Production of a polyclonal antibody to Phellinus pini and examination of its potential use in diagnostic assays

Summary In order to differentiate among Phellinus pini, Inonotus tomentosus and Inonotus circinatus a polyclonal antibody was raised to a N‐terminal part of 25‐kDa P. pini‐specific protein. The specificity of the polyclonal antibody produced against a synthetic N‐terminal peptide of this protein was investigated for diagnostic purposes using Western immunoblot, indirect enzyme‐linked immunosorbent assay (ELISA), and inhibition ELISA techniques. The N‐terminal synthetic peptide, used as the immunogen, was found to be more than 80% pure by reverse‐phase high‐performance liquid chromatography. Following immunization, antisera were collected at three different time intervals. The antibody molecules were purified from the crude antisera using immunoaffinity gel chromatography. Following one‐dimensional sodium dodecylsulfate‐polyacrylamide gel electrophoresis, Western immunoblot analysis showed that the P. pini I polyclonal‐antibody detected the immunogen, the 25‐kDa protein, in all but one of the P. pini isolates examined, but in none of the isolates of the nontarget species I. tomentosus and I. circinatus. Nevertheless, cross‐reactivity was a problem because the P. pini I polyclonal‐antibody also recognized bands at other molecular weights in nearly all of the isolates of the other species tested. With the indirect ELISA the P. pini isolates tended to have higher affinity for the polyclonal antibody than the nontarget species, but some cross‐reactivity did occur. Inhibition ELISAs, performed over a range of soluble antigen concentrations (1.56–400 ng/100 μl), failed to show a clear distinction between P. pini and the two Inonotus spp. The low level of cross‐reactivity observed for I. tomentosus isolate 52 (9%) was also apparent in the indirect ELISA analysis. All three assays indicated that P. pini isolate 41 was the most antigenic. Despite cross‐reactivity, the antibody is useful in Western immunoblots for the diagnosis of most P. pini isolates.