Bioavailability of interferon beta 1b in MS patients with and without neutralizing antibodies

Patients and control subjects.

A total of 59 MS patients receiving IFNβ-1b (8 MIU every other day) and 46 subjects without IFNβ therapy serving as a control group were included in the study. A total of 134 blood samples from patients on IFNβ-1b and 54 samples from control subjects were collected. Of 59 IFNβ patients, one sample was collected from 23 patients, two consecutive samples were obtained from 12 patients, three samples were obtained from 12 patients, four samples from 10 patients, and five and six samples from one patient. The average time between sampling was 3 months. Of the 134 samples from IFNβ–treated patients, 35 were obtained in the first year of therapy, and 99 were obtained after more than 12 months of therapy. Pretreatment samples were obtained from only two patients because most patients were on IFNβ-1b already when the MxA assay became available.

The control group consisted of eight healthy volunteers (eight samples) and 38 MS patients. Eighteen of the MS patients had no therapy (21 samples), 11 were taking azathioprine (13 samples), four received monthly IV immunoglobulin (six samples), two were taking cyclosporin A (two samples), two had methotrexate (two samples), and one patient received placebo in a phase-three study (one sample). In the IFNβ group, blood was collected within 20 to 44 hours after the last IFNβ injection. For this study two extra vials (13 mL total) were taken when the patients had blood drawn during follow-up visits, which are performed routinely every 3 months. Additional visits were performed in case of acute medical problems. All subjects gave their informed consent on entry into the study.

IFNβ Western blot (SBA).

IFNβ-1b (2.5 μg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE; 14% polyacrylamide). Proteins were later transferred to nitrocellulose membranes by electrotransfer. Then free binding sites on the membranes were blocked in 2% milk powder in phosphate-buffered saline (PBS; PBS-M) for 1 hour, and the membranes were then washed twice with PBS-0.1% Tween-20 (PBS-T) and air dried. Dried membranes were cut into 2-mm strips.

A total of 1 mL test sample diluted 1:1,000 in PBS-M was incubated with a membrane strip overnight at room temperature. A mouse monoclonal antihuman IFNβ antibody (Axell G2158, Clone MAS291P) and an internal positive control sample served as positive control subjects. For the negative control, serum from a IFNβ-naive MS patient was used. Next the strips were washed three times for 10 minutes with PBS-T and then incubated for 1 hour with phosphatase conjugated antimouse IgG antibodies (Axell G3282) or phosphatase conjugated antihuman IgG antibodies (Axell G2919) as appropriate. The strips were then washed twice with PBS-T and once with substrate buffer (100 mM Tris HCl; pH, 9.5; 100 mM MgCl₂, 100 mM NaCl). Finally, the strips were incubated with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (Boehringer-Mannheim, Mannheim, Germany). After development of the blue precipitate, the strips were washed twice with water and then were air dried. Samples with a band at the level of the mouse band were considered positive, and the SBA titer was determined by ELISA.

IFNβ ELISA.

Each test well of a microtiter plate (Nunc, Denmark) was coated overnight with 5,000 IU Betaferon in 100 μL PBS, and corresponding blank wells were coated with PBS. After decanting the coating solution, each well of the plate was blocked with 3% bovine serum albumin (BSA) in PBS for 2 hours. A total of 100 μL of sera that tested positive in the Western blot was then incubated in doubling dilutions from a baseline dilution of 1:500 in test wells and the corresponding blank wells for 1.5 hours. Plates were then washed six times with washing solution (0.15% BSA in PBS) and incubated with 100 μL horseradish peroxidase-conjugated antihuman IgG (DAKO, Carpinteria, CA) diluted 1:5,000 in washing solution. Next the plates were washed six times with washing solution, and color was developed by adding 100 μL o-phenylenediamine in 0.02 M acetate buffer with 0.0004% H₂O₂ to each well. The reaction was stopped with 50 μL 1M HCl after 20 minutes. Plates were read spectrophotometrically at 492 nm as measuring filter with a reference filter at 405 nm. Optical densities (OD) of blank wells were substracted from OD of the test wells, and titers were calculated as the inverse of the highest dilution in which the ΔOD was still greater than 0.1. On each plate a positive control (mouse IFNβ antibody Axell G2158, Clone MAS291P) and a negative control (serum from a IFNβ-naive patient) were run.

MxA ELISA.

After collection, citrate blood was lysed by adding 100 μL of lysis buffer (20% Triton X-100 in Tris 100 mM; pH, 7.2) to 900 μL of blood, and stored at −20 °C.

Each well of a microtiter plate (Nunc) was coated overnight with 100 μL (12 μg/mL) of a monoclonal anti-MxA antibody solution (MAb 1302.5, Chiron Diagnostics, Alameda, CA). The solution was then decanted and the free binding sites were blocked with 3% BSA in PBS for 2 hours. A total of 50 μL of blood lysate was added to each well for 2 hours at room temperature. Next, plates were washed six times with washing solution and each well was incubated with biotin-labeled anti-MxA antibodies (MAb 1302.34, Chiron Diagnostics) for 1 hour. Plates were then washed again and incubated with peroxidase–avidin (ExtrAvidin; Sigma Chemical Corporation, St. Louis, MO). Color was developed by adding o-phenylenediamine (Sigma Chemical Corporation) in acetate buffer for 20 minutes. The reaction was stopped with 1M HCl and plates were read at 492 nm against 405 nm as a reference wavelength. All samples were tested in duplicate. On each plate six standard dilutions of recombinant MxA (0, 15.6, 31.2, 62.5, 125, and 250 ng/mL), three internal positive control samples, and one internal negative control sample were run in duplicate. The test samples were read off a standard curve that was prepared from the MxA standard dilutions. The MxA concentration was calculated per 100,000 peripheral blood leukocytes (10⁵ PBL) because only leukocytes produce MxA in blood.10

The interassay variation for a mean MxA concentration of 13, 51, 113, and 254 ng/mL (internal control samples) was 31%, 4%, 5%, and 12% respectively. Recovery was 101%, 107%, 109%, 102%, and 106% for 15.6, 31.2, 62.5, 125, and 250 ng/mL respectively.

MxA induction (NAB) assay.

This assay was performed by MediTest Laboratories (Laupheim, Germany) according to the protocol of Berlex Biosciences (Richmond, CA). Briefly, a human lung carcinoma cell line (A549, American Type Culture Collection, no. CLL-185) was diluted in culture media (2% bovine calf serum, 2 mM L-glutamine, 50 U/mL penicillin G, and 0.05 mg/mL streptomycin sulfate in α-modified Eagle medium) at a concentration of 10⁵ cells/mL, and each well of a microtiter plate was incubated overnight with 50 μL of this solution. Test wells were then incubated with Betaferon diluted in culture media at 20 IU/mL, and an equal amount of test sera in a serial dilution resulting in a Betaferon concentration of 10 IU/mL. After 24 hours, samples were removed by aspiration and cells were lysed. The MxA concentration induced by Betaferon was determined by a chemoluminescence assay using the same pair of monoclonal MxA antibodies as in the previous MxA assay. The results are reported as the titer of serum that neutralizes 10 U/mL Betaferon activity to an apparent 1 U/mL activity as determined by a Betaferon standard curve (10, 3, 1, and 0.3 IU/mL), which was run on each plate. Titers of ≥20 are considered to be a positive test result (i.e., NAB positive). For the current study, detectable NAB titers (i.e., titer between 10 and 20, usually reported as NAB negative) were analyzed separately.