APPLICATION OF WESTERN-BLOTTING TECHNIQUE WITH CHEMILUMINESCENCE IMAGING TO THE STUDY OF HAPTOGLOBIN TYPE AND HAPTOGLOBIN COMPLEXES

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 separa tion of the proteins in serum by sodium dodecyl sulphate-polyacrylamid e gel electrophoresis (SDS-PAGE), the target proteins were transferred to a nitrocellulose membrane. The membrane was incubated with a prima ry anti-human Hp followed by secondary horseradish peroxidase (HRP) co njugated 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 a(1), a(2) and beta subunits, respectivel y, are 1.25x10(-2) 7.5x10(-3), and 1.5x10(-4) g/l Five-fold repeated a nalysis of three serum samples with different Hp phenotypes show that the relative standard deviation (RSD) is ranging from 9.8% to 18.4% fo r within-blotting and between-blotting detection, respectively. A comp arison was also carried out between ECL image and diaminobenzidine tet rahydrochloride (DAB) stain. A good correlation was obtained between t he two methods (r=0.924), but the sensitivities of ECL imaging were im proved by a factor of 20. The developed method was applied to the stud y of Hp binding to CD22, a B cell adhesion glycoprotein and Streptococ cus 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 phenotyp e-dependent. (C) 1998 Elsevier Science B.V.