应用Vero细胞检定法和家兔皮内中和法检测吸附白喉类毒素的…

Ｖｅｒｏ细胞法测定ＤＴＰ疫苗中白喉类毒素的效价与家兔皮内中和法所得结果两法之间有一种平行关系,并且有很好的相 关性,ｒ＝０．９３。但ｅｒｔｏ细胞法测得平均值低于ＮＴ法,二者之间的平均比率普０．２８７,Ｓ＝０．０８９。     

狂犬病病毒Vero细胞适应株的建立及其应用于疫苗生产的可行性探讨

将狂犬病病毒７ａＧ固定毒适应于Ｖｅｒｏ细胞,建立了稳定的ＶＲＧ适应株。病毒滴度可达７．６９ｌｏｇＬＤ５０／ｍｌ,病毒最适增殖时间５—７天,最适种毒比例１：１０￣３。应用转瓶培养Ｖｅｒｏ细胞增殖病毒,收获病毒液经β—丙内脂灭活,超离浓缩制备疫苗,效力试验结果（ＮＩＨ法）较原制疫苗成倍增高,且都大于２．５ＩＵ／２ｍｌ,表明ＶＲＧ适应株可应用于制备Ｖｅｒｏ细胞狂犬病疫苗。     

基因工程干扰素（IFN—α2b）抗柯萨奇和单纯疱疹病毒效应

本文采用微量细胞病变抑制法在Ｗｉｓｈ 及Ｖｅｒｏ 细胞上研究了ＩＦＮ—α２ｂ 抗ＣｏｘＢ３ 型及ＨＳＶ—１ 型和ＨＳＶ—Ⅱ型病毒的作用。结果表明,安达芬和干扰能ＩＨＮα２ｂ 在Ｗｉｓｈ 或Ｖｅｒｏ 细胞上５００ＩＵ／ｍｌ 分别可以抗３ｌｏｇ４ 或１０００ＴＣＩＤ５０ ,５０ＩＵ／ｍｌ 可以抗２ｌｏｇ４ 或１００ ＴＣＩＤ５０ ,５ＩＵ／ｍｌ 可以抗１ｌｏｇ４ 或１０ ＴＣＩＤ５０ 的ＣｏｘＢ３ 型或ＨＳＶ—１ 型和ＨＳＶ—Ⅱ型病毒感染细胞。提示安达芬和干扰能均显示明显的抗病毒效应,且二者无明显差别     

浓缩地鼠肾细胞狂犬病疫苗与纯化Vero细胞狂犬病疫苗接种 …

为了解国产浓缩地鼠肾细胞狂犬疫苗与法国纯化Ｖｅｒｏ细胞狂犬疫苗接种人体后中和抗体产生情况。分别用两种疫苗接种１０人,用小鼠中和试验方法检测中和抗体滴度。国产疫苗五针全程免疫后３０天（第一针按种后６０天）可１００％达到保护水平,法国疫苗在第一针接种后３０天１００％达到保护水平。第一针接种后１４、３０、６０天时,前者抗体平均滴度分别是０．０６ＩＵ／ｍｌ、１０２ＩＵ／ｍｌ、２．０７ＩＵ／ｍｌ;后者抗体平     

免疫印迹法在实验大鼠汉坦病毒监测中的应用及与间接免疫荧光法之比较

用汉坦病毒汉滩株（７６－１１８）重组核蛋白作为免疫印迹法（ＷｅｓｔｅｒｎＢｌｏｔ以下简称ＷＢ）的诊断抗原,用于实验感染大鼠血清抗体效价测定。同时与用汉城株（ＳＲ－１１）感染的Ｖｅｒｏ－Ｅ６细胞作抗原的间接免疫荧光法（以下简称ＩＦＡ）进行比较。ＷＢ法对３／４标本在大鼠接种病毒后第３天测得血清ＩｇＭ阳性,而ＩＦＡ法仅１／４标本出现阳性,ＩＦＡ效价为１：５１２０的血清,ＷＢ效价为１’：４０９６０,且在血清１：１０稀释时反应带亦清晰。两种方法分别测定６４份大鼠血清。甩ＩＦＡ法,４４份（６８．８％）出现类似阳性的荧光颗粒,而用ＷＢ法测定,无特异的反应带出现。非感染Ｖｅｒｏ－Ｅ６细胞作ＩＦＡ抗原,３０份（４６．９％）与正常细胞抗原有反应,此结果表明ＷＢ法在特异性和敏感性方面均高于ＩＦＡ法。ＩＦＡ法中的非特异性反应系血清与细胞成份之反应。     

无细胞百日咳菌苗毒性试验参考苗制备条件的探讨

将不同灭活条件下制备的无细胞百日咳毒性试验参考苗进行了检测。结果表明,以浓度０．１％Ｆｏｒｍａｌｉｎ溶液２５℃灭活９６－１２０小时制备的无细胞百日咳菌苗毒性试验参考苗,凝集效价仍可达到百日咳Ⅰ相血清原效价;不耐热毒素试验呈阴性;毒性试验ＢＷＤＵ／ｍｌ为７５．９－１２８．４;ＬＰＵ／ｍｌ为２．１－６．０;ＨＳＵ／ｍｌ为３．９－５．７;稳定性良好。该苗可标化作为无细胞百日咳菌苗毒性试验的参考标准     

影响CCID50法测定麻疹活疫苗效力的因素

对麻疹活疫苗效力测定ＣＣＩＤ５０法的某些影响因素如：滴定用细胞的敏感性、培养液的酸碱度、培养板放孵的温度等进行了试验分析。结果表明：１）滴定用细胞的敏感性对试验结果有显著影响。Ｖｅｒｏ细胞比ＦＬ细胞敏感得多（均差＝０．５０９ＬｇＣＣＩＤ５０／ｍｌ）。２）在用ＦＬ细胞滴定时,培养液的酸碱度及培养温度则对试验结果的影响不显著。３）在常规生产中,采用Ｖｅｒｏ细胞进行麻疹活疫苗的效力试验,提高了检定的敏感性和合格率。     

聚合破伤风类毒素抗原特性的研究

由戊二醛脱毒的聚合破伤风类毒素,经高压液相层析及聚丙烯酰胺凝胶电泳分析,类毒素中多聚体含量占８１．９％以上,多聚体分子量为８００ｋＤ,常规破类多聚体仅占有２．２４％。聚合破类免疫豚鼠后,平均心血抗体单位达２ＩＵ／ｍｌ,常规破类仅为０．７５ＩＵ／ｍｌ（Ｔ＝１３．１５,Ｐ＜０．００１）,聚合破类免疫马匹后所诱发的抗体水平较常规抗原的高。     

螺旋藻对小鼠SOD和GSH—Px活力的影响

采用微量测定法,观察螺旋藻对３２只昆明种小白鼠全血中超氧化物歧化酶（ＳＯＤ）和谷光甘肽过氧化物酶（ＧＳＨ－Ｐｘ）活性的影响。结果表明,灌胃螺旋藻试验组（ＳＯＤ）活性（１５７７．１６±１６９．８８ＩＵ／ｇＨｂ）,与相应对照组（１３３６．２７±１５８．２３ＩＵ／ｇＨｂ）比较,ＧＳＨ－Ｐｘ活性（２８．３３±２．３７ＩＵ／ｍｌ）与相应对照组（２４．８７±３．２６ＩＵ／ｍｌ）比较,差别均有非常显著意义（Ｐ＜０．０１）;提示螺旋藻有提高动物ＳＯＤ和ＧＳＨ－Ｐｘ活性的功效     

二氢玉米素核苷组细胞分裂素的放射免疫测定法

本文报道二氢玉米素核苷（ＤＨＺＲ）组细胞分裂素放射免疫测定法（ＲＩＡ）的研制结果。以（３Ｈ）二氢玉米素作示踪剂,测得免抗ＤＨＺＲ抗血清的效价为１：１３７０（Ｂ％＝３０％）。该抗血清主要与本组细胞分裂素发生交叉反应。回收率为９６．９％,灵敏度为１２ｆｍｏｌ ＤＨＺＲ／管,检测线性范围为０．１￣１００ｐｍｏｌＤＨＺＲ／管。批内误差ＣＶ＝４．３,批间误差ＣＶ＝２．０％。对基于同一免抗血清的ＤＨＺＲ组细胞     

Vero细胞法检测白喉抗毒素的研究

参照Ｍｉｙａｍｕｒａ等报道,建立了微量细胞培养检测白喉抗毒素的方法。以家兔皮肤试验为参照,Ｖｅｒｏ细胞培养法敏感度为９８．１１％,特异度为８４．００％,符合率为９６．２０％,相关系数ｒ＝０．９３,在白喉血清抗毒素测定中值得推广     

Immunity to diphtheria, six to 15 years after a basic three-dose immunization schedule

The results of a study of the immunity to diphtheria of 283 girls (9-18 years of age) vaccinated at the age of two years with three doses of vaccine, are reported. The rabbit skin test was used to determine the titre of serum diphtheria antitoxin. 55.8% of the subjects were found to be protected (titre greater than or equal to 0.1 IU/ml), 38.9% were only relatively immune (titre greater than or equal to 0.01- less than 0.01 IU/ml), and 5.3% were unprotected (titre less than 0.01 IU/ml). The antitoxin titres showed a tendency to decrease with time. Even so, 6-15 years after vaccination, the percentages of protected and partially protected subjects were still high (95%).     

The use of the Toxin Binding Inhibition (ToBI) test for the estimation of the potency of the diphtheria component of vaccines

The Toxin Binding Inhibition (ToBI) test, previously developed for the estimation of diphtheria and tetanus antitoxin in human sera, was adapted for the estimation of the potency of diphtheria components in vaccines. Data are presented to show that antitoxin titres of individual sera of mice obtained by the ToBI test are in good agreement with those obtained in the Vero cell test. In addition, diphtheria potency and 95% confidence interval of twelve batches of vaccine in different compositions were estimated by the ToBI test and the results were compared with those obtained in Vero cells. A significant correlation could be demonstrated. It is concluded from this study that the ToBI test is a valuable model in the potency assay of diphtheria toxoids, based on antitoxin induction in mice.     

Combined estimation of tetanus and diphtheria antitoxin in human sera by the in vitro Toxin-Binding Inhibition (ToBI) test

The use of the principle of inhibition of toxin binding to an antitoxin coated immunoassay plate as described in a previous paper for tetanus antitoxin titration, was adapted for the estimation of diphtheria antitoxin in human sera. With a few modifications, a Toxin-Binding Inhibition (ToBI) test was developed which could be used for a combined estimation of both tetanus and diphtheria antitoxin levels. The application of streptavidin-biotinylated peroxidase complex when using small serum samples (less than 50 microliters) is discussed. Antitoxin titres (both diphtheria and tetanus) of 0.002 IU ml-1 were detectable by the ToBI test, this being far below the level considered to be protective in man. Sera from 140 adults with different vaccination histories were titrated for both tetanus and diphtheria antitoxin. Good correlations were found between the estimates obtained by the ToBI test and those obtained by the toxin-neutralization (TN) test in mice (tetanus antitoxin) and those obtained in the in vitro neutralization test in VERO cells (diphtheria antitoxin). It is concluded that the ToBI test is a simple and reliable alternative to the functional models currently in use for the estimation of diphtheria and tetanus antitoxin levels. In addition, the ToBI test eliminates the need for laboratory-animal or cell-culture facilities and can be performed with small quantities of serum as required in field trials.     

Microtissue Culture Test for the Titration of Low Concentrations of Diphtheria Antitoxin in Minimal Amounts of Human Sera

A microtissue culture method for the assay of low concentrations of diphtheria antitoxin in human sera has been developed, using a monkey kidney cell (VERO) culture technique. Results obtained with sera from nonvaccinated children and with immune sera from children vaccinated with three and four injections of diphtheria pertussis tetanus vaccine were in agreement with antitoxin levels considered necessary to denote immunity to diphtheria. The use of microplates and organic buffer for culturing the animal cells improved the stability of the tissue culture system. The described method is sensitive, economical, and applicable for the titration of antitoxin in human sera particularly from infants and children from whom a minimum amount of serum is available.     

Dynamic changes of horse serum T-globulin immunization with snake venoms, tetanus and diphtheria toxoids

In course of immunizing horses with snake venoms, tetanus and diphtheria toxoids, a new serum component, T-globulin, was formed and migrated between the beta- and gamma-globulins. The T-globulin content was parallel with the antibody titre after the middle course of immunization. There were many components in snake antivenin and T-globulin was composed of most of those components. The components of diphtheria T-globulin were the same as those of crude antitoxin and tetanus T-globulin except one precipitin.     

The reduction of animal usage by the application of indirect haemagglutination in the potency testing of diphtheria toxoid in combined vaccines

Eight Adsorbed Diphtheria-Tetanus vaccines and 13 Diphtheria-Tetanus-Pertussis vaccines made by four different manufacturers were tested for the potency of the diphtheria components in guinea-pigs by the method of British Pharmacopoeia (1973). Two-hundred-and-ten guinea-pig sera consisting of ten sera related to each vaccine sample thus obtained were titrated for diphtheria antitoxin by indirect haemagglutination (IHA) and the conventional toxin neutralization (TN) tests. Statistical analysis of the results showed a good correlation between the titres obtained with the two tests. The potencies of the diphtheria components of various vaccines calculated from the antitoxin content of the respective guinea-pig sera titrated by the IHA test correlated significantly with the potencies obtained from the antitoxin content titrated by the routinely used TN test. The use of IHA in place of the TN test thus offers as an alternative that permits a reduction in animal usage.     

Potency control of diphtheria component in adsorbed vaccines by in vitro neutralization tests.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.     

Tissue Culture Method for Toxigenicity Testing of Corynebacterium diphtheriae

A method for toxigenicity testing of Corynebacterium diphtheriae in tissue cultures was developed. Results were obtained by comparing destruction of the monkey kidney or, preferably, rabbit kidney monolayer by 0.1 ml of the C. diphtheriae culture in Elek's broth containing 20% rabbit serum with the appearance after the addition of 0.2 ml of a mixture of the C. diphtheriae culture and diphtheria antitoxin. The mixture of C. diphtheriae broth culture and 10 antitoxin units per ml was incubated for 1 hr at room temperature before it was added to the tissue cultures which were then incubated as long as 5 days; most results, however, were read in 72 hr. Elek's broth medium was superior to heart infusion broth for toxin production by C. diphtheriae. Addition of 20% rabbit serum improved toxin production in either broth. Numerous toxigenic and atoxigenic C. diphtheriae cultures were tested for toxigenicity in primary rabbit and monkey kidney tissue cultures. If properly controlled, this in vitro method appeared to have an advantage over the in vitro agar gel method; its results were comparable with the rabbit intradermal test. With the wider use of tissue cultures in most laboratories, we believe that the tissue culture method for toxigenicity would be more economical and easier to perform than the animal intradermal method.     

The immunization of children with combined diphtheria and tetanus vaccine in Sweden

Two groups derived from 97 children three-four months of age were vaccinated with diphtheria and tetanus vaccines containing either a routinely prepared diphtheria toxoid or a more purified preparation. Two injections were given with an interval of one month and a third injection was given one year after the first. Prior to the third injection no child was without protection against diphtheria, i.e. had an antitoxin titre less than 0.01 IU ml-1. After the third injection 95 and 94% of the children vaccinated with the routinely and more purified diphtheria toxoids, respectively, had diphtheria antitoxin titres greater than 1 IU ml-1 (estimated to provide protection for at least ten years). Systemic reactions such as fever and malaise occurred in five children. Local reactions greater than 10 cm were observed in three children and reactions greater than 5 but less than or equal to 10 cm were seen in 14% of the children. The routinely prepared combined diphtheria and tetanus vaccine, DT, produced very good immunity against diphtheria with moderate side effects. The use of a more purified diphtheria toxoid in the combined vaccine produced the same immunity and side effects.