B-017 The Effect of Temperature on the Performance of Streptavidin-coated Paramagnetic Particles in Immunoassay Applications
Authors: Wu B , Li L , Wang D , Xiong Y , Lu W 
Published: September 2023

Abstract

Background
Streptavidin-coated paramagnetic particles (streptavidin particles) are widely used in clinical immunoassays. Streptavidin has the highest affinity with biotin among all known non-covalent interaction we know today. With biotinylated molecules, be they small or large, strong binding of such molecules can be brought to streptavidin in a reliable and robust manner. In addition, streptavidin itself is a stable molecule, more resistant to heat, proteolysis, and chemical denaturation than a typical protein (1). Many publications illustrate the binding sites, binding forces, and configurations of streptavidin with or without biotin (2, 3). From our experience working with streptavidin particles in immunoassays, we know that the challenges to their applications are mainly with baseline signal, biotin interference, and thermal stability. This abstract describes our findings on the thermal stability of assay reagents made from streptavidin particles.

Methods
Intrigued by a recent publication that reports the effect of temperature on the affinity and stoichiometry of streptavidin with biotin (1), suggesting streptavidin’s binding capacity as a function of temperature in the range of 4°C to 40°C, we coupled streptavidin particles (Dynabeads T1 type) with biotinylated thyroxine hormone (T4), estradiol (E2), folate binding protein (FBP), and anti NT-proBNP antibody at varied temperatures between 4°C and 40°C, and then evaluated their coating capacity and thermal stability at 30°C or 37°C. The detection was alkaline phosphatase conjugates with anti T4 antibody, folate analogue, anti E2 antibody, and anti NT-proBNP antibody, respectively. The evaluation used Mindray’s automated chemiluminescent analyzer, CL6000i.

Results
Our data indicate that temperature plays a critical role in enhancing the performances of streptavidin particles in these immunoassays. From pre-coupling treatment of the particle to coupling with biotinylated ligands and subsequently post-coupling blocking and aging, temperature shows great impacts on the stability and tolerance to heat of the coupled particles at elevated temperatures. In contrast to the effectiveness of pre-coupling washing at 50°C or 55°C, coupling of streptavidin particles with biotinylated ligands favors a lower temperature, for example, ambient temperature and 4°C. Coupling at 37°C to 40°C results in slightly lower RLU signals in all four assays. Coupling at lower temperatures improves signal level and, more importantly, enhances thermal resistance of coupled particles. Post-coupling aging and optimization of diluent compositions can additionally improve accelerated stability data obtained at 37°C.

Conclusion
There are two mechanisms that negatively impact the stability performance of streptavidin particles coupled with ligands: One is gradual loss of loosely bound streptavidin molecules and the other streptavidin’s configurational and structural changes triggered by temperature changes. Both mechanisms can be countered by proper control of temperature in the multiple steps of making the solid phase reagents. Our findings can help immunoassay developers to design more robust assay reagents involving streptavidin particles.