四种鸡卵黄免疫球蛋白纯化方法的研究

本文比较了分离纯化鸡卵黄免疫球蛋白(IgY)的四种方法(即水稀释法,聚乙二醇法,葡聚糖硫酸盐法和黄原胶法)。包括IgY产量,纯度,活性,简便性及扩大生产规模的潜力。试验表明:水稀释法(DS)产量最高,然后依次为葡聚糖硫酸盐法(DS),黄原胶法(Xan),聚乙二醇法(PEG);同时发现水稀释法简便、快速、高效,适用于工业化生产。     

免疫卵黄抗体提取工艺的优化

以水稀释法为基础,结合PEG6000沉淀法对卵黄中IgY进行分离提取。探讨稀释倍数、溶液pH值、静置时间、PEG6000浓度等因素对IgY提取率的影响,并通过正交试验对分离条件进行优化。通过单因素和正交试验得到最佳分离条件为稀释8倍、溶液pH值5.2、静置时间8h、PEG6000浓度8%,该条件下IgY提取率达到13.69mg/g;各因素对IgY提取率的影响顺序为PEG6000浓度>稀释倍数>静置时间>溶液pH。     

绿色高效连续分离技术制备蛋黄中活性蛋白质和磷脂

为了实现蛋黄功能性成分的充分利用，采用绿色溶剂从新鲜蛋黄中高效连续分离具有生物活性的卵黄免疫球蛋白（immunoglobulin Y, IgY）、卵黄高磷蛋白（phosvitin, PV）和磷脂（phospholipids, PL）。首先采用水稀释法（稀释倍数为1:10）分离水溶性组分与脂蛋白，通过35%饱和(NH₄)₂SO₄和0.5% NaCl（w/v）对上清液进行盐析并用8% PEG 6000纯化IgY。脂质组分中的PV在pH5.0，90 ℃条件下的NaCl溶液（w/v）中进一步纯化。采用乙醇/乙酸乙酯比例为1:2（v/v）提取蛋黄中全部脂类，进一步采用乙酸乙酯在0 ℃下提纯蛋黄总脂中的PL。经脱盐、冷冻干燥后，IgY、PV和PL的纯度分别为97.38%、78.63%和85.94%。IgY和PV的得率分别为6.15、10.15 mg/mL。蛋黄总脂中PL的含量为35.18%。因其良好的多不饱和脂肪酸/饱和脂肪酸比率（0.70）和相对较低的n-6/n-3比率（5.37），该PL产品可应用于食品加工中。本方法简便、环保并且高效，可从蛋黄中依次分离出高纯度IgY、PV和PL，并为蛋黄的综合利用提供技术支持。     

低温乙醇除脂法纯化鸡卵黄中免疫球蛋白

采用低温乙醇除脂法提取鸡卵黄免疫球蛋白 (immunoglobulinyolk,IgY)。通过低温乙醇沉淀、透析和葡聚糖凝胶等不同方法逐步纯化免疫球蛋白。研究了卵黄稀释倍数、乙醇浓度、NaCl浓度以及离心转速对纯化效果的影响。分离纯化后所得的冷冻干燥制品 ,经SDS PAGE和抑菌试验检测可知 :可溶性蛋白质得率为每毫升蛋黄 1 2 6mg ,可溶性蛋白质含量占总蛋白质量的 97% ,且保持了免疫球蛋白的活性     

蛋黄免疫球蛋白的制备与应用

介绍蛋黄免疫球蛋白(IgY)的形成、特性、免疫程序、分离、提取、检测、应用等,重点为蛋黄免疫球蛋白的制备工艺。     

卵黄免疫球蛋白的分离提取与鉴定

对氯仿法、辛酸法、水稀释法、水-辛酸法分离卵黄免疫球蛋白(IgY)进行了比较,结果表明:水-辛酸法在9倍稀释度、pH5.3、辛酸加量1%条件下,可获得较好效果,进一步用硫酸铵沉淀脱盐后,提取率达93.4%,产率达4.67mg/mL卵黄,纯度达85%。     

抗幽门螺杆菌卵黄抗体的制备及其效价的评定

本论文研究了抗幽门螺杆菌蛋黄抗体(IgY—HP)的制备方法。以HP超声粉碎上清和HP尿素酶混合抗原物免疫鸡蛋，从蛋黄中提取纯化得到了很高纯度的IgY—HP。ELISA实验表明，IgY—HP的抗HP效价和一般IgY相比提高了32倍。其可以应用于食品保健或各种HP相关胃病的抗菌免疫治疗中。     

酸奶双菌发酵过程对不同来源IgG的影响

研究了酸奶嗜热链球菌和保加利亚乳杆菌双菌混合发酵过程对牛初乳IgG，牛血IgG，猪血IgG和鸡蛋黄IgY的影响。聚丙烯酰胺凝胶电泳(SDS-PAGE)证实，常规的双茵发酵不会致使上述4种IgG分子降解，分别采用琼脂单向免疫扩散法(RID)和酶联免疫吸附法(ELISA)对牛初乳、牛血IgG及蛋黄IgY组分作定量分析，发现这些成分保持了原有免疫反应活性。虽然添加不同IgG基料后均可制备含有活性IgG的凝固型酸奶，但显著改变双茵发酵过程中乳源蛋白质的降解模式，添加牛血IgG，猪血IgG或者鸡蛋类IgY基料后，αs-酪蛋白和β-酪蛋白相应为发生降解的主要乳源蛋白。     

抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌

采用水稀释法提纯抗志贺氏菌IgY,检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320,并以此IgY为基础建立间接ELISA检测志贺氏菌,测定志贺氏菌纯培养液的检出限为105～106cfu/mL。对蜡样芽孢杆菌等10株不同菌株的检测结果表明,该方法对志贺氏菌有明显的检测特异性,对所测定的其它菌株无交叉反应。     

抗人大肠杆菌蛋黄抗体的分离纯化及理化性质研究

以人源大肠杆菌(ATCC23985)免疫产蛋母鸡所产免疫蛋为原料，通过蛋黄水提物制备、乙醇分级沉淀、离子交换色谱和凝胶排阻色谱对其中的免疫蛋黄抗体进行了分离纯化，并对其基本理化性质进行了分析。结果表明：蒸馏水-氯仿法是制备蛋黄水提物的最优方法，而35％的乙醇添加量能够很好地将水提物中的抗体沉淀出来。经DEAE-Sepharose柱和SephacrylS-200柱连续纯化的抗体在电泳检测时只有1个区带。研究获得的抗人大肠杆菌卵黄抗体的等电点为5.5，包含分子质量分别为65900u和24600u的2类亚基。该抗体的氨基酸组成与普通蛋黄抗体的氨基酸组成差异不大。     

抗大肠杆菌O157:H7的卵黄特异性IgY的研究

以大肠杆菌O157:H7为抗原免疫产蛋母鸡,从鸡卵黄中提取免疫球蛋白,建立抗大肠杆菌O157:H7的特异性IgY的效价检测方法,并研究母鸡的免疫应答性,以及抗体的提取方法和体外抑菌效果.研究结果表明,初次免疫后第6d,在卵黄中可以检测到抗大肠杆菌O157:H7 IgY,效价为1:7200;经加强免疫后效价迅速上升,至第44d达到最高效价1:230400;免疫后360 d,效价仍维持在1:7200.用水稀释法、硫酸铵分级盐析和Sephadex G-25凝胶过滤以提取IgY,提纯后IgY的效价是之前的4倍.SDS-PAGE鉴定抗体的纯度,电泳图谱中出现抗体的轻链和重链两条带.体外抑菌实验表明,IgY能抑制大肠杆菌O157:H7的生长.     

酶联免疫吸附法检测动物源性食品中恩诺沙星残留量

为检测动物源性食品中恩诺沙星残留量,评估基于卵黄抗体的间接竞争酶联免疫吸附法检测恩诺沙星的可行性,用活性脂法将恩诺沙星同卵清蛋白偶联制备免疫原和包被抗原并用紫外光谱进行验证。用聚乙二醇-6000提取卵黄抗体。五免之后效价达到峰值1∶32 000。用间接竞争酶联免疫吸附法确定包被原质量浓度、卵黄抗体的稀释倍数和IC_(50)分别为38 ng/m L、1∶64 000和18.207 ng/m L,回归曲线方程为y=0.891 1-0.016 5x(R~2=0.990)。结果表明,制备的抗恩诺沙星卵黄抗体为进一步建立检测动物源性食品中恩诺沙星的残留的免疫方法提供参考依据。     

检测赭曲霉毒素A(OTA)的酶联免疫吸附法(ELISA)体系的建立

本文分别以自制抗-OTA兔血清抗体(IgG)及鸡卵黄抗体(IgY)就影响检测OTA的ELISA(酶联免疫吸附法)体系因素进行了探讨,建立了检测OTA的ELISA方法。鸡卵黄抗体的最佳ELISA操作参数为:包被抗原浓度为7.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为1000,酶标二抗稀释倍数为14000;兔血清抗体的最佳ELISA操作参数为:包被抗原浓度为2.5μg/ml,封阻剂浓度为2.5%,抗体稀释倍数为8000,酶标二抗稀释倍数为10000。IgG和IgY的检测灵敏度分别为10ng/ml和1ng/ml。     

Growth Inhibitory Effect of Chicken Egg Yolk Antibody (IgY) on Escherichia coli O157:H7

Escherichia coli O157:H7‐specific antibodies (immunoglobulin Y [IgY]) were isolated by the water‐dilution method from the egg yolk of chickens that were immunized with E. coli O157:H7 whole cells. The specific‐binding activity of IgY against E. coli O157:H7 as determined by the enzyme immuno assay showed high levels of activity against bacterial whole cells. IgY binding activity was further demonstrated to have an inhibitory effect on E. coli O157:H7 growth in a liquid medium. The antibacterial function of IgY appeared to result from the interaction of IgY with surface components of E. coli O157:H7, as proven from observation of immunofluorescence and immunoelectron microscopy.     

Immunoglobulins from Egg Yolk: Isolation and Purification

Simple water dilution was employed for the separation of water-soluble plasma proteins from egg yolk granules. An optimum recovery of immunoglobulin Y (IgY, 93–96%) in water-soluble fraction was obtained by sixfold water dilution at pH between 5.0–5.2 with incubation time of 6 hr at 4°C. Among the factors studied, pH was found to be the most important factor affecting IgY recovery. Active IgY of high purity with good recovery was obtained by a combination of several techniques including salt precipitation, alcohol precipitation, ultrafiltration, gel filtration and ion exchange chromatography. Salt precipitation, ultrafiltration, and get filtration is the recommended sequence. Over 100 mg of electrophoretically pure IgY was routinely obtained from one egg.     

Preparation of Artificial Antigen and Development of Indirect Competitive ELISA Based on Chicken IgY for the Detection of Acid Orange II in Food Samples

To evaluate the feasibility of specific egg yolk antibodies (IgY) technology for the detection of hazardous chemical residues in foodstuffs, IgY were generated to detect acid orange II (AO2) residues. Bovine serum albumin (BSA) was made to bind with AO2 by succinic anhydride (SA), and the conjugate was used to immunize the laying chickens. PEG-6000 precipitation was used to extract IgY antibodies. The titer of anti-AO2 IgY attained the peak (1:12,800) after fifth booster immunization. Checkerboard titration showed that 1:640 dilution of anti-AO2 IgY could approximately give an optical density (OD) 1.0 at 5 μg/mL AO2-OVA coating concentration. The anti-AO2 IgY based indirect competitive ELISA (ic-ELISA) showed that the IC₅₀ value of anti-AO2 IgY was 10.72 μg/mL and regression curve equation was y?=?24.41?×?+75.11 (R ²?=?0.946). The ic-ELISA showed less cross-reactivity in range between 0.09 and 21.07 %. Recoveries from dried bean curd, sausage, and chilli powder samples were from 78.80 to 152.90 %, with relative standard deviation lower than 19.13 %. The study suggests that generated anti-AO2 IgY antibodies could be used in routine screening analysis of AO2 residues in food samples.     

GROWTH INHIBITION OF CLOSTRIDIUM PERFRINGENS VEGETATIVE CELLS AND SPORES USING CHICKEN IMMUNOGLOBULIN Y

Egg yolk antibody (IgY) was isolated by the water dilution method from the egg yolk of chickens immunized with Clostridium perfringens vegetative cells and spores. Specific binding activity of IgY against C. perfringens vegetative cells and spores remained relatively high during the immunization period (up to 9 weeks). The titer of specific IgY against C. perfringens spores was 1.4-fold less than that of specific IgY against C. perfringens vegetable cells. The specific IgY powder (10 µg/mL) was found to inhibit the growth of C. perfringens vegetative cells or C. perfringens spores in a liquid medium. The difference of C. perfringens vegetative cell growth between the treatment and control groups was 8.9  ×  10⁶ colony forming units (cfu)/mL at 8 h of incubation and 9.95  ×  10⁷ cfu/mL at 24 h of incubation. Significant cfu reductions in C. perfringens spores were also observed with specific IgY powder at 24 h of incubation.

PRACTICAL APPLICATIONS

IgY antibody exerts an antimicrobial activity against pathogens by binding, immobilizing and consequently reducing or inhibiting the growth, replication or colony forming ability of pathogenic bacteria; thus, it is proved to be a viable alternative for antibiotics and preservatives. In this study, IgY against Clostridium perfringens can be used to replace chemical preservatives in food industries. Because IgY functions well at low temperature, it can be used to inhibit the growth of Clostridia which germinate in refrigerated storage conditions, thus preventing foodborne enterotoxicity caused by such bacteria. In practical applications, natural antimicrobial IgY antibody can be applied to meat products for the improvement of food safety.     

冰乙醇分级离心法分离纯化鸡卵黄免疫球蛋白(IgY)的研究

采用冰乙醇分级离心法提取鸡卵黄免疫球蛋白 (Immunoglobulin of egg yolk,IgY).通过低温乙醇沉淀、DEAE 葡萄糖凝胶 A-50 离子交换层析、Tris-HCl 缓冲液洗脱及 Sephadex G200 凝胶过滤等逐步纯化免疫球蛋白.研究卵黄稀释倍数、pH 值、乙醇添加量以及盐浓度对纯化效果的影响.分离纯化后所得的提取物经检测 IgY 纯度可达98%,且很好地保持了免疫球蛋白的活性.