Tuberculosis (TB) is a global disease, with about one-third of the world's population infected with the etiological agent, Mycobacterium tuberculosis (6). New infections appear at the rate of about 8 million cases per year, and the annual death toll due to TB is placed at about 2 million (6). For effective control of TB, it is critical to identify infected individuals and screen their immediate contacts so that drug treatment can be administered quickly. For diagnosis of M. tuberculosis infection, more than one diagnostic test is generally applied (8, 21). The tuberculin skin test (TST) is used extensively in both humans as well as nonhuman primates. Results are variable and subject to interpretation and are thus not consistent (8,21). The sputum smear test allows direct identification of M. tuberculosis and is, therefore, highly specific, but results can be variable (8, 10). Bacterial culture for identification of M. tuberculosis infection requires a dedicated microbiology laboratory and is time-consuming (several weeks) (10).More-specific and -sensitive TB diagnostic tests have been developed by using M. tuberculosis-specific antigens and by taking advantage of recent advances in sequencing and anno-

Background Multiplex analysis allows measurements of a large number of analytes simultaneously in each sample. Based on the Luminex multiplex technology (xMAP), kits for measuring multiple cytokines and chemokines (immunomodulators) are commercially available and are useful in investigations on inflammatory diseases. This study evaluated four multiplex kits (Bio-Plex, LINCOplex, Fluorokine, and Beadlyte) that contained 27, 29, 20 and 22 analytes each, respectively, for the analysis of immunomodulators in plasma of rheumatoid arthritis (RA) patients who underwent treatment with antibody against CD20 (rituximab), a B-cell reductive therapy. Methods Multiplex kits were tested on serial plasma samples obtained from six RA patients at baseline and multiple time points (3, 6, and 9 months) post-treatment with rituximab. The RA patients included in this study had previously failed therapy with disease modifying anti-arthritis drugs (DMARD) and treatment with anti-TNFα antibody (infliximab). Results Computer modeling and hierarchical cluster analysis of the multiplex data allowed a comparison of the performance of multiplex assay kits and revealed profiles of immunomodulators in the RA patients. Conclusions In plasma of RA patients who appeared to have benefited from rituximab treatment the profile of significantly elevated immunomodulators by at least two of the three kits (BioPlex, LINCOplex, Beadlyte), is as follows: IL-12p70, Eotaxin, IL-4, TNFα, Il-9, IL-1β, IFNγ, IL-10, IL-6, and IL-13. Immunomodulator profiling by multiplex analysis may provide useful plasma biomarkers for monitoring response to B-cell reductive therapy in RA patients.

Under current practices of mouse colony maintenance, sera from mice are analyzed for antibodies against several widespread infectious pathogens by conventional immunoassays, generally enzyme-linked immunosorbent assay (ELISA). To test for multiple agents, these methods consume large volumes of mouse serum and are laborious and time-consuming. More efficient immunoassays, using small amounts of sample, are therefore needed. Accordingly, we have developed a novel multiplex diagnostic system that employs fluorescent microbeads, coated with purified antigens, for simultaneous serodetection of 10 mouse infectious agents. Individually identifiable, fluorescent microbeads were coated with antigens from Sendai virus, mouse hepatitis virus, Theiler's mouse encephalomyelitis virus/GDVII strain, mouse minute virus, mouse cytomegalovirus, respiratory enteric orphan virus (Reo-3 virus), mouse parvovirus, calf rotavirus for epizootic diarrhea virus of infant mice, vaccinia virus for ectromelia virus, and Mycoplasma pulmonis. Standard sera, singly positive for antibodies to individual infectious agents, were generated by inoculation of BALB/cj and C57BL/6j mice. Sera from these experimentally infected mice, as well as sera from naturally infected mice, were analyzed using a mixture of microbeads coated with antigens of the 10 infectious agents listed above. Results demonstrated that the multiplex assay was at least as sensitive and specific as ELISA for serodetection. Importantly, the multiplex assay required only 1 microliter of serum for simultaneous serodetection of the 10 mouse infectious agents in one reaction vessel. Thus, this multiplex microbead assay is a reliable, efficient, and cost-effective diagnostic modality that will impact serosurveillance of mice used in research.The mouse is the most widely used animal in biomedical research. Availability of specific-pathogen-free (SPF) mice for use in research is essential to obtain consistently accurate data. Experimental animals exposed to, or infected with, various infectious agents may yield questionable data, thereby confounding the findings of a given study. Mice may be screened for several important infectious pathogens (1,3,5,6,9,10,16,17). Routine screening of a large number of animals, with respect to a large number of infectious agents, is a time-consuming and tedious task under current practices. Serosurveillance of mouse colonies is usually performed indirectly by introducing sentinel mice to animal rooms. After allowing for exposure, these sentinel mice are sacrificed, and their sera are tested by conventional immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and/or indirect fluorescent-antibody assay (IFA). Because conventional immunoassays allow detection of only one infectious agent in a serum sample, large amounts of sample are consumed for the detection of multiple agents. Additionally, a composite of multiple individual tests requires much time, materials, and labor. This in turn encourages the use of sentinel mice instead of direct testing...

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