A novel method for the titration of recombinant virus stocks by ELISPOT assay.
The development of vectors for gene therapy requires the definition of quality control parameters such as titration, contamination, transduction efficiency and biological effects in defined model systems. For most viral vectors, the classical titration by plaque formation is not applicable, because vectors are defective for replication and packaging cell lines are not always available. In particular, for vectors derived from the autonomous parvovirus MVM(p), the titration method used currently is based on the amplification of the viral genome inside an infected cell, which can then be revealed with a specific radioactive probe (J. Virol. 63 (1989) 1023). In situ hybridization allows to titrate wild-type virus as well as vectors, using probes that are specific for the substituted viral genes or for the transgene, respectively. This method is, however, time consuming, making the simultaneous titration of large numbers of samples difficult. The use of a radioactive probe requires an adequate facility. An ELISPOT method that allows for rapid titration of up to 23 vector stocks in one 96 well dish was devised. This method is based on the actual expression of the transgene. Compared to in situ hybridization, titers obtained by the ELISPOT method were in general equivalent or higher. However, for some vector stocks the ELISPOT titers were repeatedly lower, indicating that in situ hybridization does not give an accurate measure of transducing units. Our model system is recombinant parvovirus MVM expressing human IL2, but the method should be adaptable to other vectors expressing transgenes that are secreted and for which antibodies are available.
Cheong
Siew Chiat
SC
IRIBHM-IBMM, Université Libre de Bruxelles, Rue des professeurs Jeener et Brachet, 12, B-6041 Gosselies, Belgium.
Clément
Nathalie
N
Velu
Thierry
T
Brandenburger
Annick
A
D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D008915:Minute Virus of Mice / Q000235:genetics*; D011995:Recombination, Genetic; D014161:Transduction, Genetic
D007376:Interleukin-2