| abstract |
A method for quantitative detection of trace amounts of an analyte in a sample is disclosed. The method in the preferred embodiment includes providing an Fab' fragment of an immunoglobulin labelled at a reactive sulfhydryl group with a fluorescent dye; combining the labelled Fab' fragment with a sample that may contain the analyte; concentrating the elements of the resulting mixture in an electric field; separating the labelled analyte/agent complex formed from any unreacted labelled agent using capillary electrophoretic methods; and detecting the fluorescent signal of the labelled analyte/agent complex. The invention also is directed to a method of producing a labelled Fab' fragment that includes cleaving an immunoglobulin molecule to obtain one F(ab') 2 fragment; reducing the disulfide-bonds of the F(ab') 2 fragment to obtain two Fab' fragments each having at least one free, reactive sulfhydryl group; and mixing an Fab' fragment having at least one free sulfhydryl group with a fluorescent dye reactive with the free sulfhydryl to form a labelled Fab' fragment. Preferably, prior to the final mixing step, intrastrand disulfide bonds are formed by oxidation within each Fab' fragment, thereby producing individual Fab' fragments each having a single reactive sulfhydryl group. The method of quantitative detection also more broadly includes using any biospecific agent to form a complex with the target analyte. |