Use of Egg Yolk-Derived Immunoglobulin as an Alternative to Antibiotic Treatment for Control of Helicobacter pylori Infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10⁸ CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60°C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Production of Egg Yolk Antibodies Specific to House Dust Mite Proteins

Purpose

House dust mites (HDMs) are an important source of indoor allergens associated with asthma, rhinitis and atopic dermatitis. Chicken immunoglobulin (Ig) Y is known to be a good alternative to mice and rabbit antibody production. In this study, we produced IgYs specific to HDMs and investigated their IgE immunoreactivities.

Materials and Methods

Total IgYs were isolated from the yolks of White Leghorn hens immunized with either Dermatophagoides pteronyssinus or D. farinae protein extract. Control antibodies were separated from the yolks of immunized hens with phosphate buffered saline. IgYs specific to HDMs were analyzed using enzyme-linked immunosorbent assay and Western blotting analysis.

Results

The concentration of egg IgY specific to D. farinae in an immunized hen increased and the highest achieved was 661.3 ug/mg (per an egg) on day 47, compared with 760 ug/mg IgY specific to D. pteronyssinus on day 16. The D. pteronyssinus or D. farinae-specific IgY was detected by binding of each mite proteins, and their immunoreactivities were elevated dependent of the specific IgY concentration.

Conclusion

IgY specific to HDMs may be a promising antibody for immunological diagnosis as well as identification of possible resistance relating to HDM allergy.     

Production of IgY anti-mouse IgG antibodies from chicken eggs

IgY technology offers several advantages over antibody production in mammals. In this study, we applied IgY technology for the production of anti-mouse IgG polyclonal antibodies and developed a FITC conjugate reagent. Two hens were immunized 3 times with mouse IgG, one via the pectoralis and the other via the calf muscles. Specific antibodies could be detected in the sera two weeks after the immunization, and maximum levels were reached at week 10. The hen which was immunized via the pectoralis muscle produced a much higher antibody response than the hen immunized via the calf muscle. In egg yolk, specific antibodies appeared 2 weeks after the first immunization, reached a plateau after week 11 and remained high until week 20. IgY were extracted from egg yolk by sodium sulfate precipitation. Approximately 40 mg of IgY could be extracted from a single egg. The extracted IgY was labeled with FITC. The so-produced antibody-FITC conjugate reacted to all mouse IgG isotypes and could be used to determine leukocyte sub-populations in blood samples by flow cytometry.     

鸡卵黄抗体的制备以及金磁微粒为载体去除人血浆部分高丰度蛋白的研究

目的:以人血浆白蛋白(HSA)和IgG为免疫原,制备特异性鸡卵黄抗体IgY(Egg yolk immunoglobulin),并将其固定于金磁微粒表面,用于HSA和IgG的去除研究.方法:用HSA、IgG以及混合成分分别作免疫原免疫Roman母鸡.制备抗HSA和IgG鸡卵黄抗体IgY,并对IgY的分离提取条件进行优化.SDS-PAGE和间接ELISA检测IgY的纯度和效价.将高效价IgY固定于金磁颗粒表面进行血浆高丰度蛋白去除研究.结果:免疫后60～120 d内,鸡血清抗体效价可达1∶15 000～1∶25 000;收获鸡蛋,提取得到的卵黄抗体IgY效价可达1∶10000～1∶25000,纯度98%以上;采用金磁微粒载体固定IgY,可对血浆中的HSA,IgG进行特异性的去除.结论:人血浆中的高丰度蛋白成分HSA和IgG免疫产蛋母鸡后,可从鸡卵黄中分离提取到高效价、高纯度的卵黄抗体IgY;IgY偶联于金磁微粒表面可特异性的去除人血浆中的HSA和IgG,作为血浆蛋白质组学研究的一种新方法,有较好的应用前景.     

抗须癣毛癣菌细胞壁蛋白的卵黄抗体制备和鉴定

目的：提取须癣毛癣菌的细胞壁蛋白作为免疫原制备特异性卵黄抗体,并鉴定其生物活性,为卵黄抗体在预防与治疗皮肤癣疾病的应用奠定基础。方法：本研究采用冷碱抽提的方法提取须癣毛癣菌的细胞壁蛋白,并用其免疫健康的产蛋母鸡。采用聚乙二醇两步沉淀法及饱和硫酸铵盐析提纯卵黄抗体;用Bradford法检测卵黄抗体中蛋白含量;SDS-PAGE凝胶电泳分析测定卵黄抗体的纯度以及相对分子质量;ELISA检测纯化的卵黄抗体的效价;Western blot分析特异性卵黄抗体的免疫反应性。结果：提取的卵黄抗体中蛋白纯度达到87.27%。由细胞壁蛋白制备的特异性卵黄抗体在初免20 d后效价开始升高,到45天达到最高值（1∶32 000）。Western blot结果显示由细胞壁蛋白制备的卵黄抗体能与其良好的特异性结合,具有较好的免疫反应性。结论：本实验提取的须癣毛癣菌细胞壁蛋白作为免疫原可以制备出特异性较强的卵黄抗体,为须癣毛癣菌感染的皮肤疾病提供了治疗新思路。     

Applied biotechnology for production of immunoglobulin Y specific to hepatitis A virus

A new protocol for producing polyclonal antibody against hepatitis A virus (HAV) is described. Twenty hens were immunized three times with a commercial HAV vaccine and HAV from a cell culture with three types of adjuvants: CpG oligodeoxynucleotides (CpG-ODN), incomplete Freund's adjuvant and an alum adjuvant. In each of the last two booster inoculations, blood from the birds was collected and tested for HAV antibodies. Egg yolk was separated from egg white and immunoglobulin Y (IgY) antibody was then purified by polyethylene glycol 6000. The mean yield of total protein in yolk was 22.62 mg/mL. Specific activity of the antibody was tested using commercial ELISA, Western blotting, and in vitro neutralization assay demonstrating that anti-HAV IgY bound specifically. After the first immunization, birds immunized with HAV from cell culture plus incomplete Freund's adjuvant with/without CpG-ODN showed highest levels of anti-HAV IgY in serum (p < 0.05). Viral combination with CpG-ODN resulted in early response of anti-HAV serum in hens, reflecting the amount of IgY transferred to the egg yolk (p < 0.05). The results suggest that egg yolk may be a large scale source of specific antibodies against hepatitis A virus. Further applications of this method have yet to be tested.     

Sensitive double antibody sandwich ELISA for the quantification of phosvitin

An effective double antibody sandwich ELISA (DAS-ELISA) method based on monoclonal (mAb) and chicken egg yolk IgY antibodies was developed to determine phosvitin (PV) content in therapeutic and functional products. Leghorn laying hens were immunized with purified PV to produce anti-PV IgY antibody in the egg yolk. High anti-PV IgY titer obtained from the egg yolks collected during 4–10 weeks of the immunization period contained approximately 6.2% of specific anti-PV IgY in total IgY. The PV detection range of the DAS-ELISA and biotinylated DAS-ELISA was 16.8–90 and 7.5–40?ng/mL, respectively. However, biotinylated DAS-ELISA was the better method for PV quantification in terms of accuracy and sensitivity. This highly efficient PV detection method may recuperate the performance of the existing protein assay methods as well as facilitate future research on PV bioactivities and applications.     

不同免疫方案制备鼠抗PBP2a多克隆抗体的比较

目的制备针对青霉素结合蛋白（PBP2a）的抗血清，比较不同免疫方案的免疫效果。方法以基因工程重组PBP2a转肽酶区蛋白作为免疫原，采用多种注射途径（足垫、腹腔、足垫＋腹腔、足垫＋皮下）免疫BALB／C小鼠，ELISA和Western-blot法检测所制备的抗血清效价和特异性。结果足垫快速免疫组、腹腔免疫组、足垫和腹腔免疫组、足垫加皮下免疫组的效价依次为1：12800、1：25600、1：51200、1：102400。Western-blot显示所制备的抗血清能有效识别原核表达及耐甲氧西林的金黄色葡萄球菌（MRSA）临床分离株中的PBP2a。结论重组PBP2a蛋白免疫BALB／c小鼠能有效刺激特异性抗体的产生，足垫加皮下多点免疫法是一种值得推崇的免疫方案。     

特异性抗内毒素鸡蛋黄抗体的制备

目的：制备特异抗内毒素鸡蛋黄免疫球蛋白（Egg yolk immunoglobulin，IgY），探索防治内毒素血症的新途径。方法：用大肠杆菌J5株、内毒素（Lipopolysaccharide，LPS）及类脂A(Lipid A)作抗原免疫25周龄Roman鸡，改良水溶法提取抗体，进行紫外分光光度计检测、SDS-PAGE及Western blot免疫印迹分析，通过酶联染色反应检测其免疫学活性。结果：大肠杆菌J5株、LPS及Lipid A抗体含量分别为11.4、9.2、9.3mg/mL蛋白黄液，质量分数分别为92.3％、87.13％、90.4％，分子质量为180000u，初步鉴定其对内毒素具有特异性结合作用。结论：用大肠杆菌J5株、LPS及LipidA免疫鸡制备的IgY效价高、特异性强、产量大，将是一种防治内毒素血症的新方法。     

产肠毒素大肠杆菌(ETEC)的两种抗原制品对蛋鸡免疫原性的对比研究

目的：采用产肠毒素大肠杆菌（ETEC）K88标准菌株，研究其不同浓度的菌毛及全菌两种抗原对蛋鸡的免疫原性，并确定最佳的抗原浓度。方法：用SDS—PAGE鉴定菌毛蛋白的纯度，通过间接ELISA法检测特异性卵黄抗体（IgY）的效价。结果：菌毛蛋白对蛋鸡的免疫原性优于全菌；纯化的菌毛蛋白对蛋鸡的免疫原性优于粗菌毛蛋白，其诱导的抗体效价最高可达1：480000，初免84天后未见有明显的下降趋势；菌毛蛋白最佳浓度为2mg／ml，全菌最佳浓度为10^10cfu／ml。结论：本研究为制备高效价特异性IgY抗体奠定了基础。     

Isolation of Viral IgY Antibodies from Yolks of Immunized Hens

Antibodies were isolated from the yolks of hens that were immunized with a variety of plant viruses by the use of polyethylene glycol (PEG). A concentration of 3.5% of the polymer caused the lipids and vitellin to separate, and the IgY was then precipitated with 12% PEG. The titre of the isolated antibody appears to remain at a high level after cessation of the course of immunization. Antibodies derived from the yolks of hens appear to have titres similar to those found in serum of rabbits immunized simultaneously. The observation made by several authors that a high salt concentration enhances fowl serum antibody precipitin titres could not be corroborated with 'yolk' antibodies directed against several plant viruses.     

Isolation of Viral IgY Antibodies from Yolks of Immunized Hens

Antibodies were isolated from the yolks of hens that were immunized with a variety of plant viruses by the use of polyethylene glycol (PEG). A concentration of 3.5% of the polymer caused the lipids and vitellin to separate, and the IgY was then precipitated with 12% PEG. The titre of the isolated antibody appears to remain at a high level after cessation of the course of immunization. Antibodies derived from the yolks of hens appear to have titres similar to those found in serum of rabbits immunized simultaneously. The observation made by several authors that a high salt concentration enhances fowl serum antibody precipitin titres could not be corroborated with ‘yolk’ antibodies directed against several plant viruses.     

Peroral immunotheraphy with yolk antibodies for the prevention and treatment of enteric infections

Oral administration of specific antibodies is an attractive approach to establish protective immunity against gastrointestinal pathogens in humans and animals. The increasing number of antibiotic-resistant bacteria emphasize the need to find alternatives to antibiotics. Immunotherapy can also be used against pathogens that are difficult to treat with traditional antibiotics. Laying hens are very good producers of specific antibodies. After immunization, the specific antibodies are transported to the egg yolk from which the antibodies then can be purified. A laying hen produces more than 20 g of yolk antibodies (IgY) per year. These antibodies also have biochemical properties that make them attractive for peroral immunotherapy: They neither activate mammalian complement nor interact with mammalian Fc receptors that could mediate inflammatory response in the gastrointestinal tract. Eggs are also normal dietary components and thus there is practically no risk of toxic side effects of IgY. Yolk antibodies have been shown in several studies to prevent bacterial and viral infections.     

抗眼镜蛇毒鸡卵黄抗体的制备、纯化和保护性实验研究

目的:探索抗眼镜蛇毒鸡卵黄抗体(IgY) 的制备方法及其生物学活性，为替代马源性抗蛇毒血清奠定基础。 方法:眼镜蛇毒原毒免疫母鸡,采用水稀释-硫酸铵盐析-阴离子交换层析三级提取法纯化IgY；SDS-PAGE 测定各级提取物的抗体纯度；间接ELISA 法和双向免疫扩散法观察效价变化；动物体外中和实验检测其生物活性。 结果:卵黄经水稀释法及经60% 硫酸铵盐析蛋白回收率为40.36%；进一步经阴离子交换层析，总蛋白回收率14.86%，效价提高24倍；IgY分子量约为220 kD，由两个亚基组成；体外中和试验表明，2.28 mg/kg 特异性IgY能完全中和2 LD50 的眼镜蛇毒，保护小鼠免受眼镜蛇毒的攻击。 结论:以眼镜蛇毒原毒为免疫源，经水提、盐析、层析三级提取，可从鸡卵黄中获得高纯度、高活性的特异性IgY。     

抗HSPC238单克隆抗体的制备和初步鉴定

目的制备抗HSPC238的单克隆抗体，为HSPC238的功能研究奠定基础。方法以纯化的重组蛋白HSPC238为抗原，免疫BALB／c小鼠，运用杂交瘤技术制备HSPC238单克隆抗体，并用间接ELISA法和Western—blot法对单克隆抗体的特性进行鉴定。结果成功建立两株稳定分泌抗HSPC238的单克隆抗体杂交瘤细胞株，分别命名为E001和E002。ELISA检测抗HSPC238单克隆抗体的腹水效价为1：12800和1：25600。两株单克隆抗体的免疫球蛋白亚类均为IgG1。通过Western—blot实验证实，两株单克隆抗体均能特异性结合真核细胞内源性HSPC238蛋白。结论成功制备了两株效价高、特异性好的抗HSPC238单克隆抗体，制备的抗HSPC238单克隆抗体可用于HSPC238蛋白的鉴定，为HSPC238蛋白的生物学功能研究奠定了基础。     

滴鼻、口服及肌肉注射途径对轮状病毒VP4重组腺病毒免疫小鼠的体液免疫效果比较

目的：比较滴鼻、口服及肌肉注射免疫途径，对重组腺病毒刺激小鼠产生的体液免疫反应效果。方法：剂量为10^8PFu的表达轮状病毒VP4的重组腺病毒经滴鼻、口服、肌肉注射分别免疫BALB／c小鼠，相同免疫剂量、免疫途径加强免疫2次，采集小鼠的血清、粪便及小肠样本，用ELISA检测轮状病毒VP4特异的IgG和IgA。实验设立了腺病毒载体对照组和PBS对照组。结果：经3种途径免疫的重组腺病毒都刺激小鼠产生了轮状病毒VP4特异的血清和肠道抗体，与对照组相比，有统计学意义（JP〈0．05）。在免疫后的第7周，滴鼻组血清VP4特异LgG达到最高值（GMT=15000），在免疫后的第8周，肠道抗体达到最高值（GMT），其中IgG为74．8，IgA为36。结论：滴鼻免疫是3种免疫途径中最有效的。     

Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants

DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1β (IL-1β) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1β adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1β adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems.     

幽门螺杆菌外膜蛋白Omp18 免疫优势表位的预测与鉴定

目的:分析预测幽门螺杆菌(Hp)外膜蛋白Omp18 的免疫优势表位,并进一步验证确定其免疫优势片段的免疫原性。方法:通过生物信息学软件DNAStar 分析预测幽门螺杆菌外膜蛋白Omp18 的潜在优势表位,并由北京赛百盛基因技术有限公司合成相应的优势片段;片段通过口服免疫的方式免疫接种雌性BALB/ c 小鼠,每周1 次共免疫4 次。末次免疫10 d后处死小鼠,取血清检测IgG、IgA 水平,取肠道灌洗液检测sIgA 水平;取小鼠脾细胞ELISA 法检测细胞上清中IFN-γ、IL-2、IL-4、IL-6 水平,并通过MTT 比色法检测脾细胞增殖情况;末次免疫10 d 后Hp 标准株攻击小鼠2 次,4 周后处死小鼠,取胃组织进行快速尿素酶实验,并计算每组的感染率。结果:ELISA 法检测各片段免疫小鼠血清IgG、IgA 以及肠道sIgA 水平均高于对照组,且差异有显著统计学意义(P<0.05);各免疫组脾细胞上清INF-γ、IL-2 水平均显著高于对照组(P<0.05),而IL-4、IL-6水平较对照组则差异无显著统计学意义(P>0.05);MTT 比色法结果显示各片段刺激后小鼠淋巴细胞均有显著增殖(P<0.05);快速尿素酶实验结果提示各片段免疫后均能显著减少小鼠胃组织中Hp 的感染(P<0.05)。结论:通过生物信息学软件分析预测的外膜蛋白Omp18 优势片段均具有较好的免疫原性,且对Hp 感染有一定的保护作用。     

含密码子优化型HPV16 L1基因重组腺病毒的构建及其免疫效果的研究

目的 构建含密码子优化型HPV16L1基因的重组腺病毒,对其经不同接种途径所诱导的系统性及黏膜免疫效果进行研究.方法 使用Admax系统包装重组腺病毒,纯化的腺病毒以不同方式免疫C57BL/6小鼠,间接ELISA及体外中和实验检测免疫小鼠血清及阴道分泌物中的特异性抗体.结果 重组腺病毒滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应,重组腺病毒肌注免疫仅能诱导系统性免疫反应,而阴道黏膜接种不能有效诱导系统性及黏膜免疫反应.结论 成功构建了含密码子优化型HPV 16 L1基因的重组腺病毒,重组腺病毒肌注可诱导高滴度的血清中和抗体,滴鼻接种可同时诱导特异性的系统性及黏膜免疫反应.