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The Luminex 100 reader is a flexible platform based on flow cytometry that enables multiplex analysis of up to 100 analytes in a single microplate well. The system is capable of analyzing various biomarkers including nucleic acids, receptor-ligand assays, immunoassays and enzymatic assays. The platform is based on the simultaneous detection of individual fluorescently dyed microspheres and antibodies conjugated to phycoerythrin (PE). In our lab, we use the Luminex 100 reader in several different assays including for HLA class I typing, KIR typing and antibody specificity determination. The assay described below uses the Luminex 100 reader to determine the antibody specificity of a monoclonal anti-HLA class I antibody.
To determine the specificity of a monoclonal antibody for HLA class I. This assay determines the binding between the antibody under test and 96 HLA class I antigens (One Lambda Labscreen single antigen beads, #LSA0104).
Luminex 100 reader, Luminex X-Y plate, Luminex sheath fluid, antibody under test, single antigen HLA class I beads.
50ul of 1ug/ml antibody stock is incubated with single antigen HLA class I beads for 60 mins at 4°C, washed and then stained with an appropriate secondary antibody, incubated for a further 60min and then transferred to a 96-well plate (Uniplate, GE Healthcare, #7701-3250). Prior to analyzing the samples under test, the Luminex reader should be primed and then flushed with a 70% alcohol solution. Following an alcohol flush cycle, two wash cycles (with Luminex Sheath fluid) should be performed prior to initiating the run proper. During the analysis phase, the Luminex reader simultaneously identifies a unique bead and the amount of secondary antibody bound to that bead. Following analysis, the system should undergo a flush cycle with 10% bleach solution prior to be placed in soak mode with distilled water.
Take care not to allow the sheath fluid resevoir to run dry as allowing air into the FACS system can lead to aberrant results and damage to the instrument. Sheath fluid may need re-filling if greater than 48 samples are being analyzed in a single experiment. Calibration should be undertaken prior to each use of the instrument to ensure the correct identity of all 96 bead types.
The Luminex reader allows for a relatively simple multiplexed assay. When testing the specificity of a monoclonal antibody, there is a clear distinction between positive and negative results and there is little to no background binding. The Luminex reader is easy to setup; an intuitive user interface forms part of the software accompanying the analyzer. The instrument and all of the assay results are controlled and viewed through this software which is loaded onto a PC. Data can be exported in a .CSV file format.
Simple Multiplex assay platform, multiple bioassays available on a single instrument, intuitive software interface
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