HPLC separation and square‐wave adsorptive‐stripping detection of human immunoglobulin G and A

Abstract

AbstractHuman immunoglobulin G and A (IgG and IgA) were studied with square‐wave voltammetry. For IgG, three reduction peaks (HIGG1, HIGG2, and HIGG3) were observed under different conditions. HIGG1 and HIGG3 are strongly related to the pH of the buffer solution. In pH 7.5 phosphate buffer, the peak potential of HIGG2 is −0.58 V (vs. Ag/AgCl). A linear response holds for human IgG below 3.3 × 10−8 M (5 ppm). As little as 1.1 × 10−10 M (16 ppb) of human IgG is detectable after only 20 seconds of adsorptive accumulation in static solution.IgA has two flat adsorptive‐stripping responses (HIGA1 and HIGA2). The sensitivity of IgA is much poorer compared to IgG. HIGA2 at −0.44 V can be observed throughout the tested pH range with the maximum response between pH 6.0–6.2. In a static solution, a linear calibration graph can be obtained for 0.8–16.8 ppm IgA.IgG and IgA can be separated from each other by using HPLC with an AB × column. The response of 32 ppb IgG can be observed clearly. This research indicates the possibility of direct separation and detection of immunoglobulins from a real sample (e.g., human serum) by simply applying HPLC‐adsorptive‐stripping analysis.