Application of Western-blotting technique with chemiluminescence imaging to the study of haptoglobin type and haptoglobin complexes

Abstract

Abstract Determination of haptoglobin (Hp) type and Hp complexes in human serum applying Western-blotting technique with enhanced chemiluminescence (ECL) imaging detection was developed in the present work. After separation of the proteins in serum by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the target proteins were transferred to a nitrocellulose membrane. The membrane was incubated with a primary anti-human Hp followed by secondary horseradish peroxidase (HRP) conjugated IgG antibody. The ECL detection process was then carried out. Under the optimum conditions, the detection limits (3 times noise of baseline), calculated for Hp α1, α2 and β subunits, respectively, are 1.25×10−2, 7.5×10−3, and 1.5×10−4 g/l. Five-fold repeated analysis of three serum samples with different Hp phenotypes show that the relative standard deviation (RSD) is ranging from 9.8% to 18.4% for within-blotting and between-blotting detection, respectively. A comparison was also carried out between ECL image and diaminobenzidine tetrahydrochloride (DAB) stain. A good correlation was obtained between the two methods (r=0.924), but the sensitivities of ECL imaging were improved by a factor of 20. The developed method was applied to the study of Hp binding to CD22, a B cell adhesion glycoprotein and Streptococcus pyogenes (S. pyogenes). The results showed that the Hp is bound to CD22 and to S. pyogenes, and the binding to S. pyogenes is Hp phenotype-dependent.