三种鸡形目雉科禽类卵黄抗体的纯化及二抗的制备

目的 纯化三种鸡形目雉科的禽类孔雀、鹌鹑、贵妃鸡的卵黄抗体IgY,并且免疫家兔制备抗血清。方法 采用了水稀释法,HiTrap IgYPurification HP免疫亲和层析法和硫酸铵沉淀法纯化IgY。免疫家兔制备抗血清,免疫双扩散法测定效价。Protein-A亲和纯化兔抗IgY血清IgG。结果 经亲和纯化和盐析纯化,得到了孔雀、鹌鹑、贵妃鸡的卵黄抗体IgY,经SDS-PAGE检测为电泳纯,孔雀、鹌鹑、贵妃鸡的卵黄抗体IgY的相对分子质量约为180×10^3。免疫后经Protein-A亲和纯化后获得了兔抗IgY的IgG。结论 证实了孔雀、鹌鹑、贵妃鸡的卵黄抗体IgY的存在及其特性。雉科鸡形目禽类卵黄抗体的纯化方法 相似,可以推广到鸡形目其它禽类的卵黄抗体的纯化中,获得的抗血清可以进一步进行标记和今后抗原的检测。     

细菌性痢疾IgY生物学活性探讨

特异性IgY(卵黄免疫球蛋白)是指从免疫母鸡的鸡蛋中提取针对特定抗原的抗体。近些年来,IgY的研究不断深入。从免疫原的种类、免疫方法I、gY的提取纯化及理化性质等均有多篇报道[1~3],口服IgY亦成为关注的焦点。口服特异性IgY能够提高机体被动保护免疫力[4,5],预防和治疗胃肠道的细菌和病毒感染。美国学者将变形链球菌IgY用于预防龋齿,国内报道应用轮状病毒IgY治疗婴幼儿腹泻均取得可喜效果。本文利用福氏志贺菌免疫母鸡,提取IgY,并在乳鼠体内测定其生物学活性,报告如下。用购自中国药品生物制品检定所的福氏志贺菌B群,福氏2a3、4型菌…     

抗HIV-1 p15(gag)IgY抗体的制备及活性检测

目的:制备具有高效价强特异性的抗HIV-1 p15(gag)鸡卵黄抗体(IgY),纯化并分析其免疫学活性。方法:用纯化的HIV-1 p15(gag)蛋白抗原免疫蛋鸡,用水稀释法对IgY抗体进行粗提取并结合乙醇沉淀和氯化钠盐析法纯化抗体,再通过SDS-PAGE、Western blot、酶联免疫吸附实验(ELISA)检测抗体的纯度、特异性及效价。结果:表达纯化的HIV-1 p15(gag)-GST融合蛋白分子量为45kDa,用于抗体检测的抗原。用纯化的His-P15蛋白作为免疫原免疫蛋鸡后,获得的卵黄抗体重链、轻链分子量分别为65kDa和25kDa。8倍体积水稀释卵黄,pH值5.1,20%冰乙醇及0.028mol/L盐溶液分离纯化,纯度可达96℅,每毫升卵黄液可得到的卵黄抗体为9.8mg。终浓度为18%～25%的冰乙醇纯化的抗体浓度高且稳定,同时具有较强的特异性和较高的效价。结论:用HIV-1p15(gag)蛋白免疫蛋鸡,可以获得高效价的特异性抗体IgY,为IgY抗体在HIV-1p15蛋白的研究奠定了实验基础。     

抗胱抑素C鸡卵黄IgY抗体的制备及鉴定

目的:制备高效价、高特异性的抗人胱抑素 C 鸡卵黄 IgY 抗体,并对其基本特性进行分析和鉴定.方法:以人胱抑素 C 为抗免疫产蛋的罗曼鸡,采用水稀释-盐析法提取及纯化 IgY 抗体,采用蛋白质定量、SDS-PAGE、West?ern 印迹和 ELISA 法对 IgY 抗体进行分析和鉴定.结果:免疫后14 d 即可从鸡冠血中检测出抗胱抑素 C 的特异性抗体,抗体效价在28 d 达最高峰(1∶32000),并可维持2个月以上;收集高效价时的免疫鸡蛋,制备鸡卵黄抗体 IgY;还性 SDS-PAGE 显示抗体 IgY 为相对分子质量分别为65×103和21×103的2条带,抗体纯度可达92%,得率为每个鸡蛋36.5 mg,抗体检出敏感度为15.63 ng/mL;Western 印迹证明该抗体具有高度特异性.结论:制备了抗胱抑素 C 的高效价、高特异性 IgY 抗体.     

基因专一性鸡卵黄抗体(IgY)的研究及应用

用抗原免疫鸡,从鸡卵黄中获取特异多克隆IgY抗体,是一条高产量、低成本的简便快速途径。IgY与哺乳动物抗体IgG相比具有许多突出的优点,使IgY在现代免疫学中已得到广泛应用。采用基因免疫方法制备的基因专一性IgY抗体,在免疫分析、寻找药靶和生物标记物、制备新抗体等方面,必有进一步的应用。     

抗绿脓杆菌鸡卵黄免疫球蛋白的制备和实验

取多价绿脓杆菌（Psetltz‘,lll‘,lltlm对川组＿,PA刚成抗原,免疫美国特种母鸡,并从其所产卵的蛋黄中分离制备抗绿脓杆菌鸡卵黄免疫球蛋白。首先采用水稀释法分离得到鸡卵黄免疫球蛋白（imrnUndeulinofyolk;IgY）组提物,进而采用离子交换层析和凝胶过滤层析得到电泳纯IgY此IgY具有抗PA抗体的特异性,fi．免疫应答迅速（免疫2周后送开始产生抗体）,持久性长（达11个月）。免疫母鸡的成功率为100％。取SD大鼠进行烧伤肠源性感染防治实验。大鼠随机分为3组（正常对照组、烧伤对照组及抗一PAlgY预防组）,烧伤后24h－c48h测户］…     

抗生殖器疱疹病毒卵黄免疫球蛋白(IgY)的研究

检测了鸡卵黄中抗生殖器疱疹病毒(HSV-2)抗体的产量、纯度、来源及稳定性。采用生殖器疱疹病毒(HSV-2)作为抗原免疫广州黄村鸡。通过改良水稀释法提取卵黄中的IgY。双紫外光波长测定抗体含量,SDS-PAGE电泳检测抗体纯度。Western blot免疫印迹法测定该抗体来源。ELISA检测IgY对温度、酸碱度的稳定性。结果,蛋黄液中抗体质量浓度13.6g.L-1,抗体纯度达96.2%。免疫印迹证明IgY与鸡血清中的IgG具有相同的分子量和抗原性。IgY具有良好的热稳定性,对酸碱具有一定的耐受力。WD水稀释法能得到高产量、高纯度的特异性IgY,而且有良好的生物学活性。     

鹭科和鸬鹚科卵黄抗体的纯化

目的 纯化鹭科具有代表性的夜鹭及鸬鹚科具有代表性的鸬鹚的卵黄抗体IgY。方法 采用了水稀释法和硫酸铵分级沉淀法粗提IgY,再过HiTrap IgY Purification HP柱子进一步纯化。结果 经两步纯化,得到了纯化的鸬鹚和夜鹭的卵黄抗体IgY,经SDS-PAGE检测为电泳纯,夜鹭和鸬鹚的卵黄抗体的相对分子质量约为180×10^3。结论 证实了鸬鹚和夜鹭的卵黄抗体IgY的存在及其特性,为这两种鸟类的卵黄抗体IgY纯化,二级抗体制备提供了参考。     

鸡卵黄免疫球蛋白研究动态

当用某种抗原免疫产卵母鸡时,鸡可产生相应的鸡卵黄免疫球蛋白（IgY）储存于卵黄中。母鸡经免疫应答后,可从其不断产出的鸡卵的卵黄中得到大量的、均一的、高效价的IgY。IgY类似于哺乳动物的IgG,但又有其独特的优点。近年来国外对IgY的研究比较活跃。本文对IgY的特点、制备提纯及其在检测诊断上和疾病防治方面的应用作了较详细的论述。可以预见,随着研究的进一步深入,IgY的应用将不断扩大。     

鸡卵黄抗眼镜王蛇毒抗体IgY的理化特性

目的 研制抗眼镜王蛇毒鸡卵黄抗体并研究该抗体的理化特性。方法 拟用减毒后的眼镜王蛇毒免疫母鸡后,从卵黄中制备抗眼镜王蛇毒抗体IgY。采用间接ELISA法对此抗体的理化特性进行研究。结果 ELISA显示免疫后12天开始出现特异性抗体,且抗体滴度逐渐升高,约在免疫后60天左右,最高可达1：100000,此最高滴度可维持30天,以后滴度逐渐降低,至免疫后100～110天,滴度仍可维持在最高滴度的一半(1：50000)。此抗体在10～65℃温度范围内活性保持稳定;在pH为4～12范围内,抗体活性稳定;此抗体经胰蛋白酶处理1h内,活性下降不明显,但1h以后活性迅速下降。结论从卵黄中制备抗眼镜王蛇毒抗体IgY,是可行的,稳定的。     

Passive immune-protection of small abalone against Vibrio alginolyticus infection by anti-Vibrio IgY-encapsulated feed

Small abalone (Haliotis diversicolor supertexta) is a high value-added shellfish. It however has been suffering Vibrio alginolyticus infections, which cause mass death of small abalone and thus great economic losses, particularly in artificial aquaculture. In this study, we attempted to treat small abalone with anti-Vibrio IgY to elicit a passive immunity directly against V. alginolyticus infections. Anti-Vibrio IgY was alginate encapsulated in egg powders as feed, which may avoid antibody inactivation in the gastrointestinal tract of small abalone. The feed was tested for the stability of anti-Vibrio IgY in a gastrointestinal mimic environment. The result showed anti-Vibrio IgY retained activity as high as 90% after 4 h exposure to pancreatic enzymes. Addition of 0, 5 or 10% anti-Vibrio IgY-encapsulated egg powders into a basal diet to form abalone diet formulae. Small abalones fed with the anti-Vibrio IgY formulae showed a relatively high respiratory burst activity than those without anti-Vibrio IgY treatments. The survival rates of small abalones fed with 5 or 10% anti-Vibrio IgY egg powders were in the range of 65–70% 14 days post-V. alginolyticus challenge (1 × 10⁶ c.f.u.), which was significantly higher than 0% of those fed without anti-Vibrio IgY. The anti-Vibrio IgY-encapsulated formulae were thus concluded to be an effective means to prevent small abalone from V. alginolyticus infection, and may be practical in use in abalone aquaculture.     

Prophylactic and Therapeutic Efficacy of Avian Antibodies Against Influenza Virus H5N1 and H1N1 in Mice

Background

Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1.

Methods and Findings

We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection.

Conclusions

The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.     

Effect of dietary anti-urease immunoglobulin Y on Helicobacter pylori infection in Mongolian gerbils

BACKGROUND AND AIM: Helicobacter pylori is known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti-urease IgY on H. pylori infection in Mongolian gerbils. METHODS: H. pylori-infected Mongolian gerbils were administered a diet containing anti-urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior to H. pylori inoculation. After 9 weeks, these animals were examined. RESULTS: In the H. pylori-infected gerbils, there were no significant differences in the level of H. pylori colonization among the different dietary and control groups. However, the MPO activity was significantly decreased in the H. pylori group administered the F + IgY diet compared with that in the H. pylori group administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition of H. pylori colonization and suppression of the elevated gastric mucosal MPO activity were observed. CONCLUSIONS: Oral administration of urease-specific IgY not only inhibited H. pylori disease activity in H. pylori-infected gerbils, but also prevented H. pylori colonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management of H. pylori-associated gastroduodenal disease.     

IgY Antibodies Protect against Human Rotavirus Induced Diarrhea in the Neonatal Gnotobiotic Piglet Disease Model

Group A Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The aim of the present work was to evaluate protection against rotavirus (RV) diarrhea conferred by the prophylactic administration of specific IgY antibodies (Ab) to gnotobiotic piglets experimentally inoculated with virulent Wa G1P[8] human rotavirus (HRV). Chicken egg yolk IgY Ab generated from Wa HRV hyperimmunized hens specifically recognized (ELISA) and neutralized Wa HRV in vitro. Supplementation of the RV Ab free cow milk diet with Wa HRV-specific egg yolk IgY Ab at a final ELISA Ab titer of 4096 (virus neutralization –VN- titer = 256) for 9 days conferred full protection against Wa HRV associated diarrhea and significantly reduced virus shedding. This protection was dose-dependent. The oral administration of semi-purified passive IgY Abs from chickens did not affect the isotype profile of the pig Ab secreting cell (ASC) responses to Wa HRV infection, but it was associated with significantly fewer numbers of HRV–specific IgA ASC in the duodenum. We further analyzed the pigś immune responses to the passive IgY treatment. The oral administration of IgY Abs induced IgG Ab responses to chicken IgY in serum and local IgA and IgG Ab responses to IgY in the intestinal contents of neonatal piglets in a dose dependent manner. To our knowledge, this is the first study to show that IgY Abs administered orally as a milk supplement passively protect neonatal pigs against an enteric viral pathogen (HRV). Piglets are an animal model with a gastrointestinal physiology and an immune system that closely mimic human infants. This strategy can be scaled-up to inexpensively produce large amounts of polyclonal IgY Abs from egg yolks to be applied as a preventive and therapeutic passive Ab treatment to control RV diarrhea.     

Pseudomonas aeruginosa infections are prevented in cystic fibrosis patients by avian antibodies binding Pseudomonas aeruginosa flagellin

Pseudomonas aeruginosa (PA) is the main cause of morbidity and mortality in cystic fibrosis (CF) patients. CF patients with chronic PA infections have a more rapid deterioration of their lung function and the bacteria become impossible to eradicate from the lungs. Antibiotic resistance among PA strains in CF patients is steadily increasing. Specific chicken (IgY) antibodies against PA have been shown to have potential to prevent PA infections in CF. Anti-Pseudomonas IgY reduces PA adhesion to epithelia, but the mechanism has not been fully elucidated. To gain further insight into the prophylactic effect of these antibodies, the immunoreactivity was investigated by 2D electrophoresis of PA strains, immunoblotting and MALDI-TOF-MS. To confirm the identity of the proteins, the tryptic peptides were analyzed by MALDI-TOF-MS to accurately measure their monoisotopic masses as well as determine their amino acid sequences. In order to facilitate fragmentation of the peptides they were N-terminally or C-terminally labeled. Several strains were investigated and anti-Pseudomonas IgY was immunoreactive against all of these strains, which strengthens its potential as a prophylactic treatment against PA. Flagellin was identified as the major antigen. Flagellin is the main protein of the flagella and is crucial for establishing infections in hosts as well as being involved in PA chemotaxis, motility, adhesion and inflammation. Furthermore, secreted flagellin elicits an inflammatory response. In conclusion, anti-Pseudomonas IgY binds flagellin, which may prevent PA infections in CF patients by hindering host invasion.     

Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution

IgY was separated from a hen's egg yolk that was immunized with Helicobacter pyroli. The anti-H. pyroli IgY activity at acidic pH and the suppressive effect of polyol on acid-induced inactivation of IgY were investigated. Sorbitol and xylitol were used as polyols. IgY was quite stable at pH 5-7. Irreversible inactivation of IgY was observed at pH below 4, and proceeded rapidly at pH below 3. The acid stability of IgY was enhanced in the presence of 30% sorbitol or above. In a 50% aqueous sorbitol solution, an acid-induced inactivation was almost completely suppressed at pH 3. However, the improvement of IgY activity was not observed in the aqueous xylitol solution. IgY showed almost the same activity as native IgY when sucrose was substituted for sorbitol. On the other hand, the xylitol replacement with sucrose did not enhance the acid stability of IgY. The acid-induced inactivation of IgY was related to tryptophyl fluorescence. Fluorescence emission spectra suggested that structural changes near the tryptophan residues may occur under acidic conditions. An increase in sorbitol concentration induced a blue shift. The fluorescence emission of IgY in a 50% sorbitol solution had a peak at 330 nm, which was the same emission peak that was exhibited by native IgY. Sorbitol could, therefore, be used as a good stabilizer of IgY under acidic conditions.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Encapsulation of Biologically Active Proteins in a Multiple Emulsion

To improve the stability of IgY antibody in oral administration, encapsulation of IgY in a W/O/W emulsion was attempted. A stable W/O/W emulsion containing 1% IgY was prepared by using polyglyceryl condensed ricinolate (PGCR) and dextran-casein conjugate as the primary and secondary emulsifier, respectively. However, the activity of IgY antibody was reduced to less than 20% by encapsulation, suggesting that denaturation/inactivation of IgY had occurred at the oil/water interface. Adsorption of IgY to the inner water droplet surface was observed by electron microscopy. Rabbit IgG, α-amylase, and lysozyme also lost their activity after being encapsulated, although the rate of inactivation was lower than that of IgY. Molecular characterization of these proteins suggested that the rate of inactivation after encapsulation is likely to be dependent on the surface hydrophobicity and molecular stability of each protein.     

The purification of IgY from chicken egg yolk by preparative electrophoresis

Chicken IgY has been purified from egg yolk by preparative electrophoresis on the Gradiflow, a system which has been employed for the purification of a wide range of proteins with high recovery and biological activity. Protein purification on the Gradiflow utilises electrophoresis with selected combinations of porous membranes and buffers. The purification of IgY was achieved by initial PEG lipid precipitation, then a single step Gradiflow run by a strategy based on the relatively high pI range of IgY compared to other egg yolk proteins. The IgY yields obtained from the delipidised supernatant are consistently greater than 80% by immunoassay. The purity of the IgY fraction compared favourably with IgY prepared using three commercial products.     

Affinity purification of egg yolk immunoglobulins (IgY) with a stable synthetic ligand

Chicken IgY (egg yolk immunoglobulin) is a functional equivalent of mammalian IgG. Traditional methods for IgY purification involve multi-step procedures that result in low recovery of IgY. After a large scale screening of our 700-member synthetic ligand library synthesized by epichlorohydrin and cyanuric chloride methods, a high efficiency ligand of IgY was found. By one-step purification with this ligand, the purity of IgY could reach 92.1%, and the recovery of IgY could reach 78.2%. This synthetic ligand had a higher binding capacity of 74.8 mg IgY/ml and had no negative effects on immunoreactivity. Remarkably, this ligand was also highly stable and could resist 1M NaOH, thus having great potential for the industrial-scale production of IgY.