B-170 Introduction of BIOLIPIDURE® for diagnostic reagents: blocking, protein-stabilizing and signal enhancing effects

Abstract

In the development of immunoassay systems, animal-derived blocking agents like BSA have been traditionally used to suppress decrease in sensitivity caused by non-specific adsorption of antibodies and detection reagents. However, due to lot-to-lot variability and risk of supply concerns by biohazard, chemically synthesized blocking agents are increasingly being used as alternatives. BIOLIPIDURE® is an additive for in vitro diagnostic reagents whose principal component is an MPC-based polymer obtained by polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC). Because MPC has a phosphorylcholine group, which is phospholipids of cell membrane, BIOLIPIDURE® exhibits multiple functions, including protein adsorption suppression (blocking) and inhibition of protein denaturation attributable to the functional group. Therefore, BIOLIPIDURE® enables to apply various assay systems such as CLEIA and lateral flow to achieve blocking, protein-stabilizing, and signal enhancement, thereby improving reproducibility, sensitivity, and specificity. In this poster presentation, we present the blocking, and signal-enhancing properties of BIOLIPIDURE®. This abstract focuses on the blocking efficacy of BIOLIPIDURE®. (Blocking Performance of BIOLIPIDURE®) Methods A: BIOLIPIDURE® (0.5wt% polymer final) was added to Tris-Buffered Saline (TBS, pH 7.4) of Magnetic beads (MagnosphereTM MX100, JSR) and the beads were blocked by incubating at room temperature for 1 h. After washing with TBS, alkaline phosphatase (AP)-labelled goat anti-human IgG diluted in TBS was added to the solution and incubated at room temperature for 1 h. Followed by washing with TBS-T (TBS + Tween-20, pH 7.4), Chemiluminescent substrate (CDP-Star®, Roche) was added to the solution to estimate the adsorbed amount of the AP-labelled antibody on the beads. Blocking performance was evaluated by comparing relative light units (RLU) under 3 conditions: (blocking agent) BIOLIPIDURE®, BSA, and none. Result A: RLU intensity with BIOLIPIDURE® was lower than that under no blocking condition and comparable to that with BSA, indicating that BIOLIPIDURE® effectively suppresses non-specific protein adsorption on the beads (Figure A). (Application to Sandwich CLEIA) Methods B: BIOLIPIDURE® (0.5wt% polymer final) was added to MES buffer solution of MagnosphereTM MX100 immobilized mouse anti-human IgG monoclonal (Fab) antibody and the beads were blocked by incubating at room temperature for 1 h. Human IgG was added to the solution and incubated at above similar condition. After washing with TBS-T, AP-conjugated goat anti-human IgG polyclonal antibody was added to detect the Human IgG bonded to the immobilized antibody. Followed by washing with TBS-T, CDP-Star® was added to measure signal intensity. Blocking performance in sandwich CLEIA was evaluated under 3 conditions: (blocking agent) BIOLIPIDURE®, and none. Result B: In the absence of human IgG, RLU intensity in case with BIOLIPIDURE® decreased similar to BSA compared in case without blocking agent. Additionally, BIOLIPIDURE® enhanced the signal intensity compared to BSA in case of using human-IgG, indicating that BIOLIPIDURE® enable to improve the S/N ratio compared to BSA (Figure B). BIOLIPIDURE® effectively suppressed non-specific protein adsorption on magnetic beads. Additionally, BIOLIPIDURE® significantly improved the S/N ratio in the sandwich CLEIA compared to BSA. In the poster, we will also present other functions of BIOLIPIDURE®, such as protein-stabilizing and signal-enhancing effects, besides blocking effect.