Background: The clinical spectrum of visceral leishmaniasis (VL), a chronic intracellular parasitic disease, ranges from a subclinical, asymptomatic infection to severe clinical disease (kala‐azar). In experimental leishmaniasis, mice that have a Th1 response to infection tend to have limited disease while a Th2 response is associated with disease progression. Humans with VL most often have mixed rather than polarized responses. However, most clinical studies have used methods that require a relatively large sample volume, thus limiting their scope. Measuring multiple cytokine levels in blood samples using a multiplexed microsphere assay (MMA) may be useful to further evaluate the Th1/Th2 paradigm in humans. Methods: Bangladeshi individuals (n = 120) living in an area endemic for VL were categorized into one of the five clinical categories. Sera from these individuals were measured for levels of IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12, IFN‐γ, and TNF‐α by multiplexed microsphere cytokine immunoassay. Results: Circulating IL‐8, IL‐10, and IL‐12 differed significantly among the clinical groups. Persons with kala‐azar demonstrated the highest median levels of IL‐8 and IL‐10 but lower median levels of IL‐12. Conclusions: The MMA for cytokines is an extremely time‐and sample‐efficient method for characterizing circulating cytokine levels in visceral leishmaniasis patients. © 2006 International Society for Analytical Cytology

Multiplexed microsphere‐based immunoassays can be developed to simultaneously measure multiple analytes in a biologic system by flow cytometric resolution of spectrally distinct microspheres coupled with capture molecules and reporter fluorochromes bound to detection antibodies. A multiplexed sandwich immunoassay is based on an ELISA format that is transferred directly to microspheres to quantitate multiple antigens. These assays require smaller sample volumes, are less expensive, and are as reproducible, reliable, and sensitive as ELISAs. However, potential cross‐reactivities between multiplexed antibodies, antigens, and specimens need to be systematically eliminated during the validation process. Sandwich and competitive immunoassays, which require only one antigen‐specific antibody, can be combined in the same multiplexed array. Antibody‐capture immunoassays are used to detect multiple antibodies from a specimen for diagnostic or surveillance purposes. The protocols for these three multiplexed immunologic assays are accompanied by methods for coupling analytes to microspheres and biotinylation of antibodies with a water‐soluble derivative.