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Rapid titration of retroviral vectors using a β-lactamase protein fragment complementation assay

Gene Therapy volume 20, pages 43–50 (2013)Cite this article

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Abstract

The availability of rapid and quantitative titration assays for retroviral vectors is important, especially in the context of clinical applications. In this report, we describe a novel assay to titrate lentiviral and gamma retroviral vectors. This rapid assay is based on protein fragment complementation involving the N-terminal (Bla1) and the C-terminal (Bla2) fragments of TEM-1 β-lactamase (BLAK). The Bla1 protein fragment is incorporated in the vector's envelope during vector production. Bla1-bearing vectors are titrated on Bla2-expressing cells. Upon transduction, Bla1 and Bla2 heterodimerize and restore BLAK's enzymatic function. The enzymatic activity of BLAK is quantified by flow cytometry using the green fluorescent CCF2/AM substrate, which is converted into a blue fluorescent product. The enzymatic conversion of the CCF2/AM substrate was found to be directly related to vector entry, as a neutralizing antibody completely blocked the conversion. The titers obtained using this rapid assay correlated well with the titers measured by functional transduction assays. The whole assay can be finished within 8 h. Thus, it is considerably less time consuming compared with other transduction-based titration assays for lentiviral and gamma retroviral vectors.

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Acknowledgements

We are grateful to Dr Douglas S Lyles for providing us with the anti-VSV-G antibody.

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  1. Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA, Bethesda, MA, USA

    W Ou, M P Marino, C Lu & J Reiser

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  1. W Ou
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  2. M P Marino
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  3. C Lu
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  4. J Reiser
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Correspondence to J Reiser.

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Ou, W., Marino, M., Lu, C. et al. Rapid titration of retroviral vectors using a β-lactamase protein fragment complementation assay. Gene Ther 20, 43–50 (2013). https://doi.org/10.1038/gt.2011.212

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