抗幽门螺杆菌rHpaA鸡蛋黄抗体的研制

目的：采用基因工程技术表达重组幽门螺杆菌黏附素rHpaA，以此蛋白为抗原制备抗rHpaA的高特异性鸡蛋黄抗体。方法：诱导重组质粒pQE30-HpaA在大肠杆菌中大量表达rHpaA蛋白，用rHpaA免疫鸡，以水稀释法联合盐析法提取纯化IgY，采用Bradford法测定含量，SDS—PAGE电泳分析纯度，Western blot鉴定抗原特异性，ELISA测定效价和巴氏消毒后的活性。结果：IgY提纯后含量为24．6mg／ml，纯度约为90％，Western blot显示在相对分子量30000处出现单一条带，ELISA效价为1：12800，巴氏消毒后未见效价损失。结论：成功获得了高浓度、高纯度、高效价、耐巴氏消毒的特异性IgY，为进一步制备预防幽门螺杆菌感染的特异性IgY制剂奠定了基础。     

抗人蔗糖酶卵黄抗体的制备及其稳定性和体外活性研究

目的:制备抗人蔗糖酶(Sucrase,SUC)卵黄抗体(egg yolk immunoglobulin Y,IgY)并对其稳定性、体外活性进行研究。方法:采用人SUC 蛋白为抗原,免疫海兰灰蛋鸡,水稀释和盐析法提取纯化IgY;间接ELISA 法监测抗体效价变化并评价其稳定性;SDS-PAGE 和Western blot 分析IgY 的纯度和特异性;PNPG 法测IgY 对琢鄄葡萄糖苷酶的体外抑制效果。结果:初次免疫10 d 后检测到IgY,40 d 后效价最高达到1 12 800;提取的IgY 重链和轻链分别在65 kD 和25 kD 处,且能够与抗原蛋白特异性结合;29 ~69益范围内水浴15 min 和pH 4 ~7 之间37益水浴4 h,抗体效价无变化;胰蛋白酶与胃蛋白酶作用IgY2 h,抗体效价降至一半,延长作用时间,IgY 对胃蛋白酶耐受力较胰蛋白酶耐受力更强;IgY 对琢鄄葡萄糖苷酶具有一定的抑制作用,呈现剂量相关性,半抑制浓度(IC50)值为0郾540 mg/ ml。结论:抗人蔗糖酶卵黄抗体(S-IgY)的成功制备为后续用于2型糖尿病大鼠模型体内实验研究奠定基础。     

双价抗蛇毒IgY灌胃对眼镜蛇、蝰蛇伤小鼠的保护作用

目的： 比较双价抗蛇毒鸡卵黄抗体(IgY) 经腹腔注射或灌胃对蝰蛇、眼镜蛇伤小鼠保护作用的差异,为其口服制剂的应用奠定理论基础。方法：2种单一抗原按顺序依次交替注入单只鸡体内,水稀释法制备双价抗蛇毒IgY;间接ELISA法观察双价抗蛇毒IgY在体外及其灌胃后小鼠血浆中的效价,通过胃排空试验确定灌胃给药的最佳时间,在此基础上,比较双价抗蛇毒IgY腹腔注射或灌胃对蛇伤小鼠的保护作用。结果：初次免疫后28 d至第42 d,以水稀释法提取的双价抗蛇毒IgY对眼镜蛇毒及蝰蛇毒的效价分别为1∶12 800和1∶6 400。不同浓度的双价抗蛇毒IgY灌胃2.5-3.5 h,小鼠的胃排空率均达68.9％以上,血浆中抗体浓度亦相应达高峰。该抗体腹腔注射或提前灌胃均能极显著延长眼镜蛇和蝰蛇毒中毒小鼠的存活时间（P<0.01）,同等剂量的双价抗蛇毒IgY提高眼镜蛇伤小鼠存活率优于蝰蛇伤,而灌胃有效剂量约为腹腔注射的10倍。结论：双价抗蛇毒IgY经2种给药途径（注射、口服）均能有效保护动物免受眼镜蛇毒和蝰蛇毒的攻击。     

特异抗内毒素鸡蛋黄免疫球蛋白效价测定及理化性质研究

目的：研究特异性抗内毒素鸡蛋黄免疫球蛋白理化性质，探索一种防治肠源性感染，治疗：内毒素血症的新途径。方法：用大肠杆菌J5株(菌体表面类脂A裸露)作抗原免疫25周龄Roman鸡，制备特异性抗内毒素IgY，用酶联免疫吸附技术检测其效价及对热、酸、碱和酶的稳定性。结果：IgY抗体效价为1：25 600，能耐受巴氏消毒，对酸、碱、酶有很好的稳定性。结论：用大肠杆菌J5株免疫鸡制备的IgY效价高，理化性质稳定，适合口服应用。     

抗咽喉细菌复合抗原的IgY抗体活性研究

目的：应用咽喉分离的细菌混合抗原免疫母鸡，提取鸡蛋黄中IgY，通过ELISA方法证实IgY抗体的特异活性。方法：采取咽喉炎相关的多种细菌作抗原免疫产蛋母鸡，收集鸡蛋，从蛋黄中提取IgY，经纯化后进行SDS-聚丙烯酰胺凝胶电泳和抗体活性的ILIAS测定。结果：电泳呈现12条带，证明IgY抗体中至少有12种抗体成分，ELISA结果表明IgY抗体效价为1：512。热稳定性试验结果显示，在37℃温度下，IgY抗体活性无改变。结论：应用多种混合的细菌抗原免疫母鸡，其鸡蛋黄中含有相应的IgY抗体成分，对热具有高度稳定性。     

融合蛋白VacA-HpaA卵黄抗体的体外研究

目的：研究VacA-HpaA融合蛋白的鸡卵黄抗体（IgY）的理化特性和生物学活性,为制备集预防与治疗为一体的抗H.pylori感染的制剂奠定基础。方法：以纯化的VacA-HpaA融合蛋白免疫产蛋鸡,水稀释法获取蛋黄抗体（VacA-HpaAIgY）。（1）用硫酸铵沉淀法纯化IgY后,用SDS-PAGE分析其纯度,Bradford法测定蛋白含量。（2）用间接ELISA法检测该IgY的耐热性、耐酸性及其对酶消化的耐受作用。（3）采用Western blot分析IgY的特异性并检测其效价。（4）用MTT法检测不同浓度VacA-HpaAIgY对细胞毒活性的中和作用。（5）用扫描电镜观察VacA-HpaA IgY对AGS细胞粘附的中和作用。结果：（1）该IgY纯度为60%,蛋白浓度为22g/L;（2）具有一定的耐热性、耐酸性和耐胃蛋白酶能力;（3）Western blot分析显示IgY的特异性,在相对分子量约27000和30000处出现在相应的条带;（4）该IgY对VacA细胞毒活性有中和作用,且具有量效性和时效性;（5）IgY可阻止H.pylori菌体蛋白的粘附作用。结论：该IgY具有良好的理化和生物学特性,可用于制备抗H.pylori感染的防治制剂。     

抗痢疾志贺氏菌卵黄抗体的制备及其稳定性

目的制备特异性抗痢疾志贺氏菌的鸡卵黄免疫球蛋白,研究母鸡对痢疾志贺氏菌的免疫应答规律及抗体的稳定性。方法以两种不同剂量的痢疾志贺氏菌作为抗原免疫产卵母鸡,应用酶联免疫吸附测定法检测抗痢疾志贺氏菌IgY的效价及其稳定性。结果初次免疫后第12天,卵黄中开始出现抗痢疾志贺氏菌的IgY,免疫应答持续时间超过25周。稳定性试验表明IgY在pH3-11,温度不超过60℃,蔗糖质量浓度不高于700 g/L的渗透压条件下活性稳定。结论用痢疾志贺氏菌免疫母鸡制备的IgY效价高,理化性质稳定。     

基于荧光纳米粒子的新型冠状病毒核衣壳蛋白免疫层析快速检测试剂的 研制

目的：建立新型冠状病毒(SARS-CoV-2)抗原快速检测试剂的制备方法，并对检测试剂的性能指标进行评价。方法：以羧基荧光纳米粒子标记的羊抗SARS-CoV-2核衣壳蛋白(NP)多克隆抗体及鸡IgY为标记抗体，硝酸纤维素膜上分别包被鼠抗N蛋白单克隆抗体和羊抗鸡IgY抗体作为检测线和质控线制备免疫荧光试纸条，对试剂最低检出限、交叉反应性、重复性、临床诊断灵敏度和特异性进行性能评价。结果：检测N全长重组蛋白及热灭活培养物的最低检出限分别为13.8pg/ml和1.6×10~2TCID₅₀/ml；测试16种常见呼吸道病原体高浓度样本均无交叉反应；高、低两个浓度参考品变异系数(CV)分别为7.68%和4.98%。临床鼻咽拭子样本及健康人群鼻拭子样本测试，诊断灵敏度为86.36%(19/22)，特异度为99.16%(355/358)，总符合率为98.42%(374/380)；一致性检验Kappa值为0.8553(P<0.05)。结论：SARS-CoV-2荧光抗原检测试剂检测灵敏度和特异性高，检测速度快，操作便携，可作为现有核酸检测法的补充手段，用于新型冠状病毒的早期筛查...     

兔抗人细胞色素P450 1A2多克隆抗体的制备及鉴定

目的：制备兔抗人细胞色素P4501A2（CYP1A2）抗体及其特性鉴定。方法：利用生物信息学方法分析CYP1A2蛋白的序列，根据亲水性、抗原性、柔韧性及表面性等指标选择多肽序列，合成CYP1A2多肽，与载体蛋白钥孔戚血蓝素（Keyhole limpet hemocyanin，KLH）偶联，免疫日本大耳白兔制备抗CYP1A2抗体。辛酸-硫酸铵法纯化抗体，间接ELISA法测定抗体的效价及相对亲和力常数，Western blot鉴定其特异性，免疫组化进行定位。结果：获得针对小分子CYP1A2的抗体。该抗体效价为1∶16000；相对亲和力常数在10^5-10^6/M，具有实用价值；可与CYP1A2合成肽及天然CYP1A2出现特异性反应；可识别正常人肝脏组织CYP1A2；Western blot的结果显示，该抗体识别的相应抗原的相对分子质量为58000。结论：合成的多肽-KLH复合物具有免疫原性，可用于制备相应的抗体。制备的兔抗CYP1A2合成肽抗体可应用于酶联免疫吸附试验、免疫印迹、免疫组化实验。     

肿瘤的抗独特型抗体免疫疗法

肿瘤的抗独特型抗体免疫疗法（文摘）［英／Ｃｈａｔｔｅｒｊｅｅ，ＭＢ…／ＣａｎｃｅｒＩｍｍｕｎｏｌＩｍｍｕｎｏｔｈｅｒ１９９４；３８：７５」将针对肿瘤抗原的抗独特型抗体疫苗偶联到载体上，可形成具强免疫原性的Ｔ细胞依赖抗原。此外，此种疫苗不含游离抗原或其...     

Development of an intranasal vaccine to prevent urinary tract infection by Proteus mirabilis

Proteus mirabilis commonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise, and leads to the subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals. To prevent these infections, we vaccinated CBA mice with formalin-killed bacteria or purified mannose-resistant, Proteus-like (MR/P) fimbriae, a surface antigen expressed by P. mirabilis during experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection by P. mirabilis (P < 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (residues 23 to 275, excluding the signal peptide), and the N-terminal receptor-binding domain of MrpH (residues 23 to 157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP) and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (residues 23 to 157) conferred effective protection against urinary tract infection by P. mirabilis (P < 0.002).     

Purification and characterization of the CFA/I antigen of enterotoxigenic Escherichia coli.

The fimbral colonization factor antigen CFA/I of enterotoxigenic Escherichia coli was purified and characterized. The initial purification step was release of these fimbriae from the bacterial cells by homogenization with a Waring blender. Common fimbriae and flagellar antigen were avoided by careful control of growth conditions and the use of a nonmotile (H-) mutant of the prototype strain H-10407 (O78:H11). The essential purification steps were membrane filtration (Millipore Corp.), ammonium sulfate fractionation, and negative diethylaminoethyl-Sephadex column chromatography. Yields were approximately 4.0 mg of CFA/I protein per g (wet weight) of bacteria. Purified CFA/I is a fimbrial molecule 7.0 nm in diameter and has an average molecular weight of 1.6 X 10(6), as determined by sedimentation equilibrium. CFA/I is a polymer of identical subunits of molecular weight 23,800 with an N-terminal valine, 37% hydrophobic amino acid residues, and 11 residues of proline per mol. The purified antigen retains its morphology, antigenicity, and biological activity. Purified antigen retains its morphology, antigenicity, and biological activity. Purified CFA/I exhibits mannose-resistant hemagglutination of human group A, bovine, and chicken erythrocytes, as do CFA/I-positive bacteria. This was demonstrated by sensitizing latex microbeads with the purified antigen since cell-free CFA/I fimbriae do not hemagglutinate erythrocytes. Thus, CFA/I detached from the bacteria are monovalent; however, purified CFA/I antigen retains an affinity for the epithelial cells of rabbit small intestine and blocks adhesion of CFA/I-positive bacteria. These results demonstrate that purified CFA/I is a good candidate for use in an oral vaccine for immunoprotection against diarrhea caused by CFA/I-positive enterotoxigenic E. coli.     

Purification, morphology, and genetics of a new fimbrial putative colonization factor of enterotoxigenic Escherichia coli O159:H4.

The ability to colonize the small intestine is essential for the pathogenesis of diarrhea caused by enterotoxigenic Escherichia coli (ETEC). Colonization is mediated by fimbriae (pili), of which there are several antigenically distinct types, including colonization factor antigen I, colonization factor antigen II (CS1, CS2, and CS3), and PCF8775 (CS4, CS5, and CS6). These fimbriae are associated with certain ETEC O serogroups. Serogroup O159 has had no known colonization factor. We found a distinct plasmid-encoded fimbria composed of 19-kilodalton protein subunits associated with ETEC serotype O159:H4. Rabbit antibody against this purified fimbria reacted with a single 19-kilodalton protein band as seen by Western immunoblot of sheared-cell preparations. The rabbit antibody, treated with colloidal-gold-labeled goat anti-rabbit immunoglobulin G, bound specifically to fimbriae when cells were examined with an electron microscope. Of 10 available ETEC O159:H4 strains from Europe, Bangladesh, and Kenya, 6 expressed this type of fimbria; its true prevalence among ETEC strains is unknown. This putative colonization factor of O159:H4 joins other ETEC fimbriae as potentially useful immunogens against human diarrhea.     

Induction of CD 14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed Gram-negative bacteria

ABSTRACT: In order to better understand the regulation of CD 14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD 14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116–54. Various cytokines such as interleukin-1β, interleukin-2, interleukin-6, interferon-γ and tumor-necrosis factor-α were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116–54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116–54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116–54 also induced strong expression of CD 14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD 14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.     

Induction of CD14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed gram-negative bacteria.

In order to better understand the regulation of CD14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116-54. Various cytokines such as interleukin-1 beta, interleukin-2, interleukin-6, interferon-gamma and tumor-necrosis factor-alpha were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116-54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116-54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116-54 also induced strong expression of CD14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.     

Enzyme immunoassay for detection of antibody-coated bacteria.

To quantitatively evaluate factors potentially affecting antibody coating of bacteria in urine, we developed an assay with enzyme-linked rather than fluorescein-conjugated immunoglobulin. Using the enzyme immunoassay (EIA) in an in vitro system in which concentrations of serotype O44 Escherichia coli and antibody titer to E. coli Orr O44 O antigen were known, we compared specimens run in parallel with a fluorescent antibody (FA) assay. At greater than or equal to 10(5) bacteria per ml, antibody titer to homologous O antigen correlated directly with absorbance in the EIA. Both tests had sensitivities exceeding 95% in specimens containing greater than or equal to 10(5) bacteria per ml, but the FA test detected 23 of 27 positive specimens with less than 10(5) bacteria per ml compared with 21 of 43 detected by EIA (P = 0.002). However, nonspecific fluorescence caused false positives in 8% of negative tests run by FA compared with 1% of simultaneous EIA tests (P = 0.05). pH alterations and pretreatment of bacteria with antibiotics did not affect either test. Heterologous E. coli strains showed no cross-reactivity with O44 antiserum, but all Staphylococcus aureus isolates tested caused false positives in both assays, and one Klebsiella strain repeatedly caused a false-positive FA assay. The EIA appears to be a simple, quantitative, and specific technique for detection of antibody-coated bacteria in this experimental system.     

Antibody-mediated enhancement of Legionella pneumophila-induced interleukin 1 activity.

The ability of antibody specific for Legionella pneumophila to enhance the induction of interleukin 1 (IL-1) production by murine peritoneal, splenic, and pulmonary macrophages in response to the bacterium was examined. Two preparations of L. pneumophila were utilized, a formalin-killed whole-cell preparation and viable bacteria. We measured both secreted (sIL-1) and cell membrane-associated (mIL-1) activities after incubation of the macrophages with the bacterial preparations in the presence or absence of the antibody. Both bacterial preparations induced sIL-1 and mIL-1 activities in each of the macrophage populations tested. These activities were generally enhanced by pretreating the bacteria with antibody, with the greatest enhancing activity observed for the formalin-killed preparations at lower doses of bacteria.     

Production of avian antibodies to three potyviruses in coturnix quail

Avian antibodies against three potyviruses were produced in a small bird, coturnix quail (Coturnix coturnix japonica Temminck et Schlegel), with 15-60 micrograms of purified virus preparations. Intramuscular injections of immunogen with Freund's incomplete or complete adjuvant into the birds did not result in higher titer of antibody compared to that of control birds given intravenous injections. Quail egg yolk antibody was as useful as hen antibody for indirect-ELISA and allowed virus to be detected in purified preparation (10-50 ng/ml) and in crude extracts (10(-6)-10(-7) dilution). The advantages of using quail to produce avian antibodies are discussed.     

Monoclonal antibodies against different epitopes on colonization factor antigen I of enterotoxin-producing Escherichia coli.

Hybridoma-secreting monoclonal antibodies (MAbs) against colonization factor antigen I (CFA/I) were produced by the fusion of spleen cells from immunized BALB/c mice with F/O myeloma cells. The 25 MAbs with the highest antibody titer against CFA/I, as determined by an enzyme-linked immunosorbent assay, were studied for specificity and the capacity to agglutinate CFA/I-carrying bacteria. Most of the MAbs agglutinated a majority of 16 CFA/I-positive strains tested but not any of a number of CFA-deficient mutants or strains carrying other colonization factors (CFA/II and CFA/IV). One MAb that agglutinated all the CFA/I-positive strains and one MAb that did not agglutinate any of these strains were compared for their reactivities with different preparations of CFA/I. Whereas both MAbs bound to or could be inhibited by isolated CFA/I fimbriae as well as the subunit protein, only the agglutinating MAb bound to CFA/I, as expressed on whole bacteria. The nonagglutinating MAb, on the other hand, bound considerably better than the agglutinating MAb to a peptide corresponding to the 46 N-terminal amino acid residues of the CFA/I subunit protein. These results suggest that the two MAbs are directed against different epitopes on CFA/I.     

人F1F0-ATP合成酶β亚基的原核表达、纯化及抗体制备

目的: 原核表达、纯化人F1F0-ATP合成酶β亚基(hATP5B)并制备其多克隆抗体.方法: 以人脐带静脉内皮细胞 (HUVEC) 总RNA为模板, 通过RT-PCR扩增hATP5B 成熟肽编码序列, 克隆入原核表达载体pET28a( ), 转化大肠杆菌E.coli BL21(DE3)并诱导表达, 表达产物用Ni2 螯合层析纯化, 纯化产物用透析复性, 产物用SDS-PAGE和 Western blot进行鉴定.复性产物免疫家兔, 制备多克隆抗体; 多抗的效价用间接ELISA法检测, 并用Western blot和细胞免疫荧光分析多抗的特异性和抗原结合性.结果: 测序证实获得hATP5B成熟肽编码序列.SDS-PAGE鉴定表明, 表达、纯化、复性产物的相对分子质量(Mr)均约55 000, 与理论值相符.灰度扫描分析重组hATP5B(recombinant hATP5B, rhATP5B)的表达量占菌体蛋白总量的36.8%, 纯化产物纯度达98.3%, 复性产物纯度达99.2%.Western blot反应阳性.多抗的平均抗体效价为1∶640 000, 可特异识别HUVEC中的天然抗原.结论: 原核表达的hATP5B具有良好的免疫原性, 其免疫家兔后获得的多抗具有高效价和特异性.hATP5B的原核高效表达和其抗体制备为进一步研究hATP5B的功能奠定了实验基础.