| 摘要 |
The invention concerns a method for the enzymatic marking of biomolecules, including peptides, hormones, antibodies and other haptens, using horseradish peroxidase (HRP). Instead of uncontrolled formation of heterogenous coupling products, the method proposed calls for the enzyme to be oxidized by sodium metaperiodate and then cross-linked intermolecularly in controlled fashion, using some of the aldehyde groups formed by the oxidation, with an alpha , omega -diaminoalkane, preferably 1,13-diamino-4,7,10-trioxatridecane. This produces an aggregate, made up of 6 to 10 HRP molecules, to which the protein to be marked can be coupled, without previously separating it from the reaction mixture, via the remaining aldehyde groups. An advantage of HRP cross-linking in this way is that a higher degree of substitution can be obtained. Since the protein is not hidden behind HRP molecules, it can exert its immunological properties without any restriction and can be determined without error using spectrophotometric techniques. The sensitivity and specificity of the HRP tracer obtained is extremely high and quite comparable to that of radioactive tracers. The HRP marking method proposed thus represents a remarkable advance in the development of high-performance enzymatic immunological detection systems.
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