Avian IgY antibodies and their recombinant equivalents in research, diagnostics and therapy

The generation and use of avian antibodies is of increasing interest in a wide variety of applications within the life sciences. Due to their phylogenetic distance, mechanisms of immune diversification and the way in which they deposit IgY immunoglobulin in the egg yolk, chickens provide a number of advantages compared to mammals as hosts for immunization. These advantages include: the one-step purification of antibodies from egg yolk in large amounts facilitates having a virtually continuous supply; the epitope spectrum of avian antibodies potentially grants access to novel specificities; the broad absence of cross-reactivity with mammalian epitopes avoids assay interference and improves the performance of immunological techniques. The polyclonal nature of IgY antibodies has limited their use since avian hybridoma techniques are not well established. Recombinant IgY, however, can be generated from mammalian monoclonal antibodies which makes it possible to further exploit the advantageous properties of the IgY scaffold. Moreover, cloning and selecting the immune repertoire from avian organisms is highly efficient, yielding antigen-specific antibody fragments. The recombinant approach is well suited to circumvent any limitations of polyclonal antibodies. This review presents comprehensive information on the generation, purification, modification and applications of polyclonal and monoclonal IgY antibodies.     

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.     

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。     

卵黄抗体在兽医寄生虫学中的应用

卵黄抗体是鸡产生的主要抗体,鸡被免疫后,IgY被持续地被合成、分泌到血液中,并被选择性地转移、富集到蛋黄中。母鸡产生的IgY可为它们的后代抵抗常见的禽类病原体提供有效的体液免疫保护。就有关寄生虫卵黄抗体的研制情况及其在兽医寄生虫病诊断、治疗等的应用情况做一综述。     

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。     

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。     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

抗MRSA-PBP2a鸡卵黄抗体的制备及乳胶凝集检测法的建立

制备抗耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a( MRSA- PBP2a)抗原的鸡卵黄免疫球蛋白(IgY),建立检测MRSA的乳胶凝集方法.采用体外诱导的方法制备PBP2a蛋白,胸部肌肉多点注射方式免疫6只海蓝蛋鸡,水稀释法提取IgY,BCA法测定蛋白含量,Western blotting进行特异性分析,用提取的IgY抗体致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法.成功诱导并制备获得纯化的PBP2a蛋白,首次免疫后1月每枚鸡蛋提纯后可获得约48 mg IgY抗体,Western blotting结果显示IgY抗体能有效识别纯化的PBP2a蛋白;成功建立检测PBP2a的乳胶凝集法,敏感性达1 mg/L.抗MRSA- PBP2a鸡卵黄抗体具有较高的敏感性和特异性,基于其建立的乳胶凝集检测方法具有较好的灵敏性.     

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。     

Refinement of polyclonal antibody production by combining oral immunization of chickens with harvest of antibodies from the egg yolk

Polyclonal antibody production in mammals is generally associated with multiple injections of antigens and adjuvants and repeated blood sampling procedures. During the past 20 yr, the use of chickens instead of mammals for this purpose has increased. A major advantage of using birds is that the antibodies can be harvested from the egg yolk instead of serum, thus making blood sampling obsolete. In addition, the antibody productivity of an egg-laying hen is much greater than that of a similar sized mammal. This article focuses on the developments in oral immunization strategies for chickens that combined with the antibodies from the egg yolk, have great potential for active implementation of the three Rs (replacing, reducing, and refining the use of laboratory animals to the extent possible) in polyclonal antibody production schemes.     

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.     

Productivity and Some Properties of Egg Yolk Antibody (IgY) against Human Rotavirus Compared with Rabbit IgG

Productivity and some properties of anti-Human Rotavirus (HRV) hen egg yolk antibody (IgY) were compared with those of anti-HRV rabbit serum antibody (IgG). The hens immunized with HRV (Wa strain, serotype 1 and Mo strain, serotype 3) were found to continuously to lay eggs without any change in the egg laying rate and the yolk of the eggs laid over a year showed a high level of neutralization titer against HRV. The production of anti-HRV IgY by a hen (one year) was at least 15 times (anti-Wa) and 120 times (anti-Mo) more effective than those by an immunized rabbit in the neutralization titer of the antibodies.

The stability of anti-HRV IgY at temperature above 70°C and low pH 2–3 was less than that of anti-HRV rabbit IgG. The temperature corresponding to the maximum of denaturation endotherm (T_max) of IgY was 73.9°C while that of rabbit IgG was 77.0°C in the analysis by differential scanning calorimetry. This discrepancy in heat and acidic pH stability found between the two antibodies as discussed with regard to their protein structures.     

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.     

A novel and efficient immunoglobulin Y extraction method using poloxamer-polyethylene glycol

Although many IgY extraction methods (such as polyethylene glycol (PEG) precipitation method, octanoic acid method, water dilution method, etc.) have been established, there is still industrial drive and real need in developing scale-up IgY production methods. Some previous studies have reported that poloxamer degreasing method shows very good result in IgY extraction from egg yolk with high degreasing speed, harmlessness, simpleness in operation and minimal effect on antibody titer. In this study, we developed a new method, poloxamer-PEG method, to obtain functional IgY with high purity and yield. First, the delipidation solution was added into the diluted yolk samples, and then the filtrates were collected from the diluted yolk samples after 3?hr in room temperature. PEG-6000 was added into the collected filtrates and the mixture was centrifuged after shaking on the roller mixer for 45?min at room temperature. Last, the precipitates were resuspended in 1?mL phosphate buffered solution (PBS) buffer and dialyzed overnight. The results showed that the total protein concentrate of extractive could reach at 30?mg/mL and the purity of the IgY could reach at 92.71% with the novel method, which was superior to the PEG precipitation method and water dilution method.     

The chicken egg yolk plasma and granule proteomes

Using 1-D SDS-PAGE, LC-MS/MS, and MS(3), we identified 119 proteins from chicken egg yolk, 86 of which were not identified in yolk previously. Proteins were roughly quantitated by calculating their exponentially modified protein abundance index (emPAI) to classify them as major or minor yolk components, and to estimate their distribution between yolk plasma and yolk granular fraction. The proteins with highest abundance were serum albumin, the vitellogenin cleavage products, apovitellenins, IgY, ovalbumin, and 12 kDa serum protein with cross-reactivity to beta2-microglobulin. In addition yolk contained many other serum and egg white proteins, the proteases nothepsin and thrombin, numerous protease inhibitors, and antioxidative enzymes, such as superoxide dismutase and glutathione peroxidase. Among the moderately abundant proteins were two alpha2-macroglobulin-like proteins different from egg white alpha2-macroglobulin, and the major biotin-binding protein of yolk. An unexpected identification was that of the eggshell matrix protein ovocleidin-116, which was previously thought to be eggshell-specific. The list of chicken egg yolk proteins provided in this report is by far the most comprehensive at present and may serve as a starting point for the characterization of less well-known yolk proteins.     

Polyclonal antibodies against rat liver cytosolic casein kinase II (CK-2) cross-react with CK-2 from other tissues and nuclear form (PK-N2) of the enzyme

Casein kinase II (CK-2) is a ubiquitous serine/threonine protein kinase, and is localized to both the cell nucleus and cytoplasm. Despite extensive biochemical similarities in their properties, there is evidence that the two forms of the enzyme exhibit certain distinctions (1). This prompted us to produce antibodies against CK-2, which could be utilized as a possible tool for investigations of the various forms of this enzyme. Specific polyclonal antibodies against the rat liver cytosolic CK-2 were raised in egg yolk of laying hens; the enzyme had repeatedly failed to elicit an immunogenic response in rabbits. The purified polyclonal antibody (egg yolk immunoglobulin, IgY) recognized all three subunits (42, 38, and 28 kDa) of the enzyme in immunoblots. The antibody when bound to a matrix was capable of removing CK-2 from solution, and the bound enzyme could be recovered from the immunoaffinity matrix with 0.1 M diethylamine. The antibody exhibited a high affinity towards CK-2 prepared from cytosol of liver, ventral prostate, and several other rat tissues, but no immunoreactivity was detected towards a number of other protein kinases tested. The subunits of the nuclear form of CK-2 (PK-N2) migrated differently when electrophoresed in parallel in the same gel. However, the antibody did cross-react with the various subunits of PK-N2 suggesting a significant homology in the immunogenic domains in the various subunits of the two forms of the enzyme.     

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.