Use and reliability of multiplex bead-based assays (Luminex) at Containment Level 4

In the UK, research on hazard group 4 (HG4) pathogens requires specialised Containment Level 4 (CL4) facilities. These differ from Biosafety Level 4 (BSL4) conditions in that work is conducted in class III microbiological safety cabinets for primary containment instead of using positive pressure suits. This presents unique challenges associated with the physical restrictions of working in a limited space, and prohibits the use of many techniques and specialist equipment. In consequence, detailed studies on the biology of HG4 pathogens and in particular their immunological relationships with the host are understudied in the UK; for example, the majority of immunological assays with which the immune system is interrogated require specialist equipment that is unsuitable for CL4. Multiplexing to simultaneously measure multiple analytes is increasingly being used in immunological studies. This assay is attractive for CL4 work because it reduces the time spent in the laboratory whilst maximising the use of valuable sample volume. The Luminex microsphere approach allows for the determination of many cytokines and chemokines, however, the detection system uses fixed aligned lasers and integrated computer systems which are unsuitable for use at CL4. Therefore, we have developed an approach in which the Luminex assay is conducted within the CL4 laboratory and a formalin-fixation stage is introduced to allow for analysis to be undertaken outside of containment. Quality control preparations allow the assay characteristics to be monitored and analysis of assay performance to be evaluated. Our data demonstrate that Luminex is an applicable tool for use at CL4 and that assays can be run reliably to generate reproducible standardised data across different plates and individual experiments.