Immunoglobulin Y Levels in Egg Yolk From Three Chicken Genotypes

Laying hens are very efficient producers of antibodies and provide an interesting alternative for large-scale production of specific antibodies. These antibodies also have biochemical advantages over mammalian antibodies (e.g. rabbit antibodies) that can be used to improve immunoassays where antibodies are used. The concentration of IgY in egg yolk is an important production parameter. The purpose of this study was to investigate the genetic variation of IgY levels in egg yolk. We have compared IgY concentrations in egg yolks from two lines, selected for egg production traits at the Swedish University for Agricultural Sciences (Single Comb White Leghorn and Rhode Island Red) and a cross between the two lines (SLU-1392). Single Comb White Leghorns have the highest mean concentration of yolk IgY, 2.21 mg ml^-1 compared to SLU-1392 1.95 mg ml^-1 and Rhode Island Red 1.68 mg ml^-1. The cross thus had an intermediate IgY concentration in relation two the two other lines. There were great differences between individual animals within each line. Our results indicate that it should be possible to increase yolk antibody production by using a high producing chicken line and by genetic selection within the line. We found three individuals with very low yolk IgY concentrations among the Rhode Island Red hens. Newly hatched chickens with limited amounts of IgY from the hen may be more susceptible to infections.     

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.     

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.     

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.     

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.     

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.     

Competitive and double antibody sandwich ELISA for the quantification of lactoferrins by using monoclonal and chicken egg yolk IgY antibodies

Two effective competitive and double antibody sandwich ELISA based on monoclonal (MAb) and chicken egg yolk IgY antibodies were developed to determine lactoferrin (LF) content in infant and milk formulas. Leghorn laying hens were immunized with purified bovine and human LFs to produce anti-bovine LF and anti-human LF IgY antibody in the egg yolk. After 5-8 weeks of the immunization, anti-LF IgY was extracted and analyzed by ELISA. Specific IgY antibodies against LFs cross reacted with human and bovine LFs, examined by ELISA and western-blot assay. Such cross-reactivity suggested the presence of common antigenic determinants between human and bovine LFs. An indirect competitive ELISA was preferred to quantify LF in milk and infant formulas, since the range of detection is 3.125-50 μg/mL, which is broader compared to the biotinylated ELISA system (5-50 ng/mL). As per the indirect competitive ELISA system, IgY at a concentration of the 150 μg/mL was incubated with various concentrations of bovine LF ranged from 0.003-100 μg/mL. The immunoassay was used to estimate the total bovine LF content in the milk samples and infant formulas, ranging from 49.28-80.96 μg/mL and 29.92-60.00 μg/mL, respectively.     

Hen egg yolk as a source of antiviral antibodies in the enzymelinked immunosorbent assay (ELISA): A comparison of two plant viruses

A method of raising antibodies against plant viruses in hen egg yolk is described. Laying hens were immunized with citrus tristeza virus (CTV) or tobacco mosaic virus-avocado isolate (TMV-A). Antiviral antibodies in the yolks of sequentially laid eggs as well as in the serum were titrated by the (heterologous) antiglobulin double antibody sandwich form of the enzyme-linked immunosorbent assay (HADAS-ELISA). Antibodies first appeared in yolk 7 days after injection and peak levels were attained on day 9–11; these levels persisted for about 6–12 days. Non-specific yolk antibodies were removed by absorption with an extract of uninfected plant tissue. Using the HADAS-ELISA technique we found that yolk titres were equal to, or higher than those in serum. The benefits of using laying hens over conventional laboratory animals as a source of antiviral antibody are discussed.     

A novel, cost-effective and efficient chicken egg IgY purification procedure

Chicken IgY antibodies have been touted to be a superior alternative to mammalian antibodies for use in various immunological, molecular biology and proteomics applications for several reasons. These include, but are not limited to, improved specificity due to maximum phylogenetic distance between host and recipient, cost effectiveness in maintaining commercial numbers of hens, IgY yield and the use of non-invasive methods used to isolate IgY from eggs as opposed to blood. Despite this, the routine use of IgY-based methodologies in the laboratory is not widespread. One reason for this reluctance may be derived from the difficulties and expense of isolating IgY antibodies from egg yolk in sufficient yield, with high purity at a realistic reasonable price. Here, we describe an extremely cost-effective ($5USD per egg), rapid (within 5 h), efficient and optimised technique to isolate high yields (60 mg) of high purity (~80%) chicken IgY from egg yolks using the common plant gums pectin and κ-carrageenan in the presence of calcium chloride to delipidate egg yolk mixtures whilst maintaining IgY in solution and then ammonium sulphate to subsequently precipitate the resulting IgY antibodies to higher purity. Our data demonstrates that this technique results in a high yield and purity of IgY that is comparable (if not superior to) existing commercial IgY isolation kits. The method also allows the isolation of immunologically active IgY which can be used for further downstream immunotechnological processes. Furthermore, it can also be easily implemented in a standard well equipped laboratory, and may be scaled up to commercial quantities (i.e., thousands of eggs).     

Anti‐idiotype and anti‐anti‐idiotype antibodies for aflatoxin from laying hens

Anti‐idiotype (anti‐id) antibodies (IgY2)for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity‐purified rabbit polyclonal anti‐aflatoxin B₁ (AFB₁) carboxymethyloxime‐bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified and then subjected to various analyses. Inhibition of the binding of pAbl to the solid‐phase AFB₁‐BSA by IgY2 and the binding of pAb1 to the solid‐phase IgY2 by free AFB₁ were demonstrated in a biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID₅₀) of the binding of pAb1 to AFB₁‐BSA was found to be 2.45 μg/assay. The ID₅₀ concentration of the binding of pAb1 to IgY2 by free AFB₁ was found to be 0.30 μg/assay. Inhibition of the binding of AFB₁‐horseradish peroxidase (HRP) to the solid‐phase pAbl by IgY2 (ID50 = 9.65 μg/assay) was also demonstrated in the direct ELISA. Egg yolk anti‐anti‐id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti‐AFB₃‐hemisuccinate‐BSA monoclonal antibody. IgY3 was subjected to affinity chro‐matography purification with Sepharose gel armed with AFB₂‐carboxymethytoxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti‐AFB antibodies induced in various experimental animals. In the direct ELISA, the ID₅₀ of the binding of AFB₁‐HRP to solid‐phase lgY3 by AFB₁ was found to be 0.12 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of IgY3 to solid‐phase AFB₁‐BSA by AFB₁ was found to be 2.2 ng ml^‐1. The IgY3‐based ELISA analysis showed higher sensitivity than that of the egg yolk antibodies directly against AFB‐protein conjugates (IgY1). A good correlation was found for the data obtained from IgY3‐based and pAb1‐based ELISAs in the analysis of AFB in the fungal culture filtrates.     

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。     

Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a major aetiological agent of childhood diarrhoea in developing countries. The structural repeating protein A subunit, BfpA, found in the bundle-forming pilus, is one of the virulent factors for EPEC pathogenesis. Recombinant BfpA in laying hens elicited sustained and vigorous antibody production. Immunoglobulin Y (IgY) anti-BfpA antibodies were recovered from egg yolk, purified and characterized. Immunoadsorption with whole extracts of the isogenic E. coli EPEC adherence factor (EAF) strain that lacks BfpA rendered the resulting IgY preparations capable of: (a) recognizing purified or recombinant BfpA proteins in a dose-dependent fashion; (b) blocking the colonization of HeLa cells by EPEC EAF+, in vitro; (c) specifically identifying E. coli bearing EAF+; and (d) inhibiting the growth of E. coli EAF+ but not the EAF strain. IgY anti-BfpA is potentially useful as a specific, low-cost immunobiological reagent to screen human faecal specimens for the presence of EPEC.     

Effects of inoculation route and dose on production and avidity of IgY antibodies

The aim of this study was to investigate the effects of route of inoculation and dose on production and avidity of IgY antibodies in chickens. The animals were inoculated with 20?µg of human IgG by intramuscular, intradermal, or subcutaneous routes, or with 2, 20, or 200?µg of human IgG via IM to evaluate the effect of dose. All routes led to significant levels of specific antibodies after the fifth inoculation. There was a significant difference in the levels of antibodies produced with 2 and 200?µg of antigen after the third and seventh inoculation. It was observed that there is no influence of route of inoculation or dose on the avidity of the antibodies produced. Furthermore, the index of avidity is influenced directly by the number of inoculations. These results are important to help future studies where antibody production of IgY from laying hens is necessary.     

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.     

抗肠道病毒71型特异性卵黄抗体的制备及鉴定

目的:制备特异性抗肠道病毒71型(Enterovirus 71,EV71)的卵黄抗体并检测其生物学性能,这对EV71感染手足口病的预防和治疗具有重大的意义。方法:用纯化并灭活后EV71作为抗原免疫健康产蛋鸡。采用本实验室水提综合聚乙二醇法提取纯化鸡卵黄抗体;用Bradford法测定卵黄抗体提取液的蛋白含量;还原性SDS-PAGE凝胶电泳分析测定提取蛋白的纯度及相对分子质量;分别用ELISA、双向免疫琼脂扩散检测纯化特异性卵黄抗体抗EV71效价;Western blot分析特异性卵黄抗体的免疫反应性。结果:卵黄中卵黄抗体含量达7.76 mg/ml,卵黄抗体的纯度为94.86%。初免10天后蛋黄中可检测到特异性抗EV71卵黄抗体,初免40天后ELISA检测抗体效价高达1∶20 480,双向琼脂扩散检测效价达1∶16。提取的卵黄抗体具有良好的免疫反应性,能与EV71特异性结合。结论:水提综合聚乙二醇法提取纯化得到的抗EV71卵黄抗体产量和纯度高、效价好且具有良好的免疫反应性。     

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.     

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。     

Comparison of four purification methods for the production of immunoglobulins from eggs laid by hens immunized with an enterotoxigenic E. coli strain

The importance of eggs as a source of specific antibodies is well recognized. Egg yolk contains 8–20 mg of immunoglobulins (IgY) per ml. However, the major problem in isolation is removal of lipids which are present in high concentrations. A method had been developed by employing water dilution to separate the yolk plasma proteins from the granules and lipids. Further purification of IgY from plasma proteins was achieved by a protocol involving salt precipitation and ultrafiltration. The water dilution method (WD) was compared with three other methods, namely, polyethylene glycol (PEG), dextran sulphate (DS) and xanthan gum (Xan) in terms of yield, purity, ease of use, potential scaling up and immunoactivity of IgY. The WD method gave the highest yield, followed by DS, Xan and PEG methods in that order. 9.8 mg IgY / ml egg yolk was routinely obtained from the WD method compared to 4.9 mg IgY / ml egg yolk with the popular PEG method with purities of 94% and 89% respectively. Purification methods had no adverse effect on the immunoactivities of IgY. WD was also found superior in terms of ease of use and large scale production of IgY. WD method therefore provides a simple, rapid and efficient means of purifying IgY with high activity.     

抗轮状病毒IgY和Fab的研制

目的对抗轮状病毒(RV)IgY和胃蛋白酶水解片断Fab进行分离与纯化.方法免疫鸡得到抗HRVIgY,用两步盐析结合凝胶过滤将其从蛋黄中分离出来,纯的IgY经胃蛋白酶水解得抗体片断Fab.结果抗HRVIgY用SDS-PAGE检测纯度可达到95%以上.抗轮状病毒(RV)IgY和抗体片断Fab经SDS-PAGE和MALDIMS法测定,其纯度达到99%以上,经ELISA法检测,Fab'的活性保持在IgY原始活性的70%以上.结论我们所设计的分离和纯化抗HRVIgY和Fab'的方法简单、有效.     

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.