CN1971275B - Method for detecting content of free polysaccharides in polysaccharides combo of pneumonia streptococcus 6B, 18C, 19F and 23F type - Google Patents

Method for detecting content of free polysaccharides in polysaccharides combo of pneumonia streptococcus 6B, 18C, 19F and 23F type Download PDF

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CN1971275B
CN1971275B CN 200610048879 CN200610048879A CN1971275B CN 1971275 B CN1971275 B CN 1971275B CN 200610048879 CN200610048879 CN 200610048879 CN 200610048879 A CN200610048879 A CN 200610048879A CN 1971275 B CN1971275 B CN 1971275B
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polysaccharide
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supernatant
streptococcus pneumoniae
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CN1971275A (en
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吴凯
黄镇
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云南沃森生物技术股份有限公司
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Abstract

The invention relates to content measuring method of free polysaccharides in pneumococcus 6B type, 18C type, 19F type, 23F type polysaccharide vinculum which belongs to biological technique field. The steps are following: the pneumococcoal polysaccharide vinculum is the sample 1. anhydrous alcohol and sodium chloride are added into another pneumococcoal polysaccharide vinculum with the same quantity, it is laid for 70-90h in -20DEG C, supernatant is collected centrifugally as the sample 2; The pneumococcoal polysaccharide vinculum is washed and precipitated adequately with proper solvent, supernatant is collected centrifugally as the sample 3; the content of phosphorus in the sample 1, 2 and 3 are detected, the content of free polysaccharides in the pneumococcoal polysaccharide vinculum is calculated as formulas. The invention can detect the content of free polysaccharides in the pneumococcus 6B type, 18C type, 19F type, 23F type polysaccharide vinculum accurately and easily to evaluate the quality of item.

Description

肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离 Streptococcus pneumoniae type 6B, 18C type, 19F type, 23F polysaccharide conjugate free

多糖含量的测定方法 Determination of polysaccharide content

技术领域: FIELD:

[0001] 本发明属于生物技术领域,更具体地,涉及一种肺炎链球菌多糖结合物中游离多糖含量的测定方法。 [0001] The present invention belongs to the field of biotechnology, and more particularly, to a method for measuring one kind of Streptococcus pneumoniae polysaccharide conjugate polysaccharide content of free.

背景技术: Background technique:

[0002] 肺炎链球菌是诱发细菌性肺炎、脑膜炎、胸膜炎、心内膜炎、中耳炎的重要病原菌, 是发展中国家儿童细菌性肺炎最常见的病因。 [0002] Streptococcus pneumoniae is an important pathogen-induced bacterial pneumonia, meningitis, pleurisy, endocarditis, otitis media, in developing countries the most common cause of bacterial pneumonia in children. 全球每年死于肺炎球菌感染者有100-200 万。 Worldwide die each year from pneumococcal infection 100-200 million. 中国肺炎发病率一直很高,近50年来,肺炎球菌感染者中菌血症死亡率一直徘徊在25-¾%之间。 China has a high incidence of pneumonia in the past 50 years, pneumococcal bacteremia infection in the mortality rate has been hovering between 25-¾%. 此外肺炎也是导致60岁以上老人死亡的主要原因之一。 In addition pneumonia is the leading cause of death in the elderly over 60 years. 由于肺炎链球菌耐药菌株高达96%以上,目前已成为世界性的公共问题。 Because drug-resistant strains of Streptococcus pneumoniae as high as 96%, has become a worldwide public issue.

[0003] 1983年美国成功研制出23价多糖疫苗,但该疫苗在2岁以下婴幼儿中诱导的保护性低,无免疫记忆反应。 [0003] In 1983 the United States successfully developed the 23-valent polysaccharide vaccine, but the vaccine in infants under 2 years of age to induce protective low, no immune memory response. 以化学方法将蛋白载体共价结合到肺炎链球菌多糖上,制备成肺炎链球菌多糖结合疫苗,将多糖由T细胞非依赖性抗原转变为T细胞依赖性抗原。 Chemically covalently bound to a protein carrier to the polysaccharide of Streptococcus pneumoniae, Streptococcus pneumoniae polysaccharide conjugate prepared as a vaccine, the polysaccharide change from T-independent antigens to T cell-dependent antigens. 接种多糖-蛋白结合疫苗后,可使2岁以下婴幼儿产生有效的免疫应答,从而获得免疫保护。 Polysaccharide vaccination - after protein conjugate vaccine in infants under 2 years old can produce an effective immune response, thus obtaining immunity protection. 初步的临床试验结果表明,肺炎链球菌多糖结合疫苗在婴幼儿中的安全性和免疫原性良好,诱导产生的抗多糖抗体高于多糖疫苗,并可诱导免疫记忆反应,所以肺炎链球菌多糖结合疫苗的研制具有重要的社会意义和价值。 Preliminary clinical trial results show that good Streptococcus pneumoniae polysaccharide conjugate vaccine safety and immunogenicity in infants, the anti-polysaccharide antibody induced by higher than polysaccharide vaccine can induce immune memory response, so the combination of Streptococcus pneumoniae polysaccharide vaccine development has important social meaning and value.

[0004] 多糖结合疫苗中的游离多糖含量超过规定的限度会对疫苗临床使用的效果产生不利的影响,因此多糖蛋白结合物中游离多糖含量测定是多糖结合疫苗质量控制中的关键指标之一,也是反映结合疫苗质量的优劣的主要指标之一。 [0004] The polysaccharide conjugate vaccine clinical effect will limit the use of the free polysaccharide content exceeds a predetermined vaccine adversely affected, thus polysaccharide-protein conjugate Determination of free polysaccharide content is a key indicator of polysaccharide conjugate vaccine quality control, one of the main indicators also reflect the quality of the vaccine in conjunction with the merits.

[0005] 因为不同细菌多糖以及不同多糖结合物性质差异较大,所以多糖结合疫苗中的游离多糖测定是一个技术难点,目前尚无多糖结合物中游离多糖含量测定的通用方法。 [0005] Since various different bacterial polysaccharides and polysaccharide conjugate is quite different, so the free polysaccharide assay polysaccharide conjugate vaccine is a technical difficulty, the general method of determination of the polysaccharide content is currently no free polysaccharide conjugate. 国外文献报道采用高效液相色谱法进行细菌多糖结合物中游离多糖测定,一种采用高效凝胶过滤,一种采用高效阴离子交换色谱。 Foreign literature using high performance liquid chromatography bacterial polysaccharide conjugate free polysaccharide assay A high performance gel filtration using a high-efficiency anion exchange chromatography. 高效凝胶过滤法基于分子大小差异来区分结合多糖与游离多糖,易受到分子大小相近杂质的影响,测定结果误差较大;高效阴离子交换色谱基于分子带电荷性质的差异而区分结合多糖与游离多糖,也易受到与多糖带电荷性质相似的杂质的影响,此外肺炎链球菌多糖在紫外区域没有强的光吸收,无法在配置紫外检测器的高效液相系统上测定肺炎链球菌多糖结合物的游离多糖含量。 High performance gel filtration based on molecular size difference to distinguish between free and bound polysaccharide polysaccharide molecular size similar susceptible impurities, the results of measurement error is large; high performance anion exchange chromatography with differences in molecular charge characteristics are distinguished based on the polysaccharide binding free polysaccharide It is also susceptible to the charged nature of the impurities similar polysaccharide, Streptococcus pneumoniae polysaccharide in addition there is no strong light absorption in the ultraviolet region, not the determination of free Streptococcus pneumoniae polysaccharide conjugate on the configuration HPLC system with UV detection polysaccharide content. 目前尚无采用乙醇沉淀法和磷含量测定法测定肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离多糖含量的报道。 Determination of Streptococcus pneumoniae type 6B is currently no method by ethanol precipitation assays and phosphorus content, type 18C, 19F type, 23F polysaccharide conjugate polysaccharide content of free coverage.

发明内容: SUMMARY:

[0006] 本发明的方法克服了现有方法无法准确测定肺炎链球菌多糖结合物游离多糖含量的不足,该方法可以准确、简单地测定肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离多糖的含量。 [0006] The method of the present invention overcomes the prior methods can not accurately measure the pneumococcal polysaccharide conjugate free polysaccharide content was insufficient, the process can be accurately and simply measured Streptococcus pneumoniae type 6B, 18C type, type 19F, 23F polysaccharide the content of free polysaccharide was bound. [0007] 本发明的技术方案及步骤: [0007] aspect of the present invention and the steps of:

[0008] (1)取所检肺炎链球菌多糖结合物3ml为样1 ;取所检肺炎链球菌多糖结合物对应衍生物溶液3ml为样4 ; [0008] (1) take the Streptococcus pneumoniae polysaccharide conjugate 3ml of sample 1 sample; Streptococcus pneumoniae polysaccharide conjugate sample collected by the corresponding derivative solution 3ml of sample 4;

[0009] (2)另取3ml所检肺炎链球菌多糖结合物置于50ml离心管;取3ml所检肺炎链球菌多糖结合物对应衍生物溶液置于另一50ml离心管;向每离心管中加入5mol/L氯化钠 [0009] (2) Another sample of the Streptococcus pneumoniae polysaccharide conjugate was placed 3ml 50ml centrifuge tube; 3ml taken by Streptococcus pneumoniae polysaccharide conjugate sample was placed in a corresponding derivative another 50ml centrifuge tube; added to each centrifuge tube 5mol / L sodium chloride

0. 75毫升,振荡混勻,再加入无水乙醇15ml,振荡混勻,置于-20°C静置70-90小时;于5°C、 4500〜6500rpm离心60〜90分钟,每离心管单独收集上清液,结合物上清液为样2,对应衍生物上清液为样5 ; 0.75 ml, oscillation mixing, and then adding ethanol 15ml, Shakers, placed in -20 ° C was allowed to stand 70-90 hours; at 5 ° C, 4500~6500rpm centrifugation 60~90 minutes, each tube The supernatant was collected separately, combined supernatant sample is 2, the corresponding derivative of the supernatant sample 5;

[0010] (3)于上述每离心管中加入0. 5ml 0〜60 %乙醇溶液,振荡混合,室温静置1小时,再加入1. 5ml相同乙醇溶液,振荡混合,室温静置2小时,于15°C、4500〜6500rpm离心40〜60分钟,每离心管单独收集上清液,结合物上清液为样3,对应衍生物上清液为样6 ; [0010] (3) above was added to each centrifuge tube and 0. 5ml 0~60% ethanol solution was mixed by shaking and allowed to stand at room temperature for 1 hour, ethanol was added 1. 5ml same mixed by shaking and allowed to stand at room temperature for 2 hours at 15 ° C, centrifuged 4500~6500rpm 40~60 minutes, the supernatant of each centrifuge tube was collected separately, combined supernatant sample is 3, the corresponding derivative of the supernatant sample 6;

[0011] (4)以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量, 分别为? [0011] (4) saline as control, determined by phosphorous assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively? 1、? 1,? 2、? 2,? 3、? 3 ,? 4、? 4 ,? 5、? 5 ,? 6 ; 6;

[0012] (5)根据公式计算回收率P : [0012] (5) P recovery was calculated according to the formula:

[0013] [0013]

Figure CN1971275BD00041

[0014] (6)根据公式计算游离多糖含量H : [0014] (6) free polysaccharide content is calculated according to the formula H:

[0015] [0015]

Figure CN1971275BD00042

[0016] 本发明的有益效果是:利用肺炎链球菌多糖6B型、18C型、19F型、23F型分子结构中含有磷的特点,以及肺炎链球菌多糖结合物在乙醇、强电解质存在条件下结合多糖与游离多糖的不同沉降特性,将游离多糖与结合多糖分离,通过测定磷含量而准确测定出肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离多糖的百分含量。 [0016] Advantageous effects of the present invention are: use of Streptococcus pneumoniae type 6B polysaccharide type 18C, 19F type, 23F type characteristic molecular structure containing phosphorus, and Streptococcus pneumoniae polysaccharide conjugate in ethanol, in the presence of strong electrolyte-binding different properties of the free settling polysaccharide polysaccharide, the polysaccharide will be isolated in conjunction with free polysaccharide, and accurately measured the percentage of Streptococcus pneumoniae type 6B, 18C type, 19F type, 23F polysaccharide conjugate free polysaccharide was determined by phosphorus content. 本发明提供了一种能准确、简便地测定出肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离多糖的含量的测定方法,从而评价制品质量。 The present invention provides a method for the determination can be accurately and easily measured Streptococcus pneumoniae type 6B, 18C type, 19F type, 23F polysaccharide polysaccharide conjugate free to evaluate the product quality.

具体实施方式: Detailed ways:

[0017] 实施例1 [0017] Example 1

[0018] 取肺炎链球菌6B型多糖结合物3ml为样1 ;取所检肺炎链球菌6B多糖结合物对应衍生物溶液3ml为样4。 [0018] Streptococcus pneumoniae 6B taken polysaccharide conjugate 3ml of sample 1; Streptococcus pneumoniae 6B polysaccharide sample collected by the corresponding derivative conjugate solution 3ml of sample 4.

[0019] 另取3ml肺炎链球菌6B多糖结合物置于50ml离心管,取3ml肺炎链球菌6B多糖结合物对应衍生物溶液置于另一50ml离心管。 [0019] Another 3ml 6B pneumococcal polysaccharide conjugate was placed in 50ml centrifuge tube, 3ml taken 6B Streptococcus pneumoniae polysaccharide conjugate derivatives corresponding to another 50ml centrifuge tube was placed. 向每离心管中加入5mol/L氯化钠0. 75ml, 振荡混勻,加入无水乙醇15ml,振荡混勻,置于_20°C静置70小时;于5°C、4500rpm离心60 分钟,每离心管单独收集上清液,结合物上清液为样2,衍生物上清液为样5。 Was added to each centrifuge tube 5mol / L sodium chloride 0. 75ml, oscillation mixing, adding ethanol 15ml, Shakers, placed _20 ° C was allowed to stand 70 hours; centrifugation at 5 ° C, 4500rpm 60 minutes , each tube was collected separately supernatant, the supernatant is combined sample 2, the sample of supernatant derivative 5.

[0020] 于上述每离心管中加入0. 5ml50%乙醇溶液,振荡混合,室温静置1小时,再加入 [0020] was added to the centrifuge tubes each 0. 5ml50% ethanol solution, mixed by shaking and allowed to stand at room temperature for 1 hour and then added

1. 5ml50%乙醇溶液,振荡混合,室温静置2小时,于15°C、4500rpm离心40分钟,每离心管单独收集上清液,结合物上清液为样3,衍生物上清液为样6 ; 1. 5ml50% ethanol solution, mixed by shaking and allowed to stand at room temperature for 2 hours at 15 ° C, 4500rpm centrifuge for 40 minutes the supernatant was collected for each individual tube, the supernatant was combined sample 3, the supernatant of derivatives sample 6;

[0021] 以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量,分别为pi、p2、p3、p4、p5、p6 ;[0022] 根据公式计算回收率P: [0021] In normal saline as control, was measured by phosphorus assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively, pi, p2, p3, p4, p5, p6; [ 0022] recovery was calculated according to the formula P:

[0023] P= P5 + P6 χ 100%,回收率应为80-120% ; [0023] P = P5 + P6 χ 100%, the recovery should be 80-120%;

p4 p4

[0024] 根据公式计算游离多糖含量H : [0024] The content of free H polysaccharide formula:

[0025] H=^2+/3 +尸χ 100%,式中ρ 为回收率。 [0025] H = ^ 2 + / 3 + dead χ 100%, where ρ is the recovery.

[0026] 运用本方法测定3批肺炎链球菌6B型多糖结合物样品,结果详见表1 : [0026] Using 3 for the determination of Streptococcus pneumoniae type 6B polysaccharide conjugate batches of samples, the results shown in Table 1:

[0027] 表1 3批肺炎链球菌6B型多糖结合物测定结果 [0027] TABLE 13 Batch type 6B pneumococcal polysaccharide conjugates measurement result

[0028] [0028]

Figure CN1971275BD00051

[0029] 实施例2 [0029] Example 2

[0030] 取肺炎链球菌18C型多糖结合物3ml为样1 ;取所检肺炎链球菌18C型多糖结合物对应衍生物溶液3ml为样4。 [0030] Streptococcus pneumoniae type 18C polysaccharide taken conjugate 3ml of sample 1; subject collected by Streptococcus pneumoniae type 18C polysaccharide conjugate 3ml solution of the corresponding derivative 4 comp.

[0031] 另取3ml肺炎链球菌18C型多糖结合物置于50ml离心管,取3ml所检肺炎链球菌18C型多糖结合物对应衍生物溶液置于另一50ml离心管。 [0031] Another 3ml Streptococcus pneumoniae type 18C polysaccharide conjugate was placed in 50ml centrifuge tube, 3ml take the subject S. pneumoniae type 18C polysaccharide conjugate derivatives corresponding to another 50ml centrifuge tube was placed. 向每离心管加入5mol/L氯化钠 Was added to each tube 5mol / L sodium chloride

0. 75ml,振荡混勻,加入无水乙醇15ml,振荡混勻,置于_20°C静置90小时;于5°C、6500rpm 离心90分钟,每离心管单独收集上清液,结合物上清液为样2,衍生物上清液为样5。 0. 75ml, oscillation mixing, adding ethanol 15ml, Shakers, placed _20 ° C stand for 90 hours; at 5 ° C, 6500rpm centrifuge for 90 minutes the supernatant was collected each tube separately, conjugate 2 like a supernatant, the supernatant of derivatives of 5 samples.

[0032] 于上述每离心管中加入0. 5ml60%乙醇溶液,振荡混合,室温静置1小时,再加入 [0032] was added to the centrifuge tubes each 0. 5ml60% ethanol solution was mixed by shaking and allowed to stand at room temperature for 1 hour and then added

1. 5ml60%乙醇溶液,振荡混合,室温静置2小时,于15°C、6500rpm离心60分钟,每离心管单独收集上清液,结合物上清液为样3,衍生物上清液为样6。 1. 5ml60% ethanol solution was mixed by shaking and allowed to stand at room temperature for 2 hours at 15 ° C, 6500rpm xg for 60 minutes and the supernatant was collected separately centrifuge tube, the supernatant was bound to sample 3, the supernatant of derivatives sample 6.

[0033] 以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量,分别为pi、p2、p3、p4、p5、p6 ; [0033] In normal saline as control, was measured by phosphorus assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively, pi, p2, p3, p4, p5, p6;

[0034] 根据公式计算回收率P: [0034] P recovery was calculated according to the formula:

[0035] P= P5 + P6 χ 100% ,回收率应为80-120% ; [0035] P = P5 + P6 χ 100%, the recovery should be 80-120%;

ρ4 ρ4

[0036] 根据公式计算游离多糖含量H : [0036] The content of free H polysaccharide formula:

[0037] Η=^^+Pχ 100%,式中P 为回收率。 [0037] Η = ^^ + Pχ 100%, where P is the recovery.

[0038] 运用本方法测定3批肺炎链球菌18C型多糖结合物样品,结果详见表2 : [0038] Using 3 for the determination of Streptococcus pneumoniae type 18C polysaccharide conjugate batches of samples, the results shown in Table 2:

[0039] 表2 3批肺炎链球菌18C型多糖结合物测定结果 [0039] Table 23 batches of pneumococcal polysaccharide type 18C conjugate measurement result

[0040] [0040]

Figure CN1971275BD00061

[0041] 实施例3 [0041] Example 3

[0042] 取肺炎链球菌19F型多糖结合物3ml为样1 ;取所检肺炎链球菌19F型多糖结合物对应衍生物溶液3ml为样4。 [0042] Streptococcus pneumoniae type 19F polysaccharide taken conjugate 3ml of sample 1; Streptococcus pneumoniae type 19F polysaccharide sample collected by the corresponding derivative conjugate solution 3ml of sample 4.

[0043] 另取3ml肺炎链球菌19F型多糖结合物置于50ml离心管,取3ml所检肺炎链球菌19F型多糖结合物对应衍生物溶液置于另一50ml离心管。 [0043] Another 3ml Streptococcus pneumoniae type 19F polysaccharide conjugate was placed in 50ml centrifuge tube, 3ml take the subject S. pneumoniae type 19F polysaccharide conjugate derivatives corresponding to another 50ml centrifuge tube was placed. 向每离心管加入5mol/L氯化钠 Was added to each tube 5mol / L sodium chloride

0. 75ml,振荡混勻,加入无水乙醇15ml,振荡混勻,置于_20°C静置75小时;于5°C、5000rpm 离心70分钟,每离心管单独收集上清液,结合物上清液为样2,衍生物上清液为样5。 0. 75ml, oscillation mixing, adding ethanol 15ml, Shakers, placed _20 ° C was allowed to stand 75 hours; at 5 ° C, 5000rpm centrifuged for 70 minutes the supernatant of each centrifuge tube was collected separately, conjugate 2 like a supernatant, the supernatant of derivatives of 5 samples.

[0044] 于上述每离心管中加入0. 5ml50%乙醇溶液,振荡混合,室温静置1小时,再加入 [0044] was added to the centrifuge tubes each 0. 5ml50% ethanol solution, mixed by shaking and allowed to stand at room temperature for 1 hour and then added

1. 5ml50%乙醇溶液,振荡混合,室温静置2小时,于15°C、5000rpm离心50分钟,每离心管单独收集上清液,结合物上清液为样3,衍生物上清液为样6。 1. 5ml50% ethanol solution, mixed by shaking and allowed to stand at room temperature for 2 hours at 15 ° C, 5000rpm centrifuged 50 minutes, the supernatant of each centrifuge tube was collected separately, combined supernatant sample is 3, the supernatant of derivatives sample 6.

[0045] 以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量,分别为pi、p2、p3、p4、p5、p6。 [0045] In normal saline as control, was measured by phosphorus assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively, pi, p2, p3, p4, p5, p6.

[0046] 根据公式计算回收率P: [0046] P recovery was calculated according to the formula:

[0047] P= p5 + p6 χ 100%,回收率应为80-120% ; [0047] P = p5 + p6 χ 100%, the recovery should be 80-120%;

p4 p4

[0048] 根据公式计算游离多糖含量H : [0048] The content of free H polysaccharide formula:

[0049] H= p2 + p3 +尸X100¾,式中P 为回收率。 [0049] H = p2 + p3 + dead X100¾, where P is the recovery.

[0050] 运用本方法测定3批肺炎链球菌19F型多糖结合物样品,结果详见表3 : [0050] Using 3 for the determination of Streptococcus pneumoniae type 19F polysaccharide conjugate batches of samples, the results shown in Table 3:

[0051] 表3 3批肺炎链球菌19F型多糖结合物测定结果 [0051] Table 33 batches of Streptococcus pneumoniae type 19F polysaccharide conjugate measurement result

[0052] [0052]

Figure CN1971275BD00062

[0053] 实施例4 [0053] Example 4

[0054] 取肺炎链球菌23F型多糖结合物3ml为样1 ;取所检肺炎链球菌23F型多糖结合物对应衍生物溶液3ml为样4。 [0054] Streptococcus pneumoniae type 23F polysaccharide taken conjugate 3ml of sample 1; Streptococcus pneumoniae type 23F polysaccharide sample collected by the corresponding derivative conjugate solution 3ml of sample 4.

[0055] 另取3ml肺炎链球菌23F型多糖结合物置于50ml离心管,取3ml所检肺炎链球菌23F型多糖结合物对应衍生物溶液置于另一50ml离心管。 [0055] Another 3ml Streptococcus pneumoniae type 23F polysaccharide conjugate was placed in 50ml centrifuge tube, 3ml take the subject S. pneumoniae type 23F polysaccharide conjugate derivatives corresponding to another 50ml centrifuge tube was placed. 向每离心管加入5mol/L氯化钠0. 75ml,振荡混勻,加入无水乙醇15ml,振荡混勻,置于_20°C静置80小时;于5°C、5500rpm离心80分钟,每离心管单独收集上清液,结合物上清液为样2,衍生物上清液为样5。 Was added to each tube 5mol / L sodium chloride 0. 75ml, oscillation mixing, adding ethanol 15ml, Shakers, placed _20 ° C was allowed to stand 80 hours; at 5 ° C, centrifuged at 5500rpm 80 minutes. The supernatant of each centrifuge tube was collected separately, combined supernatant sample is 2, derivative 5 a supernatant sample.

[0056] 于上述每离心管中加入0. 5ml注射用水(即含乙醇0% ),振荡混合,室温静置1 小时,再加入1. 5ml注射用水,振荡混合,室温静置2小时,于15°C、5500rpm离心50分钟, 每离心管单独收集上清液,结合物上清液为样3,衍生物上清液为样6。 [0056] was added to the centrifuge tube per 0. 5ml water for injection (i.e., containing 0% ethanol), and mixed by shaking and allowed to stand at room temperature for 1 hour, 1. 5ml water for injection was added, vortex mixed and allowed to stand at room temperature for 2 hours, 15 ° C, 5500rpm centrifuged 50 minutes, the supernatant of each centrifuge tube was collected separately, combined supernatant sample is 3, the supernatant of derivatives of 6 samples.

[0057] 以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量,分别为pi、p2、p3、p4、p5、p6。 [0057] In normal saline as control, was measured by phosphorus assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively, pi, p2, p3, p4, p5, p6.

[0058] 根据公式计算回收率P: [0058] P recovery was calculated according to the formula:

[0059] P= P5p/6 x 100%,回收率应为80-120% ; [0059] P = P5p / 6 x 100%, the recovery should be 80-120%;

[0060] 根据公式计算游离多糖含量H : [0060] The content of free H polysaccharide formula:

[0061] H=P2 + P3 +尸XlOO0Zo,式中P 为回收率。 [0061] H = P2 + P3 + dead XlOO0Zo, where P is the recovery.

[0062] 运用本方法测定3批肺炎链球菌23F型多糖结合物样品,结果详见表4 : [0062] Using 3 for the determination of Streptococcus pneumoniae type 23F polysaccharide conjugate batches of samples, the results shown in Table 4:

[0063] 表4 3批肺炎链球菌23F型多糖结合物测定结果 [0063] Table 43 batches of Streptococcus pneumoniae type 23F polysaccharide conjugate measurement result

[0064] [0064]

Figure CN1971275BD00071

Claims (1)

1.肺炎链球菌6B型、18C型、19F型、23F型多糖结合物中游离多糖含量的测定方法,其步骤是:(1)取所检肺炎链球菌多糖结合物3ml为样1 ;取所检肺炎链球菌多糖结合物对应衍生物溶液3ml为样4 ;(2)另取3ml所检肺炎链球菌多糖结合物置于50ml离心管;取3ml所检肺炎链球菌多糖结合物对应衍生物溶液置于另一50ml离心管;向每离心管中加入5mol/L氯化钠0. 75毫升,振荡混勻,再加入无水乙醇15ml,振荡混勻,置于_20°C静置70-90小时;于5°C、4500〜 6500rpm离心60〜90分钟,每离心管单独收集上清液,结合物上清液为样2,对应衍生物上清液为样5 ;(3)于上述每离心管中加入0. 5ml 0〜60%乙醇溶液,振荡混合,室温静置1小时,再加入1. 5ml相同乙醇溶液,振荡混合,室温静置2小时,于15°C、4500〜6500rpm离心40〜 60分钟,每离心管单独收集上清液,结合物上清液为样3,对应衍生物上 1. S. pneumoniae type 6B, 18C type, 19F type, 23F polysaccharide binding assay method was free polysaccharide content, which steps are: (1) the subject collected by Streptococcus pneumoniae polysaccharide conjugate 3ml of sample 1; collected by detecting Streptococcus pneumoniae polysaccharide conjugate 3ml solution of the corresponding derivative 4-like; (2) another sample of the Streptococcus pneumoniae polysaccharide conjugate was placed 3ml 50ml centrifuge tube; 3ml taken by Streptococcus pneumoniae polysaccharide conjugate sample solution facing the corresponding derivatives in another 50ml centrifuge tube; added to each centrifuge tube 5mol / L sodium chloride, 0.75 ml, oscillation mixing, and then adding ethanol 15ml, Shakers, placed _20 ° C was allowed to stand 70-90 hours; at 5 ° C, 4500~ 6500rpm 60~90 minutes centrifugation, the supernatant of each centrifuge tube was collected separately, combined supernatant sample is 2, the corresponding derivative of the supernatant sample 5; (3) in each of the above in the centrifuge tube was added 0. 5ml 0~60% ethanol solution, mixed by shaking and allowed to stand at room temperature for 1 hour, ethanol was added 1. 5ml same mixed by shaking and allowed to stand at room temperature for 2 hours and centrifuged at 15 ° C, 4500~6500rpm 40~ 60 minutes, the supernatant was collected separately each centrifuge tube, the supernatant was combined sample 3, corresponding to the derivative 清液为样6 ;(4)以生理盐水作对照,用磷测定法测定样1、样2、样3、样4、样5、样6的磷含量,分别为pi、p2、p3、p4、p5、p6 ;(5)根据公式计算回收率P:? 6 is a serum sample; (4) saline as control, determined by phosphorous assay sample 1, sample 2, sample 3, sample 4, sample 5, sample 6 of the phosphorus content, respectively, pi, p2, p3, p4 , p5, p6; (5) the recovery rate is calculated according to the formula P :? =ρ5 + ρ6 χ 100%,回收率应为80-120% ; ρ4(6)根据公式计算游离多糖含量H:Η=ρ2 + ρ3 +尸χ100%,式中ρ为回收率。 = Ρ5 + ρ6 χ 100%, the recovery should be 80-120%; ρ4 (6) is calculated according to the formula polysaccharide content of free H: Η = ρ2 + ρ3 + dead χ100%, where ρ is the recovery.
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