关于在微载体系统中使用VERO细胞培养生产麻疹病毒抗原效率的研究。

Studies on the efficiency of measles virus antigen production using VERO cell culture in a microcarrier system.

Mendonça R Z, Vaz-de-Lima L R, Oliveira M I, Pereira C A, Hoshino-Shimizu S

Laboratório de Immunologia Viral, Instituto Butantan, São Paulo, Brasil.

Braz J Med Biol Res. 1994 Jul;27(7):1575-87.

Abstract

A large amount of antigen is required to conduct seroepidemiologic surveys of measles. Thus, a process to obtain measles virus antigen using a bioreactor was standardized. 2. The virus was grown in a 3.7-1 culture of VERO cells using a Celligen cell culture system containing 2 mg/ml of microcarriers (cytodex I) at 37 degrees C and 60 rpm. The cultures infected with 0.5 m.o.i. of measles virus were harvested after the appearance of the cytopathic effect. The virus suspension was clarified and concentrated by ultracentrifugation. Intracellular and extracellular virus titers were determined by hemagglutination (HA) and by induction of a cytopathic effect in cell culture (TCID50). 3. Intracellular virus presented 5-7 x 10(6) TCID50/0.1 ml, HA activity per 50 microliters equal to 32, with a total HA activity of 4,480 HA units (HAU) and specific activity of 116 HAU/mg. In the concentrated supernatants, the HA titer of extracellular virus was 64, with a total HA activity of 1,024 HAU and a specific activity of 1,600 HAU/mg. 4. The antigen obtained was suitable for the detection of antibodies against measles virus in assays such as ELISA and DOT-ELISA (using 1 micrograms/well to ELISA and 2 micrograms/DOT). 5. The microcarrier system produced antigen sufficient for 26 ELISAs/ml compared to 5.7 ELISAs/ml obtained for the static culture system.

摘要

进行麻疹血清流行病学调查需要大量抗原。因此，对使用生物反应器获取麻疹病毒抗原的过程进行了标准化。2. 病毒在含有2mg/ml微载体（cytodex I）的Celligen细胞培养系统中，于37℃、60rpm条件下在3.7升VERO细胞培养物中培养。在出现细胞病变效应后，收获感染了0.5感染复数（m.o.i.）麻疹病毒的培养物。病毒悬液通过超速离心进行澄清和浓缩。通过血凝试验（HA）和在细胞培养中诱导细胞病变效应（TCID50）来测定细胞内和细胞外病毒滴度。3. 细胞内病毒呈现5 - 7×10(6) TCID50/0.1ml，每50微升的HA活性等于32，总HA活性为4480血凝单位（HAU），比活性为116 HAU/mg。在浓缩的上清液中，细胞外病毒的HA滴度为64，总HA活性为1024 HAU，比活性为1600 HAU/mg。4. 所获得的抗原适用于在ELISA和斑点ELISA等检测中检测抗麻疹病毒抗体（ELISA每孔使用1微克，斑点ELISA每点使用2微克）。5. 与静态培养系统每毫升获得5.7次ELISA的量相比，微载体系统产生的抗原足以用于每毫升26次ELISA。