[Conditional optimization and methodological evaluation of liquid protein microarray in detecting cystatin C].

OBJECTIVE: To optimize the detection conditions and evaluate of cystatin C(CysC) by liquid protein microarray. METHODS: CysC was detected by double antibody sandwich method using liquid protein microarray. On the basis of determining the optimal concentration combination of captured antibody and detected antibody, the detection conditions were optimized by determining the biological detection limit and lower detection limit, drawing the S-shaped curve and judging the linear range, and establishing the standard curve and regression equation. Methodsologically evaluate the accuracy, precision, reportable range and analytical specificity of the detection method. RESULTS: The optimal concentration combinations of CycC trapping-detection antibodies were 26.6 µg/mL-1∶800. The lower limits of detection and biologic limits of detection of the CysC were 0.037 and 0.237 ng/mL, respectively. Regression equation were as followes: y=-3.315x~2+283.04x+160.89, R~2=0.9921. The relative bias of CysC which was detected on the liquid protein microarry was 5.81%. The dilution recovery and recovery were 70.35%-84.91%(n=3)and 79.94%-122.41%(n=3)respectively. The correlation coefficient of method ology comparison experiment was r=0.616, P<0.05, and there was no significant difference between the two method(t=0.948, P=0.358); The within-run precision range from 3.54% to 4.03%(n=10); The between-run precision range from 12.07% to 15.05%(D=5, n=3); The reportable range was 0.26-3784.04 ng/mL. The analysis of interference test result showed that the both concentrations of hemoglobin(160.00, 71.11 g/L) had interference to the result of CysC detected on the chip. CONCLUSION: This study completed the optimization of conditions and methodological evaluation of liquid protein microarray in detecting CysC.

Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique.

We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 (p < 0.05), and the direct bilirubin (DBIL) of low concentration and the indirect bilirubin (IBIL) of high concentration could not interfere with the detected results of nine indicators. The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.

[Optimization of platform conditions for simultaneous detection of multiple nutritional marker proteins by liquid protein microarray].

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.