Immunoresonance Scattering Spectral Analysis Of Complement 3

PartⅠIntroductionSome basic knowledge of historical investigation of light scattering, resonance scattering and the application of resonance scattering technology in studying analytical chemistry and nanoparticles were introduced. Summarized the preparation, identification and assembly of gold nanoparticles and its application in biochemical analysis in recent years. The analytical progress of C3 was also reviewed.PartⅡRapid immune resonance scattering spectral assay of complement 3In pH 6.0 Na2HPO4-C6H8O7 buffer solutions, goat anti-human complement 3 would combine with complement 3(C3) and produce immunocomplex particles, which resulted in resonance scattering (RS) effect at 340nm, 360nm and 470nm. By the experiment, we found in the presence of appropriate concentration polyethyleneglyco (PEG), which could enhance the RS intensities and sensitivity for analysis of C3. Maxium RS intensity was produced at 60 mg·mL-1 PEG-10000. Under the optimal conditions, the intensities△IRS were proportional to the concentration C3, linear relationship was found between intensity of RS and the concentration in the range of 0.167-3.33μg·mL-1 for C3, regression equation was△I340nm=0.1603C+12.42, correlation coefficient R2=0.9926, and the detection limit reached 0.058μg·mL-1. The method was successfully applied to quantitative determination of C3 in human sera, with satisfactory results. The method was simple, rapid and reliable for human sera assay.PartⅢA rapid and sensitive immunonanogold resonance scattering spectral probe for C3.An improved trisodium citrate reduction method was used to prepare about 10 nm size gold nanoparticles and it was used to label goat anti-human complement 3 (anti-C3) in the pH 7.5 to obtain a sensitive and selective immunoresonance scattering spectral probe. It is based on the immune reaction between labeled anti-C3 and C3 in the pH 5.6 Na2HPO4-C6H8O7 buffer solutions. As added C3 the gold labeled-antiC3 would form immunogold complex in the action of polyethylene (PEG), resulting in enhancing the resonance scattering (RS) intensities at 580 nm greatly. Well linear relationships between the enhanced RS intensities (△IRS) and the C3 concentration in the range of 0.00833-0.200μg·mL-1 were obtained, with a detection limit of 0.00280μg·mL-1C3. The rapid and sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.PartⅣUltrasensitive immunoresonance spectral determination of C3 based on nano-gold labeling catalytic enhancementIn the condition of pH 7.5 goat anti-human C3 was labeled by nano-gold to obtain an immunoresonance scattering spectral probe for C3. The immune reaction between gold-labeled antibodies and the antigens took place in pH 5.6 Na2HPO4-C6H8O7 buffer solution in the presence of polyethylene (PEG). Centrifuged at 12000rpm, we obtained supernatant of unreacted gold labeled anti-C3. In pH 2.97 sodium citrate buffer solution, the immunogold in the supernatant, as a seed catalysed the immunereaction between 53.33μg·mL-1 HAuCl4 and 74.13μg·mL-1NH2OH·HCl for 3 min at 37℃, to form larger size and surface gold particles, resulting in enhancement the resonance scattering (RS) intensities at 585 nm greatly. With increasing C3, the concentration of gold labeled-antiC3 in supernatant decreased and I585nm decreased either. Linear relationships between the decreased RS intensities (△IRS) and the C3 concentration in the range of 5.0-160.0pg·mL-1 were obtained, the detection limit reached at 1.52pg·mL-1 C3. The sensitive assay was applied to quantification of C3 in human sera, with satisfactory results.