Immunoresonance Scattering Spectral Assay of Complement Factor 4(C4)
LIANG Ai-hui,WANG Su-mei
Department of Material and Chemical Engineering, Guilin University of Technology，Key Laboratory of New Processing Technology for Nonferrous Metals and Materials, Ministry of Education, Guilin 541004, China
Abstract：Based on resonance scattering (RS) effect of immune complex particles, a new resonance scattering method for the determination of C4 in the human blood serum was developed. It was based on the fact that goat anti-human C4 was combined with complement factor 4(C4) in the pH 7.3 Na2HPO4-KH2PO4 buffer solution. It is known that antibody has C-terminal and N-terminal, and the N-terminal is the binding site of antigen and it could combine with antigen. Because the stereo structure anastomoses and the charge is opposite between goat ant-human C4 and C4, they could attract and combine with each other. The attraction and combination have high idiosyncrasy and they are done by Van der Waals force, hydrophobic force, Coulomb attracting force and hydrogen bond binding force, and form immune complex particles that exhibit three resonance scattering peaks at 350, 390 and 440 nm, respectively, in the presence of PEG-6000. The laser scattering results indicate that the average diameter of the immune complex particles is about 3 440.0 nm. The influence of pH, goat anti-human C4 and PEG-6000 concentration, incubation temperature and incubation time, foreign substance such as arginine, -phenyl alanine，L-glutamic acid, L-cystine，L-threonine, L-tryptophan, L-histidine, L-leucine, glucose, EDTA，human serum albumin (HSA)，bovine serum albumin(BSA)，L-proline，L-lysine on the determination of C4 was considered in details. Under the conditions chosen, C4 concentration in the range from 0.18 to 2.60 μg·mL-1 is proportional to the resonance scattering intensity at 350 and 390 nm. Its regression equation is ΔI350 nm=28.23c+9.17 and ΔI390 nm=31.36c+11.08, the correlation coefficient is 0.993 9 and 0.992 3, and the detection limit is 0.084 μg·mL-1 and 0.11 μg·mL-1, respectively. The method has been applied to the determination of C4 in the human blood serum, and the results are in agreement with that of the immunoturbidity, with relative standard deviation of 1.88%-4.36％, and with some advantages including simplicity, rapidity, high sensitivity and selectivity.
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