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。     

AOT-异辛烷反胶束萃取技术分离牛初乳IgG的研究

在一个琥珀酸二(2-乙基己基)酯磺酸钠(AOT)-异辛烷反胶束体系中，研究了水相pH值、离子强度和有机相表面活性剂AOT浓度等因素对牛初乳乳清蛋白中IgG分离效果的影响。结果表明，在以下两组优化条件：①pH值为7．668，[Na^ ]浓度为0．270mol／L，[AOT]浓度为0．088mol／L；②pH值为6524，[Na^ ]浓度为0．205mol／L，[AOT]浓度为0．175mol／L，水相IgG的残留率或纯度分别达到最大值，分别为90．23％和98．35％．RID分析表明，反胶束萃取过程对原牛初乳乳清IgG的免疫反应活性基本无影响。     

鸡蛋黄中天然活性物质的开发与利用

鸡蛋中蛋白质、脂类、碳水化合物、维生素、矿物质等营养成分含量丰富,是人类饮食的重要组成部分。鸡蛋黄中的蛋黄油、卵磷脂、卵黄高磷蛋白、免疫球蛋白等多种成分具有抑菌、抗氧化、提高免疫力、消炎、防癌等生理功能。目前,蛋黄中生物活性成分及其生产新技术的开发已经成为国内外研究的热点,部分成果已经实现了商业化应用。如何充分利用蛋中的活性物质并将其开发成具有治疗、保健功能的原料是蛋品科学与加工技术研究的一个主要课题。本文以蛋黄油、卵磷脂、卵黄高磷蛋白、免疫球蛋白4种物质的开发为主线,就鸡蛋黄中天然活性物质的含义、制备方法、生理功能、开发利用情况进行综述,以期为蛋黄中活性物质的进一步研究开发提供参考。     

腐败希瓦氏菌卵黄抗体活性片段的制备及抑菌效果的分析

为探讨卵黄抗体活性片段的制备方法,利用胃蛋白酶对腐败希瓦氏菌卵黄抗体进行酶解,通过高效液相色谱法动态分析酶解过程,建立了一套有效的纯化方法,并对活性片段抑菌效果进行鉴定分析。结果表明,经胃蛋白酶作用6h后,卵黄抗体被酶解为43000u左右及其他更小分子量的蛋白片段,且组分不再随时间发生变化。酶解混合液经Sephacryl S-100层析纯化后,即得纯度大于90%的活性蛋白片段。此蛋白片段保留了较高的抗原结合能力,且抑菌效果明显。研究为制备高纯度的卵黄抗体活性片段提供有效方法,为进一步扩大腐败希瓦氏菌卵黄抗体应用范围奠定理论基础。      

On hen egg fractionation: applications of liquid chromatography to the isolation and the purification of hen egg white and egg yolk proteins

Liquid chromatography has been used as a means of egg protein analysis or as a method for the purification of egg proteins. Several Chromatographic methods, including gel permeation, ion-exchange, reversed-phase, hydrophobic interaction, and immobilized-ligand-affinity chromatography, have been carried out for the separation or the purification of egg yolk or egg white proteins. Ion-exchange chromatography appears to be the most frequently used method for protein isolation and it is the easiest to adapt to a process scale. From an analytical point of view reversed-phase chromatography is, at the moment, the recommended method for egg white analysis. Egg white has been fractionated more often by liquid chromatography than has egg yolk. Several Chromatographic methods have been developed on a laboratory scale, but the application of these techniques on an industrial scale remains limited.     

鸡卵黄免疫球蛋白(IgY)分离纯化的研究

对鸡卵黄免疫球蛋白(IgY)的分离纯化进行了研究.工艺流程为:卵黄→8倍水稀释并搅拌10 min→用HCl调Ph至5.2后在4 ℃静置2 h去沉淀→在上清夜中加入95 %冰乙醇(-20 ℃)使终浓度为60 %(v/v)→4 ℃搅拌20 min后离心(22000 r/min,4 ℃,25 min)→收集沉淀并加入0.028 mol/L NaCl溶液在4 ℃下过滤除去脂蛋白沉淀→收集的滤液用SDS-PAGE法进行纯化得纯度为98 %的1gY.用大肠杆菌(E.coli)对其活性进行测定,效果良好,此方法能保持免疫球蛋白的活性.     

Suppressive effect of functional drinking yogurt containing specific egg yolk immunoglobulin on Helicobacter pylori in humans

Helicobacter pylori is a human pathogen that infects over 50% of the population worldwide. It is the most important etiologic agent of gastroduodenal ulcers and malignancies. Helicobacter pylori urease enzyme is considered the main factor for the organism's colonization in the gastroduodenal mucosa. Hens immunized with the purified urease produce a highly specific anti-H. pylori urease immunoglobulin (IgY-urease) in their egg yolks. Immunoglobulin Y-urease was stable at 60 to 65 degrees C for 30 min and at pH 4.0 for 7 h. Its activity was lost at 80 degrees C for 20 min and at pH 2 for 4 h. Specially designed functional drinking yogurt containing Lactobacillus acidophilus and Bifidobacterium spp. with 1% egg yolk IgY-urease was produced commercially. Immunoglobulin Y-urease activity showed stability in the product up to 7 d, and then decreased to 85% after 3 wk of storage. A clinical study was conducted to determine the effectiveness of IgY-urease yogurt to suppress infection in humans. Forty-two volunteers who tested positive for H. pylori using a 13C-urea breath test were recruited. A total of 450 mL of IgY-urease (test group) or IgY-urease-free yogurt (control group) was consumed in 150-mL portions 3 times daily for 4 wk. Volunteers were tested after 2 and 4 wk; urea breath test values significantly decreased in the test group compared with the control group. The results indicate that suppression of H. pylori infection in humans could be achieved by consumption of drinking yogurt fortified with IgY-urease.     

利用核磁共振技术研究食盐对鸭蛋黄品质的影响

利用低场核磁共振及其成像技术研究食盐对鸭蛋黄品质影响的作用机制。在不同食盐添加量条件下,测定蛋黄质构特性、可溶性蛋白含量和出油率的变化,并分析蛋黄弛豫特性分别与质构特性和出油率之间的相关性。结果表明:食盐对蛋黄质构特性及弛豫特性有显著性影响,且随着食盐添加量的增加呈现规律性变化,而随着食盐添加量的增加,蛋黄可溶性蛋白含量先增加后下降,出油率先下降后上升,并分析得出蛋黄弛豫特性与蛋黄质构特性和出油率均呈显著相关性。表明食盐会改变鸭蛋黄内部氢质子的迁移及分布,导致鸭蛋黄质构特性及出油率发生改变,进而影响鸭蛋黄的品质。      

Thermal Stability of Bovine Milk Immunoglobulin G (IgG) and the Effect of Added Thermal Protectants on the Stability

D and z values and some thermodynamic parameters of immunoglobulin G (IgG) in phosphate buffer solution (PBS) (0.15 M NaCl/0.01 M phosphate buffer, pH 7.0) and colostral whey, with or without the presence of thermal protectants, were calculated in model systems. The D and z values for separated IgG in PBS were much lower than those for separated IgG in 20% glycerol, whey, and whey with 20% glycerol. IgG in colostral whey showed larger D and z values with the protectants. The heat denaturing rate constants at 70-82°C for separated IgG in PBS were larger than those of IgG in colostral whey; and the energies of activation for separated IgG in PBS, 0.2% glutamic acid, 10% whole milk, 20% maltose and 20% glycerol were also larger.     

牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立

将牛免疫球蛋白G（immunoglobulin G,IgG）作为免疫原免疫BALB/c小鼠,通过细胞融合、筛选获得分泌抗牛IgG单克隆抗体的细胞株。制备小鼠腹水抗体,进一步纯化获得抗牛IgG单克隆抗体。建立双抗夹心酶联免疫吸附法检测牛初乳中IgG质量浓度,该方法在7.8～1 000 ng/mL范围内有良好的线性关系,最低检出限为7.06 ng/mL,批内变异系数为4.52%,批间变异系数为4.94%,回收率为91.85%～102.45%。此法操作简便、准确度高、稳定性好,可用于实际牛初乳样品的快速检测。     

Short communication: Fractional milking distribution of immunoglobulin G and other constituents in colostrum

The provision of quality colostrum with a high concentration of immunoglobulins is critical for newborn calf health. Because first colostrum may be low in overall concentration to effectively reduce the risk of newborn infections, we tested equivalent milking fractions of colostrum for possible IgG differences. The objective of this study was to determine if the fractional composition of colostrum changes during the course of milking with a focus on immunoglobulins. Twenty-four Holstein and Simmental cows were milked (first colostrum) within 4 h after calving. The colostrum of 1 gland per animal was assembled into 4 percentage fractions over the course of milking: 0 to 25%, 25 to 50%, 50 to 75%, and 75 to 100%. The IgG concentration among the various fractions did not change in any significant pattern. Concentration of protein, casein, lactose and somatic cell count remained the same or exhibited only minor changes during the course of fractional milking colostrum. We determined that no benefit exists in feeding any particular fraction of colostrum to the newborn.     

Effect of heat treatment of bovine colostrum on bacterial counts, viscosity, and immunoglobulin G concentration

A study was conducted to identify the optimal temperature and time at which heat treatment of bovine colostrum would least change viscosity and IgG concentrations yet reduce bacterial count. First-milking colostrum with >50 g of immunoglobulins/L (measured by colostrometer) was collected from 30 Holstein cows. Aliquots of colostrum were heated for 0, 30, 60, or 90 min at 57, 60, or 63°C in a water bath. Samples were examined for viscosity, IgG₁, and IgG₂ concentrations, standard plate count, coagulase-negative staphylococci, environmental streptococci, coliform, gram-negative noncoliform, Streptococcus agalactiae, and Staphylococcus aureus counts. All heat treatments reduced counts of all bacteria groups measured compared with untreated colostrum samples. Heat treatment at ≥60°C denatured IgG₁ compared with untreated colostrum; however, colostral IgG₂ levels were not reduced when temperature was held at 60°C for <60 min. Viscosity was not affected when temperature was held at 60°C for <60 min. In this study, heat treatment of bovine colostrum at 60°C for 30 or 60 min reduced bacterial count, slightly reduced IgG concentration, and did not affect viscosity.     

Flow injection chemiluminescence determination of femtogram-level cobalt in egg yolk,fish tissue and human serum

Femtogram-level cobalt was determined based on its significantly catalyzed effect on luminol-dissolved oxygen chemiluminescence (CL) reaction in the flow system. The increment of CL signal was proportional to the concentration of cobalt, giving linear range from 10 fg ml⁻¹ to 50 pg ml⁻¹ (r² = 0.9992) with a detection limit 4 fg ml⁻¹ (3σ). At a flow rate of 2.0 ml min⁻¹, a typical analytical procedure for cobalt, including sampling and washing, could be performed in 0.5 min with a relative standard deviation of less than 3.0%. The proposed method has been successfully applied for the determination of cobalt in egg yolk, fish tissue and human serum, agreed well with radioimmunoassay.     

Effect of different heating times of high-, medium-, and low-quality colostrum on immunoglobulin G absorption in dairy calves

The objective of this study was to determine the effect of different durations of heat treatment on passive transfer of IgG from high-, medium- and low-quality colostrum. Colostrum was collected from The Pennsylvania State University dairy herd and divided by quality (high, medium, or low) based on colostrometer measurements. Colostrum was pooled by quality to create 3 unique batches. Each batch was further divided in thirds as follows: frozen to be fed without heat treatment, heated at 60°C for 30 min, or heated at 60°C for 60 min. Colostrum samples from each treatment were collected and analyzed for standard plate count, gram-negative noncoliforms, coliforms, and total IgG concentration. Serum samples were collected from 108 Holstein calves before feeding colostrum and 24 h after birth. Blood samples were analyzed for total protein, total IgG, and hematocrit. Colostrum quality (high, medium, or low), heat treatment (unheated, 60°C for 30 min or, 60°C for 60 min), and their interaction were analyzed as fixed effects, with calf sex included as a random block effect. Colostrum IgG was different between quality groups (92.5, 59.4, and 48.1 mg/mL of IgG). Heating colostrum reduced IgG concentration compared with the control by 9% when heated for 30 min and by 12% when heated for 60 min. Colostrum heated for 60 min had a lower standard plate count than colostrum heated for 30 min or not heated (1.8, 2.0, and 3.6 log cfu/mL, respectively). Serum IgG concentration at 24 h increased as colostrum quality increased (18.0, 22.2, and 24.8 mg/mL) and tended to increase as heat treatment time increased (19.7, 20.3, and 25.0 mg/mL of IgG). Apparent efficiency of IgG absorption was greater in calves that received medium-quality colostrum compared with calves fed high-quality colostrum (38.1 and 25.0%, respectively). These results suggest an upper limit may exist to the amount of IgG absorption in a given time period and that medium- or high-quality colostrum yields similar blood IgG concentration given the same volume of intake.