The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1,2 . Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications [3][4][5] . The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods [7][8][9][10][11] . TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

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Background TNF antagonists have been successfully used for the treatment of rheumatoid arthritis (RA). However, data from clinical trials and databases have shown that patients treated with these biological agents have an increased risk of reactivation of latent tuberculosis (TB), hepatitis B virus (HBV) or hepatitis C virus (HCV) infections. The Spanish Society of Rheumatology (SSR) has made some recommendations. According to these guidelines it is mandatory to exclude tuberculosis infection with an evidence level (EL) 2b, Grade of evidence (GR) B. Serology for HBV (EL4, GRC) and HCV (EL4, GRC) are recommended as complementary tests. Purpose To establish the percentage of patients who underwent the recommended tests prior to treatment in a tertiary hospital. Latent infections are ruled out by a tuberculin test and chest x-rays (tuberculosis), HBV markers and HCV antibodies. Materials and methods Retrospective observational study of patients with RA who started treatment with biological agents between January 2010 and August 2011. Data were extracted from Farmatools and Sinapsis care databases. The variables collected were: age, sex, biological agent, BCG skin tests, chest x-rays, anti HCV antibodies and HBV markers. Results A total of 36 patients (78% female, 22% male), started a biological treatment for RA. The average age was 55 years. The drugs used were: etanercept 28%, adalimumab 69% and certolizumab 3%. Tuberculosis was evaluated in all patients, HCV in 58% and HBV in 56%. HCV and HBV tests were positive in 3% and 11% of the patients respectively. Conclusions Screening for tuberculosis infections is a common practice in this hospital. Serology for HCV and HBV were tested in a noteworthy percentage of patients although these complementary tests had recently been recommended by SSR. The pharmacy department may improve this goal by making a double check before the first dose is dispensed.