Naslov
Development and Comparison of Two Cell Based Assays for Measuring Neutralizing Antibodies to Interferon-Beta - Case Study

Autori
Miller, Larisa ; Berman, Melissa ; Lerner, Michaela

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
American Association of Pharmaceutical Sciencies - National Biotechnology Conference

Mjesto i datum
Toronto, Kanada, 21.06.2008. - 26.06.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
neutralizing antibodies; qPCR; reporter gene; cell based assay; interferon beta

Sažetak
To develop and evaluate two new cell based assays for measuring neutralizing antibodies to interferon beta in human serum. First method, Mx1-qPCR assay was developed based on available literature references and further optimized in- house. Briefly, method is based on measurement of Mx1-RNA induction by interferon-beta in A549 cell line. One-step RT-PCR is employed as final read-out method and it was performed on ABI Prism 7900 HT as well as Roche LC-480 real-time PCR instruments. Mx1-gene expression is determined utilizing Delta Delta Ct method. Second method, reporter-gene assay was adapted from commercialy available NeutekBio kit and further developed in-house. Briefly, arrested reporter gene cell line is stimulated by interferon beta and response of luciferase gene is quantified on Perkin Elmer luminometer. Results of both assays show comparable sensitivity and assay range which are improved in comparisson to traditionaly employed cytopathic effect (CPE) assay. Clinical samples previously tested in CPE assay were tested in both new assays and results were compared. Furthermore, different methods of calculating the neutralizing antibody (Nab) titers were employed in this comparison. Results of all three assays showed high degree of correlation. Further discussions are needed around absolute numbers for Nab titers obtained via different computational methods. In addition, both new assays proved to be quicker and easier to perform with improved reproducibility and reliability over traditional CPE assay. Two new cell based assays for assessment of interferon- beta activity and neutralizing antibodies to interferon-beta in human serum were developed and evaluated by testing clinical samples. Both assays have improved sensitivity, linear range, reproducibility and throughput over traditional CPE assay.

Izvorni jezik
Engleski

Znanstvena područja
Farmacija, Biotehnologija

POVEZANOST RADA