PART TWO – REPORT INTERNATIONAL: TB RICC BIOMARKER – JUNIOR INVESTIGATOR TRAINING

By: Gustiani Salim

TRAINING AT SALVADOR, BRAZIL

As part of the RePORT international consortium, research at RePORT Brazil focuses on conducting experiments to evaluate the immune response of individuals, translating research findings into clinical practice, and identifying promising and effective strategies for disease treatment and prevention. Specifically, for this training program, the lab provided hands-on and theoretical training in TB biomarker development by performing high throughput cellular phenotyping and protein quantification in a diverse range of biospecimens using Luminex technology. Moreover, the facility offered training in data handling, cleaning, standardization, and analysis, in addition to designing analytical figures for manuscripts and grant applications. The training was hosted at RICC Luminex Referral Lab, managed by FIOTEC/FIOCRUZ in co-operation with the Centro Universitário Faculdade de Tecnologia e Ciências (UniFTC), in Salvador, Bahia state.

Protein-based Biomarker Study in Tuberculosis Disease

TB proteomics signature discovery has been a rapidly growing area of research that aims to identify potential protein biomarkers for the early detection, diagnosis, and treatment monitoring of tuberculosis disease. One common approach is the discovery of host-derived biomarkers, especially host immune responses to Mycobacterium tuberculosis (Mtb) infection such as identification of proteins associated with cytokine signaling, complement activation, cell adhesion, or other pathways associated with pathogen sensing and inflammatory responses, such as cytokines, chemokines, and acute phase proteins. This approach involves assessing relative differences in protein quantity, for example, which proteins are differentially expressed between treated versus untreated patients.

Luminex Technology for Assessing Protein-based Biomarker Discovery

Several different technologies have been used to profile proteins in biological samples. The gold standard and widely used assay for protein detection and quantification is the Enzyme-linked immunosorbent assays (ELISA). Although ELISA is simple and robust, its capacity is limited as it only measures one analyte at a time from each sample. It’s also time-consuming and costly when we need to perform multiple target analytes for multiple samples. Nowadays, the dynamic range of ELISA-based assays is narrower than that of other technologies such as multiplex assays like Luminex.

Luminex technology is a type of immunoassay that uses magnetic microsphere beads to simultaneously measure up to 100 analytes in a single experiment. Furthermore, the advantages and strengths of Luminex include:

• Familiarity of the process – Specific like a sandwich ELISA
• Efficiency – Analyze up to 100 analytes in one single sample
• Reduced sample volume – Much less sample requirement (25 uL)
• Time-saving
• Bespoke – Study analytes involved in a disease or a pathway in the same assay plate with the analyte profile specific to individual needs.
• Low Variability – Minimizes experimental variability with multiple data points derived from a single experiment.

Luminex assays use color-coded microspheres, or beads, that are internally dyed with different proportions of red and infrared fluorophores that correspond to a distinct spectral signature, or bead region. This allows Luminex to create up to 500 different bead colors. The unique color combination gives the bead a “Spectral Address” that can be identified by the instrument. The beads are very small, of uniform size—6.5 microns, suspended in liquid, and therefore exhibit kinetic behavior that is essentially liquid-phase.

On this opportunity, the participants were trained using the Luminex xMAP Intelliflex® system machine. The system provides fast read times for both 96-well and 384-well plate formats. Like other quantitation methods, Luminex requires a standard curve generated from a series of standard controls of known concentration to determine the concentration of tested samples. The basic principle of processing samples using Luminex technology is summarized as follows:

1. Antibodies specific to a desired analyte are coupled to color-coded beads and are incubated with sample plasma or serum. The antibodies bind to the analytes of interest.
2. After washing away unbound materials, samples are incubated with a mixture of biotinylated detection antibodies specific to the analytes of interest and Phycoerythrin (PE)-conjugated streptavidin reporter to form an antibody-antigen sandwich.
3. Using a Luminex instrument, beads are excited by one laser to determine the bead region and corresponding assigned analyte. Another laser determines the magnitude of the PE-derived signal, which is proportional to the amount of analyte bound. Multiple readings are taken at each bead region, ensuring robust detection.

For data evaluation, the quality of raw data was initially assured using Belysa software. The output data was obtained in the form of a table including the concentration of each tested analyte along with standard curve data. Subsequent data processing was performed using R studio software for quality control, statistical analysis, graph creation, and presentation needs.

Visiting the Biorepository Facility of RepORT Brazil

During the training schedule at the lab, participants had the opportunity to visit the biorepository facility that supports the storage of biological study specimens of TB from RePORT Brazil. This biorepository provides centralized storage facilities managed by the José Silveira Foundation. Located at the Bahia Rehabilitation Institute (IBR), the biorepository facility occupies an area of 24 m2 that houses nine -80C freezers and two liquid nitrogen tanks. To date, more than 275,000 specimens have been collected and stored including MTb isolates, sputum, blood, urine, DNA, and plasma at baseline and during follow-up sent from the three enrollment sites participating in the study: Rio de Janeiro, Manaus, and Salvador. All specimens are managed using the FreezerPro tracking system, a software that provides access to sample information from anywhere. The biorepository system is also connected and integrated with clinical, epidemiological, and laboratory data in a single REDcap-RePORT-Brazil database system.

After undergoing training for two full weeks, the participants returned to their respective countries. The next training will be continued at Rutgers – New Jersey Medical School (NJMS) Laboratory, Newark, New Jersey, United States of America from May 6 – 17, 2024.