Chemiluminescent imaging analysis of interferon alpha in serum samples

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence imaging analysis have been combined to develop a sensitive, simple, and rapid method for analysis of interferon alpha (α-IFN) in human serum samples. A typical “sandwich type” immunoassay was used. Reaction of o-phenylenediamine (OPD) with hydrogen peroxide (H₂O₂), catalyzed by HRP, produced 2,3-diaminophenazine (PDA), which was detected by chemiluminescence imaging analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)–H₂O₂–glyoxaline–PDA chemiluminescent system. The TCPO chemiluminescent imaging system is more sensitive and the chemiluminescence quantum yield is at least five times higher than for the luminol–H₂O₂–HRP–PIP (p-iodophenol) chemiluminescent imaging system. The results showed there was a very good linear correlation between response and amount of α-IFN in the range 1.3–156.0 pg mL⁻¹ (R = 0.9991) and the detection limit was 0.8 pg mL⁻¹ (S/N=3). The relative standard deviation (n = 9) was 4.7%. The proposed method has been used for successful analysis of the amount of α-IFN in human serum. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA. Figure Procedures of the proposed method