Emgc20

A sandwich assay to detect C-reactive protein (CRP) was employed to evaluate SH-SAW sensor measurement characteristics in specimens with different molecular sizes. The sandwich assay was performed by reacting with targeting proteins. In the sandwich assay, we used CRP with capture antibodies. Subsequently, secondary antibodies were used for amplifying the sensor output signals reacted with the CRPs captured in the previous step. The capture antibodies were immobilized onto the sensing area of the SH-SAW biosensor using a crosslinking chemical (dithiobis[succinimidylpropionate]), DSP, #22585, Thermo Scientific, Waltham, MA, USA). The HyTest (Turku, Finland) 4C28-CRP30 monoclonal antibody was used as the capture antibody. We used recombinant CRP (CRP Calibrator L-710, SHINO-TEST CORP., Tokyo, Japan) in a series of measurements. The HyTest 4C28-CRP135 monoclonal antibody was used as the secondary antibody. The secondary antibodies were conjugated with gold nanoparticles with diameters of D = 10, 15, 20, and 30 nm (EMGC10, EMGC15, EMGC20, and EMGC30, BBI Solutions, Crumlin, UK) to vary the molecular size. A structural diagram of the antigen, antibody, and gold nanoparticles on the SH-SAW biosensor surface is shown in Figure 3.

Gold nanoparticles of 20 and 80 nm diameter (EM.GC20 and EM.GC80), were purchased from BBI Solutions (United Kingdom), RPMI-1640 was purchased from Sigma-Aldrich, DMEM-F12 medium from Invitrogen as described [36 (link)], sucrose of proteomics grade was used for gradient preparation purchased from VWR Life Science.