Anal. Chem. 2008, 80, 4741–4751
Biological and Technical Variables Affecting Immunoassay Recovery of Cytokines from Human Serum and Simulated Vaginal Fluid: A Multicenter Study Raina N. Fichorova,*,† Nicola Richardson-Harman,‡ Massimo Alfano,§ Laurent Belec,| Cedric Carbonneil,|,× Silvia Chen,⊥ Lisa Cosentino,# Kelly Curtis,∇ Charlene S. Dezzutti,#,∇ Betty Donoval,O Gustavo F. Doncel,[ Melissa Donaghay,[ Jean-Charles Grivel,⊥ Esmeralda Guzman,¶ Madeleine Hayes,+ Betsy Herold,¶,∀ Sharon Hillier,# Carol Lackman-Smith,‡ Alan Landay,O Leonid Margolis,⊥ Kenneth H. Mayer,∞ Jenna-Malia Pasicznyk,† Melanie Pallansch-Cokonis,‡ Guido Poli,§ Patricia Reichelderfer,⊥ Paula Roberts,‡ Irma Rodriguez,∞ Hela Saidi,|,£ Rosaria Rita Sassi,† Robin Shattock,+ and James E. Cummins, Jr.‡ Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, Microbicides Quality Assurance Program, Southern Research Institute, Frederick, Maryland 21701, Vita-Salute San Raffaele University and Scientific Institute, Milan, Italy, Centre de Recherche des Cordeliers, Paris, France, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, Rush University Medical Center, Chicago, Illinois 60612, CONRAD, Eastern Virginia Medical School, Norfolk, Virginia 23507, Mount Sinai School of Medicine, New York, New York 10029, St. George’s University of London, London, U.K., and Brown University, Providence, Rhode Island 02912 The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were * To whom correspondence should be addressed. E-mail:
[email protected]. harvard.edu. Fax: +617-713-3018. † Harvard Medical School. ‡ Southern Research Institute. § Vita-Salute San Raffaele University and Scientific Institute. | Centre de Recherche des Cordeliers. ⊥ National Institutes of Health. # Magee-Womens Research Institute. ∇ Centers for Disease Control and Prevention. O Rush University Medical Center. [ Eastern Virginia Medical School. ¶ Mount Sinai School of Medicine. + St. George’s University of London. ∞ Brown University. × Current address: French Ministry of Health, Paris, France. ∀ Current address: Albert Einstein College of Medicine, New York, NY. £ Current address: Antiviral Immunity, Biotherapy and Vaccine Unit, Pasteur Institute, Paris, France. 10.1021/ac702628q CCC: $40.75 2008 American Chemical Society Published on Web 05/17/2008
spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal-Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected
