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A flow injection analysis (FIA) system is presented for a twostep immunoassay-based determination of the total humanprolactin (hPRL) concentration along with its degree ofglycosylation. Separate measurement of total hPRL and nonglysosylated human prolactin (nG-hPRL) were made using twoflow-through cartridges each containing immobilized antibodiesof different specificity. The antibodies are immobilized on thesurface of a carrier. Glycosylated hPRL (G-hPRL) and, thus, thedegree of glycosylation were calculated by the differencebetween the two specific determinations. Enhanced specificityfor the determination of nG-hPRL was obtained using unfavorablebinding conditions through incorporation of alkaline pH andchaotropic agents into the carrier/dispersion buffer. The assayfor total hPRL and nG-hPRL were each found to be linear withinthe relevant concentration range. The results of the two-stepFIA method were found to agree with those obtained by thestandard methods of ELISA and western blotting while offeringthe advantage of minimal analysis time (10 min) and eliminationof manual manipulations.

Flow injection analysis for simultaneous quantification of prolactin concentration and glycosylation macroheterogeneity in cell culture samples.