吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂稳定性的研究

将连续生产并已申报人体接种反应观察的三批吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂,分别于４～８℃、１８～２２℃、３７℃贮存半年、１年、１年半和３０天,然后以小鼠法或豚鼠法对该制剂进行安全稳定性、效力稳定性以及毒性逆转等测试。其毒力安全稳定性试验表明,三批混合制剂中的精制百日咳菌苗在４～８℃贮半年、１年、１年半后、它们的ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ、ＨＳＵ／ｍｌ均无显著性差异（Ｐ＞０．０５）;白喉及破伤风类毒素在４～８℃贮存半年,１年、１年半以及１８～２２℃贮存半年和３７℃贮存３０天后,三批测试组的豚鼠均健存且体重增加。在效力稳定性试验中,４～８℃贮存１年和１年半后精制百日咳菌苗的效力与贮存前（分别为２６．７３ＩＵ／ｍｌ、２１．０２ＩＵ／ｍｌ、１９．１７ＩＵ／ｍｌ）无显著性差异（ｔ＝０．１１４;Ｐ＞０．０５）;白喉及破伤风类毒素４～８℃贮存长达１年半后,效力仍在２～３．２ＩＵ／ｍｌ,动物保护率仍可达８０～１００％。三批混合制剂在３７℃贮存３０天后,通过Ｓｃｈｉｃｋ毒性试验及测定ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ、ＨＳＵ／ｍｌ各指标显示,精制百日咳菌苗、白喉和破伤风类毒素无毒性逆转。说明该吸附精制百、白、破混合     

关于吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂的现况

本文介绍了现行吸附百日咳菌苗、白喉、破伤风类毒素混合制剂的现况和新一代吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂人体接种反应和血清学效果观察的结果。     

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——吸附精制百日咳菌苗——吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂——吸附精制百日咳菌苗、白喉类毒素混合制剂     

吸附精制百日咳菌苗毒性逆转试验报告

吸附精制百日咳菌苗毒性逆转试验报告史天绪,陈爱荣,秦力,何长民（卫生部兰州生物制品研究所,兰州７３００４６）目前已知,吸附精制百日咳菌苗或吸附精制百日咳、白喉、破伤风类毒素混合制剂（简称ＡＤＴＰ）和吸附精制百日咳、白喉类毒素混合制剂（简称ＡＤＰ）,如...     

吸附精制百日咳菌苗稳定性的研究

本文对新一代吸附精制百日咳菌苗保存于不同温度条件下的安全和效力稳定性进行了实验,结果表明,９批吸附精制百日咳菌苗经贮存于４～８℃条件下半年、１年、２年和３年后,贮存于常温（１８～２２℃）半年后和贮放于３７℃４０天后,其贮存前后的ＢＷＤＵ／ｍｌ、ＬＰＵ／ｍｌ和ＨＳＵ／ｍｌ间的数值变化,均在制检规程要求的标准数值内,无显著性差异（Ｐ＞０．０５）,说明菌苗的毒性无逆转、菌苗的安全性良好。３批吸附精制百日咳菌苗经贮存于４～８℃条件下７个月和２年后、其贮存前后的效力单位间的数值变化,均在制检规程要求的标准数值内,无显著性差异（Ｐ＞０．０５）,说明菌苗的效力无下降,菌苗的质量稳定。     

吸附精制百日咳菌苗、白喉类毒素混合制剂人体接种反应和血清学效果观察报告

在陕西省大荔县三个村222名4～6岁健康儿童中进行吸附精制百日咳菌苗。白喉类毒素混合制剂加强免疫。结果显示,新一代的吸附精制百、白混合制剂接种后没有出现局部硬结反应,而老百、白混合制剂则出现3例,占接种人数的2.86％。血清学测定,新百、白混合制剂接种后产生的四种免疫抗体水平,其中百日咳凝集素水平免后较免前升高33倍、抗LPF抗体升高468倍、抗FHA抗体升高104倍、抗白喉抗体分布于1.0～8.0HAU/ml之间,≥1.0HAU/ml者为100％。上述四种抗体用平均超过抗百日咳和抗白喉感染的保护水平,说明新研制的吸附精制百、白混合制剂是对人体接种反应轻微、免疫效果良好的优质制剂。     

百日咳、白喉、破伤风、乙型肝炎联合疫苗的实验研究

对制备无细胞百日咳菌苗、白喉、破伤风、乙型肝炎联合疫苗的实验室条件进行了初步探索,实验结果表明,联合疫苗的配方以每毫升无细胞百日咳组分１５－１８μｇ．ＰＮ、白喉类毒素３０ｌｆ、破伤风类毒素７－１０ｌｆ和基因工程乙肝表面抗原２０μｇ为宜,稀释缓冲液选用０．８５％ＮａＣｌ溶液吸附效果较好,动物实验证明联合疫苗中各组分均安全有效。     

吸附无细胞百白破(DPTa)的稳定性试验

采用半综合液体培养基（含０．０５％ＭｅβＣＤ）,大罐培养百日咳Ⅰ相ＣＳ菌制备的吸附无细胞百日咳菌苗、白喉、破伤风类毒素混合制剂（ＤＰＴａ）置４－８℃分别保存一年、保存两年,测三种抗原成分的效力及毒性,以及将该制剂于３７℃分别放置三周、三个月,测定有无毒性逆转。结果表明,该制剂质量稳定,无毒性逆转     

吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂人体接种反应和血清学效果观察报告

在陕西省大荔县所辖的五个乡(镇)、二个工厂319名3～6月龄婴幼儿中进行吸附精制百日咳菌苗、白喉、破伤风类毒素混合制剂人体接种反应及血清学效果观察。从人体接种反应观察结果中看出,新研制的吸附精制百、白、破混合制剂的全身反应中没有出现中、强反应,而老百、白、破混合制剂则出现中反应9例(占接种人数的8.6％)、强反应2例(占0.92％);局部反应中前者中反应1例(占0.96％),后者为10例(占9.28％);局部硬结反应中,前者没有出现中、强反应,而后者出现5例(占4.58％)。说明新研制的吸附精制百、白、破混合制剂的接种反应极为轻微,安全性上明显地优于老百、白、破混合制剂。血清学测定结果表明,吸附精制百、白、破混合制剂免疫后产生出五种抗体应答,百日咳凝集素水平≥1:320者为73％,免疫后较免疫前增长167.18倍(P相似文献   

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     

精制破伤风类毒素产毒及精制工艺的改进

用超滤、硫酸铵二段盐析法取代等电点沉淀法后,精制破伤风类毒素（精破类）的纯度由８０７Ｌｆ／ｍｇＰＮ提高到１８８３Ｌｆ／ｍｇＰＮ,纯度提高一倍以上。使用双胨培养基取代酪素培养基后,产毒水平由４７Ｌｆ／ｍｌ提高到８８Ｌｆ／ｍｌ（ｔ＝６．４６,ｐ＜０．００１）;用新法精制后,精破类纯度分别为１９４９Ｌｆ／ｍｇＰＮ及１７８５Ｌｆ／ｍｇＰＮ（ｔ＝０．３３４,ｐ＞０．０５）,引用双胨培养基后可提高产毒水平,但不影响精破类的纯度。     

Subacute toxicity of adsorbed 250 Lf tetanus toxoid in rats

Since the naturally acquired tetanus antitoxin titre in Indian population is mostly less than protective levels, to minimise the incidence of morbidity, Glaxo Laboratories (India) Ltd., Bombay have prepared a single dose potent tetanus vaccine of 250 Lf as compared to 5 Lf previous tetanus toxoid. Subacute toxicity study revealed that this toxoid (250 Lf) injected at different dose levels (1-2 ml/rat/injection x 4) is non-toxic to rats.     

The immunization of adults against diphtheria in Sweden

Two hundred and three women who disclaimed vaccination against diphtheria were divided into four groups and injected with either 2.0 or 6.25 Lf of a routine diphtheria toxoid or of a more purified preparation. One hundred and twenty-six of these women who did not show a secondary antibody response were given a second and a third injection one month and one year, respectively, after the first injection. Prebooster (third injection) antitoxin titres of greater than or equal to 0.01 IU ml-1 (the minimum level for protection) were found in 22 and 37% of those who received 2.0 and 6.25 Lf, respectively. Postbooster titres of greater than or equal to 1.0 IU ml-1 (calculated to give a protection of at least ten years of duration) were found in 23 and 58% of those who received 2.0 and 6.25 Lf, respectively. The rate of untoward reactions was low. Fever of short duration occurred in five women. Four out of the five women received 6.25 Lf of the more purified diphtheria toxoid and one 2 Lf of the routine toxoid. Local reactions greater than 10 cm were observed in three women. All received the higher dose, 6.25 Lf of diphtheria toxoid. Local reactions greater than 5 but less than or equal to 10 cm occurred in up to 13% (6.25 Lf of diphtheria toxoid). No significant difference between the groups of women vaccinated with routine or more purified toxoid was found. It was concluded that the diphtheria toxoids in the two doses of 2 Lf and 6.25 Lf did not induce a satisfactory immune response. To induce adequate protection the dose of diphtheria vaccine needs to be the same for adults and children, i.e. 12.5 Lf.     

Optimization of diphtheria toxin production by <Emphasis Type="Italic">Corynebacterium diphtheriae</Emphasis> using a casein-based medium in a fermenter

The Td-based combined vaccine contains only small amounts of the diphtheria toxoid antigen. However, a high level of purity is necessary for this antigen. The diphtheria toxin is produced by growing Corynebacterium diphtheriae in a semisynthetic, casein-based medium in a fermenter. In order to obtain a highly pure diphtheria toxoid, the optimal conditions to express the toxin at 300 Lf/mL in a fermenter culture were determined. When C. diphtheriae was cultivated in a fermenter and a high concentration of toxin was obtained, specific patterns for the pH and dissolved oxygen levels identified. Overall, the fermenter cultivation process was divided into four stages according to variations in the pH. A specific range of K _La in the fermenter (0.0092 ~ 0.0093/sec) was required to produce high level expression of diphtheria toxin. The amount of toxin expression varied significantly according to culture conditions. Agitation and aeration in the fermenter affected toxin expression, even when the optimal K _La value for toxin production was maintained. A previous study has reported that the amounts of agitation and aeration are important factors when cultivating fungus in the fermenter to produce chitinolytic enzyme. A mass production of diphtheria toxoid with a purity level greater than 2,500 Lf/ mgPN was obtained through purification and detoxification from this optimized toxin production.     

Optimization of the formulation for a candidate lyophilised tetanus toxoid reference preparation

Tetanus toxoid is a vital primary reference material used for standardization of assays required to establish the antigenic purity of tetanus toxoid for vaccine production. Several formulations were assessed and ampouled fills of each formulation lyophilised. The relative Lf content determined by Ramon flocculation, SRD, and ELISA assays was measured. The stability of the tetanus toxoid activity in each formulation was assessed by accelerated degradation studies. Formulations containing glycine were not suitable in flocculation tests but both sorbitol and trehalose formulations were. The trehalose/sodium chloride formulation had a good appearance, showed good activity in all assays and maintained its activity best under stress conditions. This formulation has been applied to a large scale batch of ampoules prepared as a WHO candidate replacement standard, evaluated in a collaborative study and accepted as a replacement WHO IS for use in flocculation test (WHO ECBS, October 2007, ref no BS/07.2061). The stability of this formulation was also excellent for the large scale batch. The benefits of using thermal analysis and freeze drying microscopy coupled with small scale lyophilisation trials in order to screen formulations for the preparation of batches of biological reference materials are demonstrated.     

Comparative immunologic effectiveness of variants of the DTP vaccine

The immunological effectiveness of two batches of adsorbed DPT vaccine, the batch with the normal content of antigens (control) and the batch with the content of diphtheria and tetanus toxoids reduced to 20 Lf/ml and 5 BU/ml respectively (test batch), has been studied under the conditions of controlled trial. As a result, the reduction of the antigenic content of adsorbed DPT vaccine has been found to exert no negative influence on the immunological effectiveness of the diphtheria, tetanus, and pertussis components of this preparation under the conditions of the new immunization schedule.     

Use of monoclonal antibodies against horse immunoglobulin in an enzyme immunoassay of bacterial toxins and toxoids

Immunization of BALB/c mice by horse antiserum against diphtheria made it possible to obtain IgG₁ monoclonal antibodies (MoAbs) 2B7E4 specific for light chains of horse immunoglobulin (Ig). Unlike commercial preparations of anti-horse immunoglobulin antibodies, which are specific for the whole Ig molecule or its Fc-fragment, the peroxidase (HRP) conjugate of the MoAb, 2B7E4-HRP did not interact with human, mouse, rabbit, and sheep Igs, or horse albumin. The conjugate obtained was used with MoAbs against bacterial toxins and commercial horse antitoxins, as a universal reagent in sandwich enzyme immunoassay (ELISA) for bacterial toxins and toxoids. The detection sensitivity of diphtheria toxin/toxoid equaled 0.0005 Lf/ml; tetanus toxin and toxoid were detected with sensitivities of 20 LD₅₀/ml and 0.005 UI/ml, respectively. A similar sandwich ELISA for botulinum toxoids (group measurement) allowed types A, B, and E to be detected at 0.02, 0.002, and 0.001 UI/ml, respectively; selective measurement was only possible in the case of type E toxoid (0.001 UI/ml).     

On the relation between toxin production and protein synthesis byCorynebacterium diphtheriae and the iron content of the culture medium

Summary An investigation of the quantitative relationship between toxin production and protein synthesis byC. diphtheriae and the iron content of the medium has been reported on. Both functions can be expressed quantitatively,viz., Lf/ml and Lf/mg P.N.Both functions appeared to be narrowly related to the iron concentration of the medium. The optimal Fe-concentration for the greatest purity of the toxin (Lf/mg P.N.) lies, however, lower than that for the maximal yield of toxin (Lf/ml).In connection with the fact that for the subsequent purification of the toxoid to be obtained from the diphtheria toxin, starting from as pure as possible raw material is essential, a somewhat lower production of toxin than an optimal iron concentration might yield, has to be accepted.