Objective: To optimize the technical conditions for simultaneous detection of serum ferritin(SF), soluble transferrin receptor(sTfR), C-reactive protein(CRP)and retinol-binding protein four(RBP4)by liquid protein microarray.
Methods: The trapping antibodies of the four proteins were coupled to magnetic beads with different codes, and the samples were added to the 96-well plate. The antibodies were detected by double antibody sandwich method. The serum of 5 patients were diluted with commercial diluent, 1% albumin from bovine serum(BSA) and phosphate buffer saline(PBS) to detect 4 target proteins, and the results were compared. The antibody specific binding ability was tested by antibody specific validation test. The interference between proteins was verified by the paired t test of the signal values of the single reaction system and the mixed reaction system. The lower limit of detection and the limit of biological detection of each protein were found by using multiple dilution method. The standard curve and regression equation were established.
Results: 1%BSA and PBS were selected to replace commercial diluent as diluents for the detection of 4 proteins in this experiment. The cross-reaction rate of the four antigens with other capture antibodies and detection antibodies was less than 2%. There was no significant difference in the signal value of each protein in the single reaction system and the mixed reaction system. The limit of detection and the limit of biological detection of SF were 1.155 and 1.625 ng/mL, respectively. The lower limit of detection and the limit of biological detection of sTfR were 2.682 and 5.208 ng/mL, respectively. The detection limit and biological detection limit of CRP were 0.302 and 0.391 ng/mL, respectively. The lower limit of detection and the limit of biological detection for RBP4 were 1.814 and 3.540 ng/mL, respectively. The standard curve and regression equation of the four proteins within the common linear range were as follows: SF y=172.5x-39.65, R~2=0.9968;sTfR y=60.10x+77.38, R~2=0.9972;CRP y=-6.000x~2+210.3x+246.1, R~2=0.9063;RBP4 y=-0.6998x~2+64.31x+134.8, R~2=0.9748.
Conclusion: The conditions of the detection platform for four proteins such as SF, sTfR, CRP and RBP4 were optimized by using liquid protein chip technology.
Keywords: C-reactive protein; liquid protein microarray; retinol-binding protein four; serum ferritin; soluble transferrin receptor.