Immunonanogold Catalytic Spectrophotometric Determination of Trace IgG
DENG Jun-yao1,2,ZHANG Sheng-sen1,JIANG Zhi-liang1,3*
1. School of Environment and Resource, Guangxi Normal University, Guilin 541004, China 2. Children-Women Health Hospital of Guilin City, Guilin 541001, China 3. Department of Material and Chemical Engineering, Guilin University of Technology, Guilin 541004, China
Abstract：Gold nanoparticles the size of about 10 nm were prepared by improved trisodium citrate reduction procedure, and were used to label goat anti-human IgG to obtain a sensitive spectral probe for IgG in the condition of pH 6.5. The immune reaction of nanogold-labeled IgG antibody (anti-IgG) with the antigen IgG took place to form the nanogold immune complex in pH 7.0 Na2HPO4-C6H8O7 buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 7.0, 10.76 μg·mL-1 nanogold-labeled anti-IgG, 8.0% PEG 6000 and incubation time of 30 min under the ultrasonic irradiation. After centrifuging for 15 min at 16 000 rpm, the excess nanogold-labeled anti-IgG in the upper solutions was obtained, and was used to catalyze the colored particle reaction of HAuCl4 with NH2OH·HCl to produce gold particles with bigger size. The influence of pH value, HAuCl4 and NH2OH·HCl concentration, and reaction temperature and time on the immunonanogold catalytic reaction was considered spectrophotometrically. A pH 2.27 Na3C6H5O7-HCl buffer solution, 0.094 mmol·L HAuCl4, 1.92 mmol·L NH2OH·HCl, and reaction time of 6 min at 30 ℃ water bath were chosen for use. Results demonstrated that with increasing IgG, the concentration of gold labeled anti-IgG in the upper solution decreased, and the absorbance decreased linearly. Linear relationships between the decreased absorbance at 700 nm and the IgG concentration CIgG in the range of 0.10-10 ng·mL-1 were obtained. Its regress equation was ΔA760 nm＝0.014 4ｃIgG+0.004 2, the related coefficient was 0.992 6, and the detection limit reached 0.06 ng·mL-1 IgG. The influence of foreign substances on the determination of 3 ng·mL-1 IgG was examined, with the relative error ±10%. Results showed that the following coexistent substances had no impact on the assay: 6 000 ng·mL-1 HSA, 6 000 ng·mL-1 gluocose, 6 000 ng·mL-1 Zn(Ⅱ)，3 000 ng·mL-1 IgA, 3 000 ng·mL-1 Ca(Ⅱ), 3 000 ng·mL-1 L-arginine, 3 000 ng·mL-1β-phenylalanine，2 400 ng·mL-1 Cu(Ⅱ)，2 400 ng·mL-1 EDTA, 2 400 ng·mL-1 L-cystinol etc. This showed that the assay has high selectivity. The sensitive, rapid and highly specific assay was applied to the quantification of IgG in human sera, with satisfactory results.
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