A method for selective conjugation of an analyte to enzymes without unwanted enzyme-enzyme cross-linking

The conjugation of a ligand to an enzyme is often a necessary step in the development of enzyme-linked immunoassays. Such conjugation is typically accomplished by reacting an amine with a carboxyl functional group in the presence of an activator such as a carbodiimide. However, one enzyme's free carboxyl groups often react with another's free amino groups and a large amount of cross-linking between enzyme molecules occurs; few discrete enzyme molecules conjugated only to the ligand of interest are produced. Hence, it is necessary to carry out laborious chromatographic purification steps or to make an activated ligand such as an N-hydroxysuccinimide ester. This too can be a difficult task because N-hydroxysuccinimide esters are not stable in protic solvents and many biological ligands that would be of interest are poorly soluble in organic solvents. This difficulty may limit the quantity and yield of product. We describe a method that eliminates enzyme-enzyme cross-linking by blocking the solvent-accessible carboxyl groups of horseradish peroxidase and alkaline phosphatase, with dialysis being the only purification step necessary. We are consequently able to produce enzyme-ligand conjugates in high purity and in large quantity with little effort and in a relatively short period of time.