Poster U14 - Development of a point of care sample preparation-integrated vertical flow immunoassay for the detection of intracellular leukocyte proteins

Introduction: The detection of intracellular blood biomarkers is pivotal in various medical diagnostics, enabling rapid and accurate assessment of health status in numerous clinical scenarios. For instance, intracellular blood proteins have been identified as radiation dosimetry biomarkers for assessing absorbed radiation dose after radiobiological accident. The use of point-of-care (POC) assay for the detection of such biomarkers is then critical to diagnose and monitor exposed individuals anywhere and at any time. However, the development of a complete “sample-in-answer-out” platform stays to date challenging due to the numerous steps involved in the workflow, from the sample collection to the data analysis. Here, we present a self-sustained POC device which includes a sample preparation module (SPM) to process whole blood (WB) from fingerstick collection and obtain a leukocyte lysate that can be directly transferred to a vertical flow immunoassay (VFI) module to detect biodosimetry (DDB2 and BAX) and housekeeping (β2M) proteins.

Materials and

Methods: The SPM is meant to perform red blood cell (RBC) lysis, leukocyte binding and lysis steps. We thus tested Ammonium-Chloride-Potassium (ACK) and Erythrocyte Lysis (EL) buffers, as well as water for RBC lysis. Fingerstick-relevant volume (100μL) of WB was incubated in 500, 1000, and 800μL of ACK, EL buffers, and water, respectively. RBC lysis efficiency was assessed by the hemoglobin release measured at 404 nm absorbance. For leukocyte-antibody binding assessment, a 96-well ELISA plate was coated with 5μg/mL anti-CD45 antibody, and samples were resuspended in different buffers. After 30 minutes, to assess binding efficiency, wells were washed twice with RPMI, then 100μL RPMI and 15μL MTT were added per well, followed by a 4 h incubation at 37Co. Absorbance was measured at 570nm after adding stop solution. For the bioassay, Commercially antibodies for DDB2, BAX and β2M were screened par standard ELISA to determine the best antibodies pairs for each protein. Then, selected captured antibodies were printed on VFI membranes using a Nano-plotter. Assay buffer was spiked with DDB2, BAX and/or β2M recombinant proteins after incubation with the appropriate detection antibodies and 40 nm gold conjugated secondary antibodies. Sample was then dispensed into the device, and the results was acquired after 15 minutes diffusion by capillarity within the device.

Results, Conclusions, and Discussions: To identify the best RBC lysis buffer, we first examined RBCs lysis with ACK buffer, EL buffer, and water. After 15 min, all the three buffers showed the ability to lyse the RBCs effectively. Furthermore, water could lyse the RBCs as efficiently in 10 s as in 15. Next, leukocytes resuspended in ACK, EL buffer, water and 1X PBS showed no binding after 30 min incubation with anti-CD45 antibody except for 1X PBS, suggesting that none of the buffers used for RBC lysis could be suitable for a one step RBC lysis-leukocyte binding, thus complexifying the workflow integration in the SPM. Therefore, we then opted to test RBC lysis with water for 10 s supplemented by 10% 10X PBS to restore isotonic condition, preserve leukocytes and provide appropriate condition for the binding. The results demonstrated that such approach effectively allowed RBC lysis while preserving the leukocytes as shown by the high number of retained CD45+ cells and cell viability. This workflow has been then effectively implemented and tested on the SPM. We then tested 3 different candidate materials for the SPM manufacturing, Poly-carbonate (PC), -propylene (PP), and -styrene (PS) to determine the optimal surface for immobilizing anti-CD45 Abs. FTIR measurements showed that PP and PS were able to immobilize anti-CD45 Abs while PC did not retain any. Finally, the ELISA screening to identify Abs for the detection of DDB2, BAX and β2M proteins showed that a sandwich format allowed DDB2 detection with a limit of detection (LOD) of 3.664 ng/mL, a competitive format allowed β2M and BAX detection with LODs of 17.289 ng/mL and 12.5 ng/mL, respectively. DDB2 and β2M assays were then successfully integrated into a paper-based format using the VFI with LODs of 1.892 ng/mL and 33.5 ng/mL, respectively. Interestingly, both proteins were then multiplexed on the same VFI membrane and demonstrated good specificity with no cross-reactivity. Next steps will involve assessing the combined workflow using the SPM and VFI modules.

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