[Immunonanogold catalytic spectrophotometric determination of trace complement 3].

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 Na2 HPO4-C6H8O7 buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 5.6-9.7 microg x 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 x 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 NH2 OH x 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 microg x mL(-1) HAuCl4, 74.13 microg x mL(-1) NH2OH x HCl, and reaction time of 3 min at 37 degrees C 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 x mL(-1) were obtained by spectrophotometry at 760 nm. The regress equation was deltaA760 nm = 0.276c+0.025 4, the correlation coefficient was 0.990 3, and the detection limit reached 0.007 2 ng x mL(-1) of C3. The influence of foreign substances such as HAS, BSA, and ammonia acid on the determination of 0.2 ng x mL(-1) of C3was 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.