ELISA 法用于马抗狂犬病毒抗体的检测

本文建立了ＥＬＩＳＡ间接法,用以检测精制（马）抗狂犬病血清（或血浆）制造过程中的中和抗体效价,并与传统的小鼠脑内中和法比较。结果表明（１）两种检测方法对不同水平抗体的检出是一致的。它们之间有很好的线性关系（ｙ＝２．１７ｘ＋２１,ｒ＝０．９９,ｐ＜０．０１）。（２）应用ＥＬＩＳＡ间接法测定马抗狂犬病血清效价简便、快速、特异性及重复性好,可代替小鼠脑内中和法。     

流行性出血热免疫球蛋白的研制

本文首次报告采用纯化灭活流行性出血热（ＥＨＦ）Ⅰ型疫苗免疫健康献血员,采集ＥＨＦ抗体高滴度的血浆,用低温乙醇法及盐析法分离纯化三批ＥＨＦ免疫球蛋白。结果表明：（１）采用０,１,３,４（月）或０,１,４,５（月）免疫程序,疫苗剂量１～２ｍｌ（０．１５～０．３０ｍｇ蛋白）,受免献血员血清平均抗体滴度可达１∶４０６（ＥＬＩＳＡ）或１∶１１２（ＲＰＨＩ）。（２）通过生化检定,三批制品的电泳纯度为９７．０５％,９６．８４％,９９．２６％;ＩｇＧ单体和二聚体含量为８９．５５％,９１．３０％和９８．２１％。（３）用空斑抑制中和试验及免疫印染试验证明所纯化的免疫球蛋白具有抗ＥＨＦ病毒特异性。（４）三批ＥＨＦ免疫球蛋白的效价测定,结果为ＥＬＩＳＡ滴度≥１∶５１２,ＲＰＨＩ滴度≥１∶１０２４,ＰＲＮＴ（中和抗体）滴度１∶４０。按１６％蛋白计,ＥＨＦ免疫球蛋白的效价可达原料血浆的１０倍以上。（５）无菌、安全、毒性及热原质试验检定结果,全部通过《中国生物制品规程》要求。     

破伤风类毒素抗体酶联双抗原夹心法定量检测试剂的研制及评价

研制破伤风类毒素抗体酶联双抗原夹心法定量检测试剂,用于破伤风免疫血浆抗体效价检测。以精制破伤风类毒素经Sephacryl S-300柱层析纯化后作为包被抗原,用辣根过氧化物酶以改良过碘酸钠法标记精制破伤风类毒素作为酶标记抗原,以破伤风人免疫球蛋白国家标准品采用小鼠中和试验法标定试剂盒定量标准品,制备双抗原夹心法定量检测试剂;进行试剂盒检测范围、特异性、重复性、精密度及稳定性考核,并与小鼠中和试验法、琼脂双扩散法及国外破伤风类毒素抗体酶联试剂盒进行比较。结果显示,试剂盒的检测范围为10~150mIU/ml,灵敏度为10mIU/ml,线性好(r>0.996),板内孔间变异度小(CV<8%),特异性强(100%),重复性好(CV<13%),于37℃放置6天测定结果无明显差异,与小鼠中和试验法、英国Biding Site酶联试剂有良好的一致性。试验证明所研制的试剂盒适用于破伤风免疫血浆中的破伤风抗体效价定量检测。     

抗绿脓杆菌外毒素 A 血浆的研制

试验制备了抗绿脓杆菌外毒素－Ａ血浆,使用马匹免疫抗原为精制ＰＡ－１０３菌株的外毒素－Ａ。抗血浆效价第一程采血达到了２０８８单位／ｍｌ,第三程采血达到５１８６单位／ｍｌ。     

絮状单位测定与小鼠体内中和试验法检测TAT效价的比较

目的为了更好地进行生产质量控制,快速准确地测定破伤风抗毒素(TAT)效价,以统计分析絮状单位测定法(体外法)和小鼠中和试验法(体内法)测定TAT效价的相关性。方法对2010—2013年破伤风抗毒素生产过程中的原料血浆、原液的絮状单位值与小鼠中和效价进行了统计分析和比较。结果体外法和体内法检测TAT原料血浆和原液效价的相关系数分别为r血浆=0.794 3,P0.001和r原液=0.901 6,P0.001,均呈显著性正相关。结论体外法可指导或替代体内法TAT效价测定应用于生产过程中间品的质量控制。     

中枢应用β-内啡肽对大鼠血浆唾液酸水平的影响以及与免疫功能的关系

实验选择体重２００－２５０ｇ健康Ｗｉｓｔａｒ大鼠６４只,采用麻醉大鼠中枢微量注射,分光光测定法及免疫组织化学法,研究侧脑室,弓状核（ＡＲＣ）区注射β－内啡肽（β－ＥＰ）对大鼠血浆唾液酸（ＳＡ）水平的影响及与免疫功能的关系,结果表明：（１）侧脑室注射β－ＥＰ可明显降低血浆ＳＡ水平;（２）血浆ＳＡ水平在ＡＲＣ区注射β－ＥＰ后明显降低,此效应可被Ｍ胆碱受体阻断剂阿托品或切断双侧颈迷走神经所阻断;（３）Ａ     

绿脓杆菌外毒素A及其抗毒素

绿脓杆菌外毒素Ａ（ＰＥＡ）是绿脓杆菌的主要致病因子之一。本文仅就ＰＥＡ的产生、纯化、分子结构、生物学活性、检测方法，ＰＥＡ抗毒素产生，ＰＥＡ及其抗毒素的应用前景做了简要介绍。     

神经毒素原性酪酸梭菌与E型肉毒梭菌毒素的精制及特性比较

对首次自Ｅ型肉毒中毒食品中分离到的一株神经毒素原性酪酸梭菌（ＬＣＬ１５５）所产生的神经毒素,同Ｅ型肉毒梭菌（Ｅ１５３）所产生的神经毒素进行了精制及特性比较,发现（１）两菌神经毒素的分子量,Ｎａｔｉｖｅ－ＰＡＧＥ测试均为３２０ｋＤａ;ＳＤＳ－ＰＡＧＥ测试则均为１４７ｋＤａ,非毒性非血凝素部分均为１２８ｋＤａ;用胰蛋白酶激活神经毒素后发现两菌神经毒素均由分子量为１０３ｋＤａ的Ｈ链和４８ｋＤａ的Ｌ链组成。（２）两菌神经毒素柱层析图像基本一致,但在菌体毒素提取效果及精制效果诸方面,分离的酪酸梭菌却都较差。（３）胰蛋白酶激活试验表明：两菌神经毒素达到最大毒力所需激活时间不等。在相同温度下,分离的酪酸梭菌毒素只需５ｍｉｎ,而Ｅ型肉毒梭菌毒素却需３０ｍｉｎ,提示两菌神经毒素激活动力学上存在差异。（４）琼脂双扩散试验结果表明两菌神经毒素的抗原性是一致的,没有发现沉淀线呈交叉或部分交叉现象。     

黄曲霉毒素B_1的免疫检测 Ⅱ.抗体的产生及应用

将黄曲霉毒素Ｂ１肟（ＡＦＢ１Ｏ）与牛血清白蛋白（ＢＳＡ）的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第６０天开始有较明显抗体产生,第１２０天达到高峰,维持１５天左右后开始下降;抗体的ＥＬＩＳＡ效价高达１：３００００;和黄曲霉毒素Ｂ１（ＡＦＢ１）的结构类似物的竞争ＥＬＩＳＡ表明,抗体有很好的特异性。运用该抗体,以ＥＬＩＳＡ分析检测了几种农产品及饲料中污染ＡＦＢ１的含量,并和薄层层析法的分析结构进行了比较,结果表明当ＡＦＢ１的含量大于等于５ｎｇ／ｍｌ时,两者间有很好的相关性。     

蛇毒蛋白C激活物的初步研究

目的：研究蝮蛇毒蛋白Ｃ激活物的分离纯化与理化性质。方法：经ＤＥ５２－纤维素、ＣＭ－Ｓｐｈａｄｅｘ Ｃ－５０、Ｇ－７５柱层析,从安徽芜湖产蝮蛇（Ａｇｋｉｓｔｒｏｄｏｎ ｈａｌｙｓ）蛇毒中纯化一种均一的蛋白Ｃ激活物（ＰＣＡ）。结果：ＳＤＳ－ＰＡＧＥ测定分子量约为１５５０００Ｄａ,ＩＥＦ－ＰＡＧＥ测定等电点为４．８。它能使人血浆的ＫＰＴＴ明显延长,显示出强烈的抗凝活性。通过中和试验与显色肽定量实验表明,     

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30–50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab′)₂ fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl). A peak containing primarily the F(ab′)₂ antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.     

Improved method for purification of Xenopus laevis vitellogenin and demonstration of cross-reactivity among wide-range species by radioimmunoassay

This study was performed to improve the purification of Xenopus vitellogenin and establish the radioimmunoassay. The procedure of purification consisted of ammonium precipitation, DEAE-Sephadex chromatography and Sephadex G-200 gel chromatography. Using this procedure, 934 mg vitellogenin was purified from 49 ml of estradiol treated female Xenopus plasma (about 19 mg/ml). Vitellogenins purified from male and female plasma after a single injection of estradiol showed good correspondence in electrophoretic patterns and amino acid compositions, indicating that vitellogenin synthesis in the male occurs in four different genes as in the female. The radioimmunoassay for vitellogenin was established using an antibody in the plasma obtained from rabbits injected with purified Xenopus female vitellogenin. The titer was 20,000 times dilution of the plasma, and the minimum detectable amount of vitellogenin was 0.1 microgram. The cross-reactivity of this antibody with newt vitellogenin was about 65% and that of chick 6%. The cross-reaction was also observed in female bullfrog plasma. Vitellogenin content was increased gradually during the first 6 days after injection of estradiol in female and the elevated level of vitellogenin dropped afterward.     

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.     

A型肉毒毒素Hc结构域在大肠杆菌中的高水平表达及免疫原性

对A型肉毒毒素受体结合区Hc基因的全序列进行优化和人工合成,获得了全长1287bp,编码429aa的Hc基因。以pTIG-Trx为原核表达载体,实现了Hc在大肠杆菌中的高效可溶性表达及纯化,该表达水平可占可溶性全菌总蛋白的36%～53%,经一步亲和层析纯化可获得电泳级纯度的目的蛋白,在常规培养条件下,产量达到30mg/L以上。然后,纯化的重组蛋白Hc免疫小鼠后能够诱导产生高滴度特异性的抗体,也能诱导产生特异性的细胞免疫应答反应。小鼠体内A型肉毒毒素中和试验结果表明免疫组小鼠血清中含高滴度的体内中和抗体。结果表明,利用本实验的原核表达系统不仅能够高水平可溶性地表达A型肉毒毒素受体结合区Hc,而且重组Hc具有良好的免疫原性,可以用于制备治疗性抗毒素和作为亚单位候选疫苗用于预防A型肉毒毒素中毒。     

小麦根质膜H^＋—ATPase的部分纯化

以小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）根为材料,采用不连续蔗糖密度梯度离心法制备高纯度质膜微囊。质膜经ＴｒｉｔｏｎＸ１００和ＫＣｌ处理后,再用Ｚｗｉｔｅｒｇｅｎｔ３１４增溶Ｈ＋ＡＴＰａｓｅ,最后用硫酸铵沉淀得到部分纯化的质膜Ｈ＋ＡＴＰａｓｅ。ＳＤＳＰＡＧＥ结果表明,经过上述步骤纯化,分子量为９４ｋＤ的膜蛋白组分得到富集;与质膜相比,其含量提高１５．７倍。部分纯化的质膜Ｈ＋ＡＴＰａｓｅ可以水解ＡＴＰ,受Ｋ＋刺激,并被Ｎ,Ｎ′ｄｉｃｙｃｌｏｈｅｘｙｌｃａｒｂｏｄｉｍｉｄｅ（ＤＣＣＤ）抑制;ＡＴＰ水解活力被Ｎａ３ＶＯ４抑制９５％,但不被ＮａＮ３、ＮａＮＯ３和Ｎａ２ＭｏＯ４抑制。     

Titration of Cholera Antitoxin in Human Sera by Microhemagglutination with Formalinized Erythrocytes

Microtiter hemagglutination tests employing formalinized sheep erythrocytes sensitized with either crude or purified cholera toxin were used to assay the cholera antitoxin content of human sera. Comparable results were obtained with either crude or purified toxin-sensitized cells with the exception of two sera that gave unusually high hemagglutination titers with the crude toxin. Sera from 13 convalescent cholera patients showed a high degree of correlation between antitoxin levels as determined in vitro by the hemagglutination test and in vivo by the skin permeability factor neutralization test. Fourfold or greater rises in antitoxin levels between acute and convalescent sera were detected in 9 of 15 patients with bacteriologically proven cholera. No significant increases in titer were observed in 14 cases of noncholera diarrhea. Cholera antitoxin was detected by hemagglutination in only 1 of 33 sera, obtained from eight countries, containing vibriocidal antibodies. Formalinized sheep erythrocytes sensitized with toxin and stored at 4 C in the presence of 1:10,000 thimerosal were stable and sensitive for at least 6 months (the longest time tested).     

Monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase and the application of enzyme purification

A monoclonal antibody against the catalytic subunit of bovine heart cAMP-dependent protein kinase has been prepared. The antibody, M73/PK1, belongs to the heavy-chain subclass IgG3 and has a titer of 20,480. Purified M73/PK1 was obtained by ammonium sulfate precipitation and Protein A-agarose affinity chromatography and was coupled to CNBr-activated Sepharose 6MB. The catalytic subunit was purified by this affinity column. A 75% yield and 4746-fold purification were obtained. These results show that this is a suitable and effective method for the purification of the catalytic subunit of bovine heart cAMP-dependent protein kinase.     

Purification of equine IgG using membrane based enhanced hybrid bioseparation technique: a potential method for manufacturing hyperimmune antibody

Hyperimmune equine IgG is widely used as antivenom and anti-rabies agents. This article discusses a membrane based enhanced hybrid bioseparation technique for efficient and scalable purification of equine immunoglobulin G (IgG) from horse serum. This technique is an improved version of a standard hybrid bioseparation technique developed within our group earlier for fractionation of human plasma proteins (Ghosh. 2004. J Membr Sci 237: 109-117). In the presence of a high antichaotropic salt concentration, equine IgG is selectively and reversibly captured within a stirred cell membrane module from horse serum, partly due to precipitation and microfiltration, and partly due to hydrophobic interaction based membrane adsorption, while the impurities are washed out from the device. The reversibly sequestered IgG is then released by lowering the salt concentration which favor both dissolution of the precipitated IgG and desorption of the membrane bound IgG. The enhanced hybrid bioseparation technique improves the IgG recovery from the membrane module by switching from a stirring to non-stirring mode during the IgG release phase. It also reduces membrane fouling by an appropriate pH switch. The effects of operating conditions on equine IgG capture were first systematically studied. The enhanced hybrid bioseparation technique was followed by an ultrafiltration step to remove ammonium sulfate and low molecular weight impurities. The equine IgG purity obtained under optimized conditions was 88% and its recovery was over 90%, both being significantly higher than corresponding values obtained using currently used purification techniques.     

Mode of inhibition of diphtheria toxin by ammonium chloride

Kim, K. (University of Washington, Seattle), and N. B. Groman. Mode of inhibition of diphtheria toxin by ammonium chloride. J. Bacteriol. 90:1557-1562. 1965.-The inhibition of diphtheria toxin by ammonium salts was independent of toxin concentration over a 100-fold range of toxin. Inhibition by minimal concentrations of ammonium chloride was abolished by lowering the pH, indicating that free ammonia is the active form of inhibitor. A single addition of ammonium chloride inhibited toxin for a limited period of time, but periodic readdition of the ammonium salt was required to sustain inhibition indefinitely in the absence of antitoxin. Toxin was not destroyed and its adsorption occurred equally well in the presence or absence of ammonium chloride. Preadsorbed toxin was also effectively inhibited by the addition of ammonium chloride. Inhibited toxin remained accessible to antitoxin neutralization. Attempts to reverse ammonia inhibition by the addition of succinate or reduced nicotinamide adenine dinucleotide were unsuccessful. Attempts to inhibit toxin by interfering with active transport were also unsuccessful.     

Progress in Tetanus Prophylaxis: The Advent of Human Antitoxin

Injections of tetanus antitoxin of animal origin frequently cause serious disability and sometimes death. Despite world-wide knowledge of these effects, millions of prophylactic injections of equine tetanus antitoxin are given annually, and it is continually proposed that the dosage be increased in order to obtain higher “protective” levels in the serum, a procedure which would increase the incidence and severity of reactions. Furthermore, equine antitoxin frequently fails to prevent tetanus.Tetanus antitoxin of human origin is available which carries no risk of complications and confers a higher degree of immunity more quickly than equine antitoxin. The cost of treating reactions to horse serum, together with the financial loss incurred by work-absence, far outweighs the cost of human antitoxin. In the author''s opinion, the use, in this country, of antitoxin of animal origin is no longer medically acceptable and may well prove legally indefensible.