Abstract
The immunoblot technique is an enzymatic immunoassay for the detection of antigens at picogram (10–12) levels. Like the indirect enzyme-linked immunosorbent assay (ELISA), the method described here can be used with a commercially prepared secondary anti-body-enzyme conjugate. In principle, the ELISA and the immunoblot assay are similar. In the immunoblot assay, the antigens are immobilized on a nitrocellulose membrane and reacted with strain-specific (primary) antibodies. After a washing step, the fixed primary antibodies bound to the membrane are reacted with alkaline phosphatase conjugate of antibodies specific to the primary antibodies. Reaction with an enzyme substrate causes appearance of purple dots on a white background, indicating the presence of the rhizobial strain assayed. Like the ELISA, but unlike most other serological methods, the immunoblot assay can be used to analyze a large number of samples simultaneously. This makes this method especially useful for establishing strain occupancy in root nodules. In this chapter, the use of the immunoblot assay is demonstrated in a strain com-petition experiment between two strains of Bradyrhizobium japonicum.
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