Antimicrobial Agents and Chemotherapy, September 2004, p . 3610-3612, Vol . 48, No . 9

Convenient Biological Assay for Polyethylene Glycol-Interferons in Patients with Hepatitis C

Anne Boulestin,^1* Nassim Kamar,² Florence Legrand-Abravanel,¹ Karine Sandres-Saune,¹ Laurent Alric,³ Jean-Pierre Vinel,⁴ Lionel Rostaing,² and Jacques Izopet¹

Laboratoire de Virologie EA 2046-IFR 30,¹ Service de Médecine Interne,³ Service d'Hépato-Gastro-Entérologie, CHU PURPAN,⁴ Unité de Transplantation d'Organes, CHU RANGUEIL, Toulouse, France²

Received 9 January 2004/ Returned for modification 18 March 2004/ Accepted 11 May 2004

Detection of IFN- levels in serum can be performed by immunoassay or bioassay (12) . The immunoassay measures the physical quantity of material but does not differentiate between active and inactive molecules . Bioassays for IFN- are based on the protection of cultured cells against the cytopathic effect of a challenge virus (6, 10, 11) .

In this study, we tested the suitability of the vesicular stomatitis virus (VSV) cytopathic effect reduction assay for PEG-IFN- quantification .

The entire procedure of the bioassay was performed with a 96-well microtitration plate . Samples were placed in the first row of wells . Twelve serial twofold dilutions were carried out with Eagle minimal essential medium (MEM) supplemented with 10% fetal bovine serum . An aliquot (0.1 ml) of a suspension of Madin-Darby bovine kidney cells (150,000/ml) was placed in each well . After incubation for 18 h at 37°C, a confluent cell monolayer was obtained . The culture supernatant was drawn off and replaced with a suspension of virus in MEM without fetal bovine serum; 100 50% tissue culture infective doses of VSV was placed in each well in a volume of 0.1 ml . After an additional 24 h of incubation, the cytopathic effects were estimated by visual examination of the monolayers under a microscope . The greatest dilution of sample that still protected the cells was evaluated by comparison with the standard dilution range . The following controls were included in each test: two cell controls, five virus controls (100 50% tissue culture infective doses and dilutions of this viral suspension of 10^–1, 10^–2, 10^–3, and 10^–4) .

Solutions of PEG-IFN-2a (Roche, Neuilly sur Seine, France) or PEG-IFN-2b (Schering-Plough, Levallois-Perret, France) in MEM ranging from 0 to 500 IU/ml were assayed as described above . The measured titer correlated well with the solution titer for both PEG-IFN-2a (r² = 0.99) and PEG-IFN-2b (r² = 0.99) . The dose-response line (Fig . 1) was used as the working standard for the subsequent experiments .

 
The bioassay detected 5 IU of PEG-IFN- per ml in 95% of the assays . No false positive was observed when assaying a concentration of 0 IU/ml, indicating the high specificity of the test . The reproducibility of the result was verified by repetition of six determinations of five titers (Table 1) .

 
Human serum from naive patients containing concentrations of PEG-IFN-2a or PEG-IFN-2b ranging from 0 to 500 IU/ml was assayed . Measured titers and solution titers were highly correlated (PEG-IFN-2a, r² = 0.97; PEG-IFN-2b, r² = 0.99) . The slope of the dose-response line in human serum was not different from that of the working standard (PEG-IFN-2a, P = 0.44; PEG-IFN-2b, P = 1) . Serum samples from 10 normal volunteers and 18 chronic HCV carriers who were not on treatment were assayed; the titer was 0 IU/ml in all cases .

Concentrations of ribavirin (Schering-Plough, Levallois-Perret, France) ranging from 1,000 to 5,000 ng/ml were assayed as were solutions of PEG-IFN- . The cell monolayers were always completely destroyed . Those concentrations of ribavirin were not cytotoxic .

Human serum containing 3,000 ng of ribavirin per ml and PEG-IFN- titers ranging from 0 to 500 IU/ml were assayed . The measured titers correlated well with solution titers (PEG-IFN-2a r² = 0.98; PEG-IFN-2b r² = 0.95) . The slopes of the dose-response line in human serum plus ribavirin were similar to those of the working standard (PEG-IFN-2a, P = 0,77; PEG-IFN-2b, P = 0.31) .

Serum samples from 25 patients treated with PEG-IFN-2b plus ribavirin and from 9 patients treated with PEG-IFN-2a plus ribavirin were assayed . Estimated concentrations in serum ranged from 0 to 125 IU/ml for PEG-IFN-2b and from 0 to 190 IU/ml for PEG-IFN-2a (Fig . 2) .

 
The VSV cytopathic effect reduction assay appears to be suitable for quantifying PEG-IFN-2a and PEG-IFN-2b . Variations can be kept to a minimum by strict standardization of the assay . Controls for cell viability and for the total destruction of the cell monolayer by VSV in the absence of IFN- are included in each plate . Every sample is assayed twice under the same conditions . Hence, our data show good reproducibility of the assay .

Human serum was reported to protect Madin-Darby bovine kidney cells against the cytopathic effect of VSV even without IFN- in the sample (11) . The similarity of the slope of the dose-response line in serum to that of the working standard confirmed that the inhibition observed was truly due to PEG-IFN- . Comparison of the slope of the dose-response line in samples containing ribavirin to that of the working standard also excluded distortion of the test results by ribavirin .

HCV-induced endogenous IFN-, which would lead to overestimation of the sample titer (9), was not found in the blood of chronic HCV carriers who were not on treatment (1, 2) . Growth or cytotoxic factors in serum were observed in a limited number of cases, but they did not hamper interpretation of the assay .

The VSV cytopathic effect reduction assay was used to quantify the antiviral activities of PEG-IFN-2a and PEG-IFN-2b in serum samples from patients on combination therapy . The data variability was great for both types of PEG-IFN- . The elapsed time from the last dose of PEG-IFN- could be a factor in titer variability, but postdose levels of PEG-IFN- in serum were reported to be essentially flat for most subjects (3) . Discrepancies in PEG-IFN- titers in serum could also be attributed to interindividual variability in PEG-IFN- pharmacokinetics .

This study shows that the VSV cytopathic effect reduction assay is suitable for assaying PEG-IFN-2a and PEG-IFN-2b . The concentration of PEG-IFN- in serum samples from patients on combination therapy can be measured with this bioassay .

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11. Vrolijk, J . M., A . Kaul, B . E . Hansen, V . Lohmann, B . L . Haagmans, S . W . Schalm, and R . Bartenschlager. 2003 . A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C . J . Virol . Methods 110:201-209.
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