieved and that this knowledge can be applied in clinical or academic research.     

The use of chicken IgY in a double antibody sandwich ELISA for detecting African horsesickness virus.

An indirect sandwich ELISA that can detect as little as 8 ng of African horsesickness virus (AHSV) was developed. Viral antigen was captured from suspension using an immobilized monoclonal antibody specific for an epitope on VP7, a protein that is a major constituent of the virus core. Egg-yolk derived chicken IgY directed against AHSV (serotype 3) was used as the secondary antibody. Since IgY and mouse IgG do not cross-react serologically, the secondary antibody was not labelled, but was instead detected with enzyme-coupled sheep antibodies directed against avian immunoglobulins. The assay recognized all nine AHSV serotypes, but not the Cascara isolate of equine encephalosis virus, a related orbivirus that also infects horses. In addition to being able to detect and quantify whole AHSV, the ELISA could show the presence of VP7 produced by recombinant baculoviruses.     

Preparation and Identification of N-glycolylneuraminic Acid IgY Antibody

The experiment was carried out to establish a method for preparation and identification of N-glycolylneuramic acid IgY antibody.Neu5Gc was linked to carrier proteins by carbodiimide(EDC)method.After the immunization of immunogen to laying hens,the IgY antibody was gained.Neu5Gc-IgY was analyzed and identified by ELISA.The results of UV and agarose gel electrophoresis showed that the artificial antigens of Neu5Gc were successfully synthesized.The development of the IgY antibody titer was monitored by indirect ELISA.The result showed that the IgY antibody was generated at 7th day after the first immunization.The titer was gradually increased and reached its peak (1:10 000) at 63th day after the first immunization.The high titer of the IgY antibody maintained through the whole observation period.The sensitivity of anti-Neu5AGc IgY antibody was detected by the competitive blocking ELISA.The linearity about the first chicken of the standard curve showed good,the liner regression equation was y=30.28x+16.923,R²=0.9581.The IgY antibody gained in this study laid the foundation for the establishment of indirect ELISA immunoassay method for detecting Neu5Gc in red meat,milk and tumor tissues.     

N-羟乙酰神经氨酸IgY抗体的制备及鉴定

本试验旨在建立N-羟乙酰神经氨酸(Neu5Gc) IgY抗体的制备及鉴定方法。通过碳二亚胺法将Neu5Gc和载体蛋白进行偶联,制备Neu5Gc人工完全抗原,用制备的完全抗原免疫海兰褐蛋鸡制备Neu5Gc-IgY抗体,经ELISA检测分析鉴定抗体活性。经紫外光谱扫描、琼脂糖凝胶电泳法初步判断Neu5Gc人工完全抗原制备成功,获得的特异性Neu5Gc-IgY抗体经ELISA检测分析,首次免疫后第7天产生抗体,抗体效价呈动态变化,随着免疫次数的增多,血清效价和抗体效价逐步上升,至初次免疫后63 d达到高峰,效价达1:10 000,停止加强免疫后抗体效价可持续一个月左右,1号鸡产生的IgY抗体具有很好的抗原竞争活性,获得竞争回归方程y=30.28x+16.923,线性系数R²=0.9581。以上结果表明,本试验制备的Neu5Gc IgY抗体取得了理想效果,为红肉、牛奶及肿瘤组织中Neu5Gc的检测奠定了基础。     

猪硒蛋白W的原核表达及IgY多克隆抗体制备

试验旨在优化硒蛋白W （selenoprotein W，SelW）的原核表达系统，并评估SelW的免疫原性和基于IgY抗体检测猪体内SelW的可行性。将获得的猪SelW基因序列密码子进行优化、合成并连接至pET-32a （+）表达载体中，构建原核表达重组质粒pET32a （+）-SelW，转化E.coli BL21（DE3）宿主菌中，进行IPTG诱导表达，SDS-PAGE鉴定重组猪SelW的表达情况，并免疫产蛋鸡制备抗SelW的IgY多克隆抗体，通过间接ELISA检测IgY抗体滴度，采用Western blotting检测IgY识别抗原的特异性。SDS-PAGE结果显示，SelW基因在原核细胞中成功表达，得到了大小约为33 ku的重组蛋白，IPTG最佳诱导浓度及时间分别为0.5 mmol/L和6 h；可溶性分析结果显示，重组蛋白SelW主要以包涵体形式存在。间接ELISA检测结果显示，免疫后45 d的IgY多克隆抗体的效价可达1：51 200。Western blotting检测结果显示，重组蛋白具有良好的免疫原性，所制备的IgY抗体与SelW的亲和力较高。本试验成功构建并优化了SelW蛋白的原核表达系统，提高了SelW蛋白的表达量，获得了具有良好免疫原性的SelW蛋白，制备的IgY抗体能特异性识别猪肌肉组织中的SelW，可用于检测猪组织中SelW的表达、预防硒中毒和缺硒性疾病的监测等，为进一步探究SelW的生物学功能奠定基础。     

In vitro reactivity and growth inhibition of EPEC serotype O111 and STEC serotypes O111 and O157 by homologous and heterologous chicken egg yolk antibody

IgY is a chicken egg yolk antibody which has been used for treatment and prophylaxis of gastrointestinal infections. Our aim was to verify if IgY obtained from chickens immunized with EPEC O111, STEC O111 and STEC O157 is able to show in vitro reactivity and biological activity towards the three bacteria. IgY was obtained from eggs laid before and after immunization with each bacterium. The preparations of IgY anti-EPEC O111 and anti-STEC O111 shared high reactivity detected by ELISA and growth inhibition ability towards both bacteria EPEC O111 and STEC O111. Nevertheless, the preparation of IgY anti-STEC O157 showed high reactivity and growth inhibitory effect only towards the homologous strain. Our results showing in vitro biological activity of IgY reinforce its use as an alternative for the treatment or prophylaxis of E. coli infections and encourage the development of in vivo studies for a possible future human therapeutic use.     

鸡新城疫卵黄抗体IgY的分离提纯研究

本文对母鸡经鸡新城疫疫苗免疫接种后产生并富集在卵黄中的抗体IgY的提取纯化工艺条件进行了研究与优化，确定了去脂、提取抗体最佳工艺为：卵黄液用0．05mol／L,pH5.0的乙酸一乙酸钠缓冲液1：9倍稀释，搅拌15min，4℃静置10小时，抗体回收率为90％，去脂率为99％；该提取液用40％饱和度的硫酸铵盐析，得到的盐析液抗体回收率为82％，纯度达到69％。盐析液经截留分子量为100KD的有机陶瓷膜超滤后，截留液抗体回收率达到92％。纯度为85％。     

IgY在免疫检测及治疗中的应用进展

卵黄抗体(IgY)是存在于卵黄中的免疫球蛋白,是鸟类、爬行动物和两栖类动物体内的主要抗体,具有强大的免疫功能。论文介绍了卵黄抗体的分子结构和理化特性,综述了IgY在寄生虫(弓形虫、血吸虫、球虫)、细菌(肠产毒素大肠埃希菌)、病毒(B型流感病毒、狂犬病病毒)等病原检测、抗生素(卡那霉素、庆大霉素)残留检测及相关疾病如雏鸡球虫病、猪大肠杆菌病、鳗鱼弧菌和利斯顿氏菌病、轮状病毒病治疗中的应用,并指出目前IgY应用中存在的诸如可能存在禽类未知病原、尚无生产标准规程以及稳定性不够理想等问题。因卵黄抗体具有安全、高效、无公害的特点,其在疫病检测和防治中具有广阔的应用前景。     

Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae

A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera.     

卵黄抗体提取方法及其在畜禽细菌性肠道疾病防治中的应用

细菌性肠道感染是畜禽养殖过程中的常见疾病，易引起畜禽采食量下降、生产性能降低等问题，严重者甚至造成死亡。在养殖过程中多采用抗生素对其进行治疗，但由于抗生素长期大量使用，特别是不合理使用，使药物残留和细菌耐药性问题日益凸显。因此，发展安全可靠的抗生素替代品已然成为当今畜牧行业的研究热点。卵黄抗体（egg yolk immunoglobulins，IgY）是通过免疫接种产蛋鸡后，在蛋黄中表达的一种免疫球蛋白，可用于相应疾病的预防和治疗。IgY具有产量大、性质稳定、安全高效等诸多优点，目前IgY作为抗生素替代品已被广泛用于畜禽养殖过程中，并取得了显著的效果。文章介绍了IgY的分子结构、理化性质和作用机理，总结并比较了5种IgY提取方法的去脂率、抗体效价、总蛋白含量等指标，最后详细介绍了IgY在防治畜禽常见细菌性感染中的研究现状和应用前景，以期为IgY的高效生产及疾病防治提供研究依据和技术支持。     

Influence of enrofloxacin and chloramphenicol on the level of IgY in serum and egg yolk after immunostimulation of hens with Salmonella enteritidis antigens

In the chicken, maternal antibodies are transferred into the egg and subsequently transported into the developing embryo. IgG (called IgY) is the primary immunoglobulin isotype of the egg yolk. Their level in serum depends on the correct function of immunological system in laying hens. Many factors have a direct or indirect influence on antibody level in fowl. One of them is a commonly used antibiotic, but its influence on avian immune system is still unknown. The objective of the study was to determine the effect of enrofloxacin and chloramphenicol on the level of IgY antibody in serum and egg yolk after immunostimulation of hens with living cells of Salmonella enterica subsp. enterica serovar Enteritidis and lipopolisaccharide. Forty adult egg-laying Arbor Acres and Isa 215 hens (32 and 50 weeks old) from the reproductive flocks and 1640 of their eggs were used for the investigation. No clinical symptoms of any diseases were observed in birds during the entire breeding period. Additionally the birds were checked as free from Salmonella spp. in the beginning of the experiment. The birds were divided into 6 experimental and 2 control groups (5 birds in one group). The hens in the experimental groups were immunized with S. Enteritidis antigens: living bacteria and lipopolisaccharide and treated with enrofloxacin or chloramphenicol. Antibiotics were administered in drinking water for 10 days (from 3rd to 13th day of experiment). To indicate anti-S. Enteritidis, antibodies in sera and egg yolk were used indirectly on ELISA based on lipopolisaccharide from S. Enteritidis. As conjugate these were applied anti-chicken IgY with horseradish peroxidase and ABTS with H2O2 as obtained. Additionally, to detect antibody in serum, a rapid slide test was used with Pullognost and Enterognost standard antigens made in the laboratory. The study revealed that both antibiotics tested decreased the level of specific IgY in laying hens immunized with living bacteria and lipopolisaccharide. It seems that antibiotics have a suppressive effect on the immunological system. The strongest immunosuppressive effect was exerted by chloramphenicol.     

A Comparison of Three Methods for Extracting IgY from the Egg Yolk of Hens Immunized with Sendai Virus

Hens were immunized with partially purified Sendai virus that had been grown on chicken embryos. The titres of specific antibodies to Sendai virus varied from log₂ 13.0 to 15.5 during the 4 months after immunization and the immunoglobulin Y (IgY) concentration varied from 2 to 10 mg per ml of egg yolk. A method has been developed employing dextran blue to isolate IgY from the egg yolk. The dextran blue method was compared with two other methods (poly(ethylene glycol) and chloroform) in terms of yield, total protein content, IgY concentration, specific activity of IgY and SDS-PAGE analysis. The specific activity and the IgY content obtained by these three methods proved to be identical, in the range log₂ 12.9–14.1, and 4.6–12.8 mg/egg, respectively. However, the total protein content when purified by chloroform was 2-fold to 4-fold higher than that of the others. The analysis of IgY by SDS-PAGE demonstrated that IgY purified with dextran blue contained three major protein components with molecular weights of 34.7 kDa, 41 kDa and 66 kDa and one minor protein of 45 kDa. IgY that was extracted with chloroform contained two major proteins of 45.7 kDa and 75.2 kDa and that extracted with PEG-6000 contained only one major protein of 66 kDa. The IgY obtained by these latter methods also contained several minor proteins in the range 41–80 kDa.     

Cross-reactivity of anti-chicken IgY antibody with immunoglobulins of exotic avian species

Background: A major challenge in the serologic diagnosis of infectious diseases in exotic birds is the limited availability of species-specific antibodies. Objectives: The purpose of the current study was to determine if there is cross reactivity between commercially available anti-chicken IgY antibodies and immunoglobulins of several avian species, with particular emphasis on psittacines. Methods: To quantitate the reactivity with anti-chicken IgY, Western blot analysis was performed using plasma samples from many different avian species. Results were compared with gamma globulin fraction quantitation obtained by protein electrophoresis. Results: By Western blot, 2 protein bands corresponding to the heavy and light chains of chicken IgY were identified in species from 21 avian orders using 1 of 2 rabbit anti-chicken IgY antibodies. Densitometric analysis showed that the amount of immunoglobulin estimated from Western blots correlated strongly with data from protein electrophoresis assays. Conclusions: The results demonstrate that some commercially available anti-chicken IgY antibodies exhibit good cross-reactivity with most avian species.     

Egg yolk IgY against RHDV capsid protein VP60 promotes rabbit defense against RHDV infection

VP60 capsid protein is the major structural and immunogenicity protein of RHDV (Rabbit hemorrhagic disease virus, RHDV), and has been implicated as a main protein antigen in RHDV diagnosis and vaccine design. In this report, egg yolk antibody (IgY) against N-terminal of VP60 was evaluated and developed as a new strategy for RHDV therapy. Briefly, N-terminal of VP60 (～250aa) fragment was cloned and inserted into pET28a expression vector, and then the resultant plasmid, pET28a/VP60-N, was transformed into E. coli BL21(DE3) for recombinant VP60-N protein (rVP60-N) expression. Next, the rVP60-N was purified by Ni⁺-affinity purification chromatography and identified by Western blotting with RHDV antiserum. After immunizing the chickens with rVP60-N, the anti-rVP60-N IgY was isolated, and the activity and specificity of the IgY antibody were analyzed by ELISA and Western blotting. In our results, the rVP60-N could be expressed in E. coli as soluble fraction, and the isolated anti-rVP60-N IgY demonstrated a high specificity and titer (1:22,000) against rVP60-N antigen. For further evaluation of the IgY efficacy in vivo, rabbits were grouped randomly and challenged with RHDV, and the results showed that anti-rVP60-N IgY could significantly protect rabbits from virus infection and promote the host survival after a sustained treatment with anti-rVP60-N IgY for 5 days. Taken together, our study demonstrates evidence that production of IgY against VP60 could be as a novel strategy for the RHDV therapy.     

禽白血病病毒ELISA检测方法的建立与初步应用

本研究以在毕赤酵母系统中表达并纯化的p27重组蛋白为包被抗原,HRP标记的兔抗鸡IgY为酶标二抗,建立了一种快速有效的禽蛋白血病病毒ELISA检测方法,并对其反应条件进行优化。结果显示,该方法具有良好的特异性和重复性;与市售商品试剂盒相比,符合率为96.92%,且更为敏感。应用建立的ELISA方法检测来自吉林省内鸡场314份疑似样品,阳性检出率为75.15%。本研究为禽白血病病毒的检测与诊断提供了一种简便、有效的检测方法。     

Immunoglobulins and anti-Marek's disease virus antibody synthesis in chickens after passive immunization with immunoglobulin Y anti-Marek's disease virus antibody.

The effect of passive immunization with immunoglobulin Y (IgY) antibody against Marek's disease virus (MDV) was examined in MDV-susceptible chickens. The production of IgY, immunoglobulin M, and probably also immunoglobulin A was depressed in passively immunized chickens when compared with that in MDV-exposed chickens which had not been given IgY anti-MDV antibody. In passively immunized chickens, the synthesis of immunoglobulin M and IgY anti-MDV antibodies in response to MDV infection also was delayed as determined by agar gel precipitin and indirect fluorescence antibody tests.     

对鸡卵黄免疫球蛋白两种提取方法的比较

本文比较了水稀释-饱和硫酸铵沉淀法（法一）和水稀释-冰乙醇分级沉淀法（法二）分离提纯鸡卵黄免疫球蛋白（IgY）的产率、纯度及抗体效价。结果表明：两种方法提取的IgY纯度均较高,法一纯化的IgY的产率及蛋白活性相对更好。     

牛支原体间接免疫酶标组织化学检测方法的建立

为建立检测牛支原体的间接免疫组织化学(简称免疫组化)方法,分别以鸡抗牛支原体(Mycoplasma bovis)IgY、羊抗鸡IgG-HRP为一抗和二抗,对牛支原体菌体涂片和牛支原体阳性肺脏组织切片进行免疫组化染色,并对染色条件进行优化,确定组织触片及组织切片的最佳免疫组化条件。在最佳条件下对病死犊牛病料切片进行免疫组化检测,并以细菌分离培养和PCR方法进行验证。结果显示,免疫组化法阳性标本与分离培养和PCR结果相符,而阴性对照和替代试验均为阴性。结果表明本试验建立的间接免疫组化方法可用于检测临床病例组织样本及培养物中的牛支原体,具有特异性和可靠性,为探究该病原在机体内的定位、动态分布规律及致病机理提供了手段。     

猪圆环病毒2型Cap全基因的克隆与原核表达

Cap蛋白是猪圆环病毒2型(porcine circovirus type 2,PCV2)的主要结构蛋白,能诱导免疫保护,是临床上利用血清学诊断PCV2的主要依据。本研究根据PCV2 TJ株全基因核苷酸序列(GenBank登录号:KC751546)设计特异性引物,利用PCR从PCV2 TJ株扩增获得Cap全基因,将该基因片段连接到原核表达载体pET-32a(+)中,获得重组质粒pET32a-Cap,经IPTG诱导获得约48 ku的重组融合蛋白,Western blotting分析结果表明其与小鼠抗6×His单克隆抗体和PCV2阳性猪血清均呈阳性反应。本研究成功克隆Cap全基因,构建原核表达载体,并实现Cap融合蛋白的表达,这为PCV2 Cap蛋白的功能研究及诊断方法的建立和亚单位疫苗的研制奠定了基础。