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.     

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。     

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.     

Preparation of specific anti-Helicobacter pylori yolk antibodies and their antibacterial effects

Objective: To study immunization procedures and preparation methods of specific IgY antibodies (IgY-Hp, IgY-IB) produced by hens immunized with Helicobacter pylori (Hp) bacterial antigen and recombinant Hp specific antigen IB, detect the inhibition effects on Hp growth and Hp urease activity, and study the effects of oral administration for treating Hp infection. Methods: By using recombinant cholera toxin subunit B (rCTB) as an adjuvant, hens received intramuscular injection immunization for continuous 7 times at an interval of 14 days. Then, the eggs were collected; IgY was purified. Results: On day 49 after hens were immunized, levels of two antibodies all reached 1:12800; after they were purified by Ammonium sulfate precipitation, their purity was over 80%. IgY-Hp could inhibit Hp growth and inhibit Hp urease activity; although in vitro, IgY-IB could not inhibit Hp growth but could inhibit Hp urease activity. The experiments in vivo found that when IgY-Hp or IgY-IB with sucralfate dual oral therapy was used to treat Hp infected mouse model, the cure rate all could reach 83.3%. Conclusion: According to immunization procedure, high titer specific IgY antibody (1:12800) can be obtained in 49 days and its titer remains stable. Oral administration of the specific IgY antibodies in Hp infected mice can reach a cure rate of 83.3%, and the antibodies are expected to become new drugs and therapeutic methods of targeted therapy against Hp infection.     

Comparative Analysis of Methods of Purification of Egg Yolk Immunoglobulin

This study is a critical comparison of reported methods of purification of IgY from hen egg yolk. Five methods of lipid removal were compared, followed by a comparison of three methods of immunoglobulin precipitation. Each of these methods was tested three times. Lowry assays were used to measure the protein content of the various purified fractions, and densitometric analysis of SDS-polyacrylamide gels was used to quantify the proportion of IgY. Peak IgY yields of 15.6 and 15.1 mg of IgY per ml of egg yolk, with greater than 60% purity, were obtained after lipid removal using dextran sulphate and calcium chloride or phosphotungstic acid and magnesium chloride, respectively. Further precipitation of IgY from these fractions using 12% PEG, the most effective method of immunoglobulin precipitation, recovered pure IgY preparations, with mean yields of 8.80 and 8.62 mg per ml of egg yolk. Alternatively, a simpler and more cost effective method of lipid removal by freezing and thawing of egg yolk at neutral pH recovered 13.1 mg of IgY per ml of egg yolk at a purity of 71.1%. Subsequent Ig precipitation also recovered a pure IgY preparation with a mean yield of 7.49 mg per ml of egg yolk.     

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.     

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.     

Limited Day to Day Variation of IgY Levels in Eggs from Individual Laying Hens

Laying hens are highly efficient producers of polyclonal antibodies (PAbs). These antibodies are transported to the egg yolk in large quantities from the blood of laying hens. The IgY concentration in the egg yolk is important to protect the newly hatched chick against infections and it is also an important factor for the production of yolk antibodies for commercial purposes. A single egg yolk contains approximately the same amount of IgY as 30 ml of blood. This is a significant loss of antibodies for an animal the size of a hen. We have studied the IgY levels in egg yolk. We found low day to day variability. There was no decrease in IgY levels at the end of the egg laying cycle and there was no correlation between IgY concentration and egg production.     

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。     

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。     

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。     

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.     

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.     

Neutralization of enterotoxigenic escherichia coli heat‐labile toxin by chicken egg yolk immunoglobulin Y and its antigen‐binding fragments

The protective effect of chicken egg yolk immunoglobulin Y (IgY) and its antigen‐binding FAb’ fragments was studied by their neutralization of heat‐labile toxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) strain H10407. High levels of specific antibodies against LT were produced and maintained over a period of 8 months in eggs of hyper‐immunized hens. The FAb’ fragment produced by peptic digestion was found to be as effective as the whole IgY molecule in neutralizing LT. Removal of the enterotoxin‐specific antibodies eliminated the neutralization activities of both IgY and the FAb’ fragment. Heat pasteurization at 65°C for 15 min and presence of high salt (1.5 M‐NaCl) at pH 7.0 did not change the neutralization activities of either IgY or FAb’. Although incubation at pH of 2.0 for 4 h led to a 16‐fold decrease in the neutralization activities of IgY and FAb’, substantial neutralization activities were retained. The combination of low pH and high salt content, however, resulted in the complete destruction of the neutralization activity of IgY, but not the FAb’ fragment.     

Oral ingestion of egg yolk immunoglobulin from hens immunized with an enterotoxigenic Escherichia coli strain prevents diarrhea in rabbits challenged with the same strain.

White Leghorn hens were immunized with enterotoxigenic Escherichia coli B16-4 with heat-labile enterotoxin and colonization factor antigen I in Freund's adjuvant. Specific antibodies were detected by an enzyme-linked immunosorbent assay in the serum after 8 days and in eggs after 10 days, with levels reaching peaks at 15 and 20 days after the first immunization, respectively. The protective effects of the egg yolk antibodies were tested in the rabbit reversible ileal tie model of diarrhea. Five control rabbits developed severe diarrhea within 72 h after inoculation with enterotoxigenic E. coli B16-4. Oral ingestion of egg yolks from immunized hens for 4 days prior to inoculation protected five rabbits from diarrhea after challenge with the same strain of E. coli. The rabbits showed no adverse effects from the ingestion of the egg yolks. Four rabbits fed control eggs were also afforded some protection in that three rabbits developed mild diarrhea and one rabbit remained entirely well. In vitro experiments showed that immunoglobulin from egg yolks interfered with the binding of E. coli to purified small bowel mucins; immunoglobulin from immunized hens reduced binding more than immunoglobulin from nonimmunized hens. These findings indicate that eggs from hens immunized with appropriate antigens have potential as a useful source of passive immunity.     

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.     

Preparation and determination of immunological activities of anti-HBV egg yolk extraction

To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. Its chemical characteristics were identified by ultraviolet spectrum, HPLC, Lowry analysis and pharmacopocia-raleted methods. The specific immunological activity was examined by leukocyte adherence inhibition （LAI） in vitro and delayed type hypersensitivity （DTH） in vivo. Anti-HBV EYE was a small dialyzable substance with molecular weight less than 12 kD containing 18 kinds of amino acids. The preparation could obviously inhibit LAI and DTH which was similar to hepatitis B virus-specific transfer factor of pig spleen. However, there were no similar effects observed in the nonspecific transfer factor （NTF） group, control egg yolk extraction （CEYE） group and hepatitis A virus （HAV） group. The results suggested that anti-HBV EYE contained hepatitis B virus-specific transfer factor （STF） and had the antigen-specific cell immune activity similar to PSHBV-TF. The STF obtained from egg yolk of the hens immunized with specific antigen, might be a potential candidate for immunoregulation in hepatitis B prevention and treatment.     

Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand

Due to the peculiar composition of the egg yolk and the lack of specific affinity ligands, Y immunoglobulins are normally purified using complex and time consuming procedures involving a combination of precipitation and chromatographic steps first to extract and capture and then to polish IgY. In this study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screening of combinatorial libraries, and already characterized for its capability to purify immunoglobulins of class G, M, E and A. Soluble proteins were separated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the ligand on the commercially available support Emphaze. In a single chromatographic step TG19318 affinity columns led to an efficient capture of IgY directly from crude samples, and with a purity degree higher than 90%, as determined by densitometric scanning of SDS-PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4x excess IgY to 1 ml bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per ml of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivatized matrix was found to be very stable, in terms of ligand leakage and maintenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage.     

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.