CN1259207A - 滴定复合病毒组合物的方法 - Google Patents

滴定复合病毒组合物的方法 Download PDF

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CN1259207A
CN1259207A CN98805813A CN98805813A CN1259207A CN 1259207 A CN1259207 A CN 1259207A CN 98805813 A CN98805813 A CN 98805813A CN 98805813 A CN98805813 A CN 98805813A CN 1259207 A CN1259207 A CN 1259207A
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C·格尔迪尔
J·F·萨鲁佐
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Abstract

本发明涉及一种确定包含不同种或型的活病毒的组合物中各种型或种的病毒量的方法,由下面步骤组成:在不诱导病毒干扰的允许细胞中繁殖各种型或种的病毒;使用特异性的单克隆抗体分析各种型或种的病毒。

Description

滴定复合病毒组合物的方法
本发明涉及病毒组合物领域；更具体地说本发明涉及一种确定包含不同活病毒种或型的组合物中的各个种，或更具体地说指定的病毒种的各种血清型的病毒数量的方法。
现有技术已知分析病毒组合物中各种病毒种或病毒型的量的方法。通常采用的方法由使用多克隆抗体中和不希望分析的病毒，并接着确定剩余病毒的量组成。当需要确定在用于抗脊髓灰质炎疫苗接种的组合物中I、II、III型减毒活病毒的数量时情况尤其如此。然而，这些方法并不总是可行的，因为某些时候中和存在于组合物中的一些病毒，而不干扰希望分析的病毒是困难的，甚至是不可能的，尤其是在欲滴定包含特定病毒，如登革病毒的几种血清型的病毒组合物时。这是因为没有型特异性多克隆抗体；能识别特异性指定型的单克隆抗体通常不够中和；甚至有时没有已知的中和单克隆抗体。然而，在一些情况下，尤其是在疫苗工业，必须制备以完全确定的比例包含不同病毒种，或某一病毒种不同血清型的病毒组合物。而且，药物学要求使得必须随后能以可靠的方式定量地控制所制备的产物的组成。
因此希望能够获得使复合病毒组合物无需修饰就能被滴定的方法。
为此目的，本发明涉及一种确定包含不同活病毒种或型的组合物中的各种病毒型或病毒种的病毒量的方法，特征在于它包含下面步骤：
在不诱导病毒干扰的允许细胞中繁殖各种型或种的病毒，
使用特异性单克隆抗体分析各种型或种的病毒。
根据本发明的方法被用于包含一些不同种的病毒和/或同一种的一些血清型的组合物。病毒尤其可为导致脊髓灰质炎、风疹、流行性腮腺炎、麻疹或登革热的病毒或其它轮状病毒。包含这些病毒的组合物可为疫苗组合物，其中虽然病毒毒性被减弱，但病毒被维持在活的状态。因此，组合物可为例如包含脊髓灰质炎病毒的三种血清型的疫苗组合物或包含登革病毒的四种血清型的组合物。当病毒的抗原性极为相关并且一种血清型的中和通过交叉反应导致其它血清型的中和时，根据本发明的方法尤为令人关注。
根据本发明，存在于待检测的组合物中的病毒以各种稀释度在不诱导病毒干扰的允许细胞中繁殖。因此可以使用Vero细胞。
将细胞置于适宜于培养细胞的平板的各孔中并接着接种病毒悬液。
用于病毒繁殖的培养基为根据采用的细胞以及待滴定的病毒的性质而加以调整的常规培养基。在CO2气氛下，在病毒的最适生长温度下，将病毒温育，温育时间取决于具体病毒(例如在登革病毒的情况下为一周)，然后除去细胞培养物上清；接着使用例如预冷的丙酮固定细胞。
接着根据进行的滴定使用种或血清型特异的单克隆抗体确定各种稀释度存在的病毒量。使用荧光素标记的抗一种抗体或使用适宜于ELISA检测的底物使反应显色。通过Spearman和Karber法确定病毒效价并以感染50％细胞培养物的剂量表示(CCID50)。
使用特异于病毒组合物中希望滴定的病毒种或型的各种单克隆抗体平行进行相同的过程。
因此，使用该方法可令人惊奇地滴定存在于没有一种血清型占优势的病毒组合物中的各种血清型。
实施例
滴定四种一价疫苗组合物，其中每一种包含一种登革病毒的血清型，以及在分析时通过混合等量的四种检测的一价组合物制备的组合物。
以下面的方式在96孔微量平板中进行滴定：
-使用包含5％胎牛血清和2g/l碳酸氢钠的MEM培养基制备各种组合物的连续稀释物，
-将由此获得的病毒悬液以10孔/稀释度的比例接种至已建立一天的Vero细胞层(参考ATCC：CCL81VERO)上。在至少一个96孔平板中滴定一价悬液和四价组合物的效价，
-接着36℃下，5％CO2中，将平板保温一周，
-除去细胞培养物上清，用冷却至-20℃的丙酮将细胞固定于平板上，
-使用特异于疫苗组合物中血清型的单克隆抗体检测病毒的存在。采用的抗体来自由CDC(疾病控制中心，Atlanta，美国)提供的杂交瘤。
国际1血清型用来自杂交瘤D2-1F1-3的抗体标记
国际2血清型用来自杂交瘤3H5-1-12的抗体标记
国际3血清型用来自杂交瘤5D4-11-24的抗体标记
国际4血清型用来自杂交瘤1H10-6-7的抗体标记
-使用荧光素标记的抗小鼠IgG抗体使反应显色。
使用荧光显微镜读取结果。计数各种稀释度情况下具有至少一个感染灶(具荧光)的孔数目。
产物的效价相当于导致50％细胞层受影响的稀释度(即为50％的孔)并通过Spearman和Karber法确定。它以CCID50的log10以表示。
借助于平行进行的各种分析，获得下面的结果：
    1型     2型     3型     4型
  一价组合物：分析1分析2 3.63.8 4.74.8 5.15.7 2.62.9
  四价混合物：分析1分析2 3.03.3 4.34.6 5.65.2 1.92.4
注意到获得的结果与预期的结果一致；对于四价混合物中各种病毒型所观察到的效价的差异变化为约0.5 log10 CCID50，它相当于制备混合物时各种血清型的1/4稀释度。
因此，根据本发明可使用非中和单克隆抗体分析存在于病毒组合物中的各种血清型，而不诱导不同血清型之间的干扰。

Claims (6)

1.确定包含不同活病毒种或型的组合物中的各种病毒型或病毒种的病毒数量的方法，特征在于它包含下面步骤：
在不诱导任何病毒干扰的允许细胞中繁殖各种型或种的病毒，
使用特异性的单克隆抗体分析各种型或种的病毒。
2.根据权利要求1的方法，特征在于繁殖在Vero细胞中进行。
3.根据前述任一项权利要求的方法，特征在于包含不同活病毒种或型的组合物为疫苗组合物。
4.根据前述任一项权利要求的方法，特征在于包含不同活病毒种或型的组合物为包含四种减毒活登革病毒血清型的组合物。
5.根据权利要求1-3任一项的方法，特征在于包含不同治病毒种或型的组合物为包含三种活的减毒脊髓灰质炎病毒血清型的组合物。
6.根据权利要求1-3任一项的方法，特征在于包含不同活病毒种或型的组合物为包含不同型轮状病毒的组合物。
CN98805813A 1997-04-04 1998-04-03 滴定复合病毒组合物的方法 Expired - Fee Related CN1130465C (zh)

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FR9704371A FR2761698B1 (fr) 1997-04-04 1997-04-04 Methode de titrage d'une composition virale complexe
FR97/04371 1997-04-04

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EP (1) EP0974055B1 (zh)
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AT (1) ATE235685T1 (zh)
AU (1) AU737465B2 (zh)
BR (1) BR9808482A (zh)
CA (1) CA2285647C (zh)
DE (1) DE69812601T2 (zh)
DK (1) DK0974055T3 (zh)
ES (1) ES2191299T3 (zh)
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US8589667B2 (en) 2010-04-19 2013-11-19 Apple Inc. Booting and configuring a subsystem securely from non-local storage

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FR2511702B1 (fr) * 1981-08-24 1985-09-13 Pasteur Institut Anticorps monoclonaux antipoliovirus pour l'identification du type et la differenciation intratypique de souches isolees de poliovirus, hybrides cellulaires pour leur fabrication et procede de diagnostic in vitro mettant en jeu des anticorps
GB8703696D0 (en) * 1987-02-18 1987-03-25 Oxford J S Influenza vaccine
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NZ500005A (en) 2001-07-27
ATE235685T1 (de) 2003-04-15
DE69812601D1 (de) 2003-04-30
AU737465B2 (en) 2001-08-23
DE69812601T2 (de) 2003-10-09
FR2761698B1 (fr) 1999-05-07
EP0974055A1 (fr) 2000-01-26
DK0974055T3 (da) 2003-07-21
AU7055298A (en) 1998-10-30
FR2761698A1 (fr) 1998-10-09
CA2285647A1 (fr) 1998-10-15
CA2285647C (fr) 2008-11-18
EP0974055B1 (fr) 2003-03-26
KR20010006045A (ko) 2001-01-15
US6890711B1 (en) 2005-05-10
BR9808482A (pt) 2001-07-31
ES2191299T3 (es) 2003-09-01
KR100543344B1 (ko) 2006-01-20
CN1130465C (zh) 2003-12-10
WO1998045709A1 (fr) 1998-10-15

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