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.     

IgY抗体作为导向载体对消化系统肿瘤的选择性识别作用研究

用生物治疗方法治疗肿瘤已被视为肿瘤治疗的第四大疗法,其主要是免疫治疗．包括抗体与偶合物,肿瘤疫苗等。而要想获得有效的肿瘤治疗药物．其关键是要寻找小型化的、高效的导向载体。我们的实验室以低分化腺胃癌的P110癌蛋白作为抗原,免疫SPF鸡,从卵黄中提取抗体IgY作为植物毒素的导向性载体;实验结果证明,IgY能特异性地选择识别消化系统的肿瘤组织,为进一步研究肿瘤的导向药物提供了另一理论依据。     

艰难梭菌毒素B羧基端的表达与卵黄抗体制备简

目的：在大肠杆菌中可溶性表达艰难梭菌毒素B羧基端（TcdB-e）,免疫产蛋鸡,获得针对TcdB-c的卵黄抗体（IgY）。方法：人工合成TcdB-c的基因,将其克隆至pET32b（＋）载体中,转化大肠杆菌BL21（DE3）,诱导表达产物经金属螯合层析纯化,凝血酶酶切后得到目的蛋白TcdB-c;利用兔红细胞凝集和兔肠袢实验检测目的蛋白活性,用TcdB-c免疫产蛋鸡制备鸡卵黄抗体,分离纯化卵黄抗体并经ELISA测定抗体效价,用兔肠袢实验检测抗体的中和活性。结果：构建了TcdB-c的重组表达载体,诱导表达的融合蛋白相对分子质量约为79000,经凝血酶酶切后的相对分子质量约65000;目的蛋白免疫产蛋鸡后获得效价为1：20000的抗TcdB-C卵黄抗体,且该抗体可以中和TcdB-c对兔小肠的毒性作用。结论：获得了具有生物学活性的TcdB-C,并制备了针对TcdB-c的鸡卵黄抗体,为利用基因工程方法防治艰难梭菌感染打下了基础。     

抗甲型流感鸡卵黄抗体的制备及效价测定

目的:研究抗甲型流感卵黄抗体的制备与纯化,并探讨其效价随免疫时间的变化关系。方法:用灭活甲型流感病毒复合抗原免疫蛋鸡,用PEG6000对卵黄抗体进行分离提取,SDS-PAGE法对其进行分子量测定,考马斯亮蓝法对其含量和纯度进行测定,用微量凝集法检测蛋鸡血清抗体和卵黄抗体的效价。结果:提取得到的卵黄抗体重链分子量为66 kDa、轻链分子量分26 kDa,每毫升卵黄液可得到纯度为95.80%的卵黄抗体9.98mg,回收率93.01%;高效价持续时间90 d以上;免疫蛋鸡血清和卵黄中3种特异性抗体的消长规律基本相似,但抗体水平之间存在明显的差异。结论:采用灭活甲型流感病毒复合抗原免疫蛋鸡可制备高效价、高纯度抗甲型流感卵黄抗体,为卵黄抗体在甲型流感防治中的应用研究奠定了基础。     

抗肺炎支原体卵黄抗体的制备及其免疫特性

目的制备抗肺炎支原体卵黄抗体,并研究其免疫特异性。方法以超声粉碎法制备肺炎支原体抗原;以ELISA法测定卵黄抗体的效价及免疫特异性;以水稀释法联合疏水层析的方法分离纯化卵黄抗体;应用SDS-PAGE法测定分子量及鉴定抗体纯度;改良Lowry法测定蛋白含量。结果低、高剂量组均诱导母鸡产生有效免疫应答,高剂量组免疫效价高于低剂量组。高剂量组于初免疫后约50d抗体效价达高峰,持续约2个月;而低剂量组在初免疫后约60d抗体效价达高峰,持续约1个月。之后效价逐渐下降,在免疫约120d,高剂量组由13log2下降到10log2;而低剂量组则由11log2下降到7log2。以水稀释法联合疏水层析法制备了电泳纯抗肺炎支原体IgY,分子量约178KD,平均每1ml卵黄液可获得较纯抗体6.4mg。制备的IgY与肺炎支原体具有较高特异性,与解脲支原体和人型支原体无明显交叉反应,与生殖支原体有轻度的交叉反应。结论本研究初步制备了抗肺炎支原体卵黄抗体,为肺炎支原体的防治与检测提供新的途径。     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.     

Preparation of Immunoglobulin Y (IgY) Against Lipopolysaccharide using Gel Chromatography from the Yolks of Eggs Laid by Immunized Hens

The objective is to prevent and treat injuries caused by lipopolysaccharide (LPS) from gram negative bacteria in animals and humans, we produced antibodies against LPS from egg yolk. LPS from E. coli (O111:B4) mixed with Freund’s Adjuvant was used as the immunogen to immunize Roman hens. Immunized eggs were collected, and immunoglobulin Y (IgY) was purified using a water solution, salt precipitation and gel chromatography. The molecular weight and purity were determined by SDS–PAGE, the antibody titer by noncompetitive enzyme-linked immunosorbent assay (ELISA) and antibody activity against LPS by the mortality of mice intraperitoneally injected with LPS or LPS-IgY solutions. IgY against LPS showed two protein bands at 68 and 26 kDa on the gel; the antibody titer was almost 1:25,600. After incubation with LPS, IgY decreased the mortality of mice challenged with LPS. This study provided an efficient way to produce high-titer egg yolk antibodies, which could attenuate lethal effects of LPS, by immunizing hens. Furthermore, the LPS antibody was purified well using a water solution, salting-out and gel chromatography.     

Efficient production, purification, and application of egg yolk antibodies against human HLA-A*0201 heavy chain and light chain (beta2m)

The importance of eggs as a source of specific antibodies is well recognized. Egg yolk contains 8--20mg immunoglobulins (IgY) per milliliter. However, the major problem in separating IgY is to remove the high concentrations of lipids in egg yolk. We first used water dilution method to get the supernatant containing IgY, then purified the antibody by caprylic acid-ammonium sulfate method, and obtained specific antibody with satisfactory purity and activity. By comparison of these several methods, each has its advantages, one can be chosen to purify IgY according to practical need. The purified IgY produced by the immunized chickens can stain the human peripheral blood mononuclear cell effectively when labeled with fluorescent FITC.     

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20°C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).     

Production and purification of IgY antibodies from chicken egg yolk

The isolation of polyclonal antibodies from the serum of immunized mammals has significantly contributed to scientific research and diagnosis. The fact that recent technologies allow the production of antibodies in the yolk of eggs laid by hens, has led to the development of an alternative method for antibody generation that is less stressful to animals. As hens are kept under almost all their natural conditions, antibodies are isolated from the collected eggs; this technology is expected to become an interesting alternative to the conventionally serum-based techniques that eventually require to sacrifice the animal.Here we present a modified protocol for the isolation of IgY antibodies from immunized chickens and provide comparison between two chicken breeds in relative to IgY yield per egg. Our results show the possibility of generating large quantities of highly pure IgY from chicken eggs and also show large differences in the yield of IgY production between the two studied breeds. The results of this work indicate that IgY technology can be used for the production of primary antibodies for immunological work and disease diagnosis.     

Chicken egg yolk antibodies (IgY-technology): a review of progress in production and use in research and human and veterinary medicine

The production of antibodies (Abs) in chickens and the extraction of specific Abs from egg yolk (IgY Abs) are increasingly attracting the interest of the scientific community, as demonstrated by the significant growth of the IgY literature. This review offers detailed and comprehensive information about IgY-technology, including: a) possibilities for hen keeping in accordance with the Three Rs principles; b) new insights into the IgY transfer mechanism from blood to yolk as a biological basis for the technology; c) the comparative characteristics of IgY Abs and IgG Abs; d) the high efficacy of the technique, in view of the extraordinary amount of IgY Ab produced by one hen in one year (between 20 g and 40 g IgY in total); e) comparisons between the efficacies of IgY Abs and IgG Abs (rabbit, sheep, mouse) in several immunological assays; f) immunisation protocols, as well as the most commonly used IgY-extraction procedures; g) new possibilities for application in human and veterinary medicine, including strategies for the treatment of Helicobacter pylori infection or fatal intestinal diseases in children, particularly in poor countries, for reducing the use of antibiotics, and, in Asia and South America, for producing Abs against snake, spider and scorpion venoms; and h) the use of IgY Abs in various fields of research, also taking into consideration recent developments in South America (particularly Argentina and Cuba) and in Asia.     

Molecular stability of chicken and rabbit immunoglobulin G.

Molecular stability of chicken egg yolk immunoglobulin G (IgY) and that of rabbit IgG were compared by measuring antibody activities and conformational changes. Stability of rabbit IgG to acid denaturation was much higher than that of IgY. Conformation of the IgY molecule was readily changed in acidic conditions, resulting in a rapid loss of antibody activity. Much less stable natures of IgY to heat-treatment and guanidine-HCl denaturation than rabbit IgG were also observed. Differences in the structure between the two immunoglobulins that might participate in their different stability were inferred from their amino acid sequence data. Importance of the intramolecular disulfide linkage in the rabbit light chain and some other structural differences were suggested.     

Egg Yolk antibody and its application

A hen transfers her serum immumnoglobulin G to the egg yolk (IgY) and gives im|munity to her offspring. Therefore, the hen egg can be an effective supplier of a large amount of antigen specific antibody that accumulates in the egg yolk. Antigen specific antibody has been widely used for immunological analysis in the field of diagnosis as well as pure scientific research. The production and separation technology of IgY is demonstrated in the present study.     

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。     

Generation and application of chicken egg-yolk antibodies

Despite the fact that the use of chicken as immunization host brings many advantages to the production of polyclonal antibodies, the generation of egg yolk immunoglobulins (IgY) is rarely chosen. In this review, we report on the fast and efficient method for generation and affinity purification of IgY, in this case raised against the alpha-subunit of hypoxia-inducible factor-1 (HIF-1). The IgY antibody was successfully applied in a variety of methods and a number of different species for HIF-1alpha detection. In electrophoretic mobility shift assays, the IgY antibody recognized the native HIF-1 complex. The IgY antibody also detected HIF-1alpha protein on Western blots with extracts derived from human, monkey, pig, dog and mouse cell lines grown under hypoxic conditions. Immunofluorescence and immunoprecipitation experiments using the IgY antibody allowed detection and subcellular localization of HIF-1alpha in the nuclei of hypoxic cells. Chicken antibody production brings great benefit concerning the welfare of the immunized animals, due to non-invasive antibody harvesting with the added convenience of simple egg collection. An additional advantage is the fast and simple IgY isolation from egg yolk. IgY technology is a great improvement and should be considered as a good alternative to conventional polyclonal antibody production in mammals.     

Eliciting antigen-specific egg-yolk IgY with naked DNA

Immunization with naked DNA was used to elicit chicken egg yolk antibodies (IgY). Layer hens were inoculated with plasmid DNA encoding the enhanced green fluorescent protein, the fusion protein of Newcastle disease virus, and VP2 of African horse sickness virus. IgY was extracted from egg yolks by polyethylene glycol precipitation. Specific antibodies were present in the yolks of eggs from hens immunized with each of the three different plasmids. This approach to raising polyclonal antibodies obviates the need to produce and purify large quantities of proteins for immunization and can potentially yield large amounts of diagnostically or therapeutically useful reagents.     

IgY在胃肠感染性疾病中的应用

卵黄抗体（1mmunoglobulinofyolk,IgY）是通过使用特种抗原免疫产蛋鸡,由其卵黄中提取的抗体,与哺乳动物的IgG相似,IgY性质稳定,在控制胃肠道感染性疾病方面存在许多独特的优点,可用于相应疾病的预防和治疗。研究表明,口服特种的IgY对大肠埃希菌、沙门菌、轮状病毒等引起的胃肠感染性疾病可起到免疫保护作用,有望成为抗生素的替代制剂。目前IgY正成为生物技术领域和医药研究中的新热点。     

In Vitro Inhibition of Oral Candida albicans by Chicken Egg Yolk Antibody (IgY)

This study was conducted to measure Candida albicans-specific chicken egg yolk antibody (IgY) inhibition of fluconazole-sensitive and resistant strains of C. albicans in order to assess potential use in the prevention and treatment of oral candidiasis. In this study, laying hens were immunized, and IgY was extracted by water dilution. The Minimal Inhibitory Concentrations (MICs) of IgY for inhibiting C. albicans growth were determined using the broth microdilution method from the CLSI M27-A2 protocol. Fluconazole (FLC) was used as the control. The results were analyzed with the chi(2) test. The anti-Candida titer of anti-C. albicans IgY was 1:12,000. The concentration of the IgY extract that effectively inhibited the growth of C. albicans was between 1.25 g/l and 5.0 g/l, and the efficacy rate was 82.98% during the observed 24-48 h time period. No correlation was recorded between the drug resistance of FLC and growth inhibition by IgY. It was concluded that anti-C. albicans IgY inhibited the growth of C. albicans in vitro and there was no correlation between the drug resistance of FLC and the growth inhibition by IgY (P > 0.99).     

抗哇巴因鸡蛋黄IgY与兔抗体IgG在酶联免疫检测中的比较

目的：探讨提高内源性哇巴因（EO）检测方法的准确性及特异性,为哇巴因的研究打下基础。方法：分别制备出鸡蛋黄抗哇巴因多克隆抗体及兔抗体。然后采用酶联免疫吸附法（ELISA）比较两种抗体检测内源性哇巴因的准确性及特异性。结果：采用IgY检测EO的平均批内变异系数为2．03％,批间变异系数为2．34％。兔抗体IgG则分别为2．83％、3．29％;两者均与氢化可的松及地塞米松无交叉结合反应,与西地兰和地高辛分别存在3．45％vs5．95％、3．20％vs5．20％的交叉反应。结论：IgY比兔抗体ELISA检测内源性哇巴因的特异性及准确性较高。     

Evaluation of Shiga toxin 2e‐specific chicken egg yolk immunoglobulin: Production and neutralization activity

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e‐specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti‐Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost‐effective agent to develop for prophylactic foods or diagnosis kits for edema disease.