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Quantification and characterization of anti-interferon beta antibodies in multiple sclerosis patients undergoing interferon beta-1b therapy

University of British Columbia

The increasing recognition of antibody induction in patients undergoing treatment with biologically active recombinant proteins, makes these antibodies of concern. In the case of Relapsing Remitting Multiple Sclerosis (RRMS), administration of interferon beta (IFNβ) - lb can lead to the development of anti- IFN β antibodies in some patients. These antibodies may attenuate the effects of IFNβ, resulting in resistance to therapy or to disease relapses. On the other hand, anti-IFNP antibodies may have carrier or stabilizing functions. The purpose of the current study was the quantification of anti- IFN β antibodies in MS patients' sera, and to further characterize the frequency and serial profile of the antibody response. This was achieved by a sandwich enzyme-linked immunosorbent assay (ELISA) specifically developed for this study. In the process of developing the sandwich ELISA, an anti- IFN β antibody reference pool was established by screening serum samples from 50 RRMS patients undergoing IFNβ therapy, using a qualitative direct ELISA method. Ten serum samples, identified as anti- IFN β antibody positive, were selected for the reference pool which was arbitrarily assigned an anti- IFNP antibody concentration of 100 Laboratory Units / mL. Serial serum samples of 20 patients on IFNβ -1β therapy were assayed using a standard curve generated from the reference pool, and each assay included a set of sera from 5 healthy individuals. The intraassay coefficient of variation (CV) was 6-10% and inter-assay CV was 3-8%. The validity of the assay was assured through binding specificity, recovery and inhibition tests. Of 20 patients treated for 1-24 months, approximately 16 developed antibodies within the first three months. Two antibody profiles were observed: one profile consisted of a rise and gradual decline of antibodies to pretreatment levels, and a second profile characterized by a rise which was followed by a decline to lower antibody levels that were maintained over the course of treatment. Additionally, supernatants of cultured lymphocytes were assayed, using a biotin-streptavidin amplified version of the sandwich-ELISA, and results indicate that some patients secrete anti- IFNβ antibodies in vitro. These findings confirm that IFNβ -1b is immunogenic in MS patients and that the appearance of anti- IFNβ ' antibodies is an early phenomenon. The sandwich ELISA is a specific, sensitive and reproducible method for the quantification of anti- IFNβ ' antibodies, and can be easily adapted in any laboratory for the large-scale throughput monitoring of anti- IFNP antibodies. The assay can also be employed as a tool to study the effects of anti-IFNP antibodies on the therapeutic efficacy of IFNJ3 in MS patients, and the mode of action of IFNp.

Thesis/Dissertation

application/pdf

2009-08-05

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http://hdl.handle.net/2429/11731

Pathology

University of British Columbia

UBCV

DSpace