[Immunoresonance scattering spectral assay for the determination of antithrombin-III]

Abstract

A new sensitive, selective and simple immune resonance scattering spectral assay was proposed for the determination of trace amounts of Antithrombin-III (AT-III). It was based on the immune reaction of AT-III with the goat-anti-human AT-III antibody and the resonance scattering effect of the immunocomplex particles in pH 7.2 Tris-HCl buffer solution and in the presence of polyethelene glycol (PEG)6000. The results showed that the resonance scattering signal of AT-III and goat-anti-human AT-III antibody was very weak. However, AT-III was combined with goat-anti-human AT-III antibody specifically, and aggregated to form immunocomplex particles, which enhanced the resonance scattering intensity greatly and produced three resonance scattering peaks at 368 nm, 491 nm and 536 nm respectively. The strongest resonance scattering peak was at 491 nm. In the present paper, the influences of pH, goat-anti-human AT-III antibody and PEG-6000 concentration, incubation temperature, reactive time, and foreign substances were investigated. The result showed that the resonance scattering intensity at 491 nm (I(RS)) is linear to the AT-III concentration in the range of 62.5 to 850 ng x mL(-1), under the optimum conditions of 0.30 mL goat-anti-human AT-III antibody, 30 mg x mL(-1) polyethelene glycol-6000, being incubated at 37 degrees C for 15 min, the voltage at 400 V, and the excitation and emission slit width both at 5.0 nm. Its regress equation is deltaI(RS) 0.062 5c + 1.36, and a relative coefficient of 0.996, with a detection limit of 29.4 ng x mL(-1). The results of co-existing substance tolerance test showed that 1.40 x 10(5) ng x mL(-1) glycine, 9.0 x 10(3) ng x mL(-1) L-glutamic acid, 5.0 x 10(5) ng x mL(-1) glucose, 5.0 x 10(4) ng x mL(-1) urea, 1.5 x 10(4) ng x mL(-1) IgG, 3.0 x 10(4) ng x mL(-1) human serum albumin, and 1.5 x 10(4) ng x mL(-1) bovine serum albumin did not interfere with the resonance scattering determination of 2.50 x 10(2) ng x mL(-1) AT-III, when the relative error was within +/- 10%. Four polyethelene glycols, i. e. polyethelene glycol-4000, polyethelene glycol-6000, polyethelene glycol-10000 and polyethelene glycol-20000, on the immune resonance scattering spectral system were examined in details. The results show that polyethelene glycol-6000 has low blank and high deltaI(RS) value, and was chosen for use. The new resonance scattering spectral method features high sensitivity, good selectivity, simplicity and rapidity, and was applied to the quantitative analysis of AT-III in plasma and serum samples with satisfactory results. Its recovery is in the range of 90.2%-108.9%.