Immunonanogold Catalytic Spectrophotometric Determination of Trace Complement 3
HUANG Wen-xin1,JIANG Zhi-liang1, 2*,LIANG Ai-hui2
1. School of Environment and Resource, Guangxi Normal University, Guilin 541004, China 2. Department of Material and Chemical Engineering, Guilin University of Technology, Guilin 541004, China 3. Environmental Monitoring Station of Santou City, Santou 515041, China
Abstract：Gold nanoparticles about 10 nm in size were prepared by improved trisodium citrate reduction procedure, and were used to label goat anti-human C3 to obtain a sensitive spectral probe for complement 3 (C3) in the condition of pH 7.5. The immune reaction between nanogold-labeled C3 antibody (anti-C3) and the antigen C3 took place to form the nanogold immune complex in pH 5.6 Na2HPO4-C6H8O7 buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 5.6-9.7 μg·mL-1 nanogold-labeled anti-C3, 6.0% PEG 6000 and incubation time 15 min under ultrasonic irradiation. After centrifuging for 10 min at 12 000 r·min-1, the excess nanogold-labeled anti-C3 in the upper solutions was obtained, and was used to catalyze the colored particle reaction between HAuCl4 and NH2OH·HCl to produce gold particles with bigger size. The influence on the immunonanogold catalytic reaction was considered spectrophotometrically. A pH 2.97 Na3C6H5O7-HCl buffer solution, 53.33 μg·mL-1 HAuCl4, 74.13 μg·mL-1 NH2OH·HCl, and reaction time of 3 min at 37 ℃ water bath were chosen for use. Results demonstrated that with increasing C3, the concentration of gold labeled anti-C3 in the upper solution decreased, and the absorbance decreased linearly. Linear relationships between the decreased absorbance and the C3 concentration in the range of 0.025-0.60 ng·mL-1 were obtained by spectrophotometry at 760 nm. The regress equation was ΔA760 nm＝0.276c+0.025 4, the correlation coefficient was 0.990 3, and the detection limit reached 0.007 2 ng·mL-1 of C3. The influence of foreign substances such as HAS, BSA, and ammonia acid on the determination of 0.2 ng·mL-1 of C3 was examined. Results showed that this assay has high selectivity. The sensitive, rapid and highly specific assay was applied to the quantification of C3 in human sera, with satisfactory results.
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