Immunoprecipitation for Rapid Detection of Macroprolactin in the Form of Prolactin–Immunoglobulin Complexes

Abstract

The 3 major forms of serum prolactin (PRL), identifiable by gel-filtration chromatography (GFC), are monomeric PRL (23 kDa), big PRL (45–60 kDa), and big, big PRL or macroprolactin (150–170 kDa) (1). The common macroprolactin is PRL complexed with human IgG, but aggregates of PRL, some extensively glycosylated, may also occur (2)(3)(4)(5)(6). Macroprolactin has slower serum clearance than monomeric PRL and little or no apparent in vivo bioactivity, probably because it does not cross capillary walls (7)(8). Macroprolactin causes diagnostic confusion in evaluating hyperprolactinemia (9)(10). Polyethylene glycol (PEG) precipitation is a rapid screening method for macroprolactinemia (11)(12)(13)(14)(15)(16)(17), but PEG interference with PRL assays limits its general use. The method, moreover, shows nonspecific precipitation (up to 15%), necessitating use of a gray zone. We recently validated a screening method based on recognition of the IgG component of macroprolactin by anti-IgG-agarose (18); this method, however, had insufficient PRL assay sensitivity for use in moderate hyperprolactinemia (PRL <1000 mIU/L). A more rapid screening method based on PRL-IgG precipitation with protein A-Sepharose was described recently (19). We report a simple, rapid method, also based on precipitation of PRL-IgG complexes, with protein G-agarose (PGA) suspension and high IgG binding capacity (20 mg/mL of resin). The protein G polypeptide binds the Fc region of all subclasses of the human IgG molecule and also binds the IgG3 fraction (20). Results with this method were compared with those from GFC and PRL-PEG precipitation. For GFC, we used the fast FPLC system (Pharmacia) with a Superdex 200 HR10/300 prepacked column (Amersham Pharmacia) (11). Serum (100 μL) was applied and eluted with phosphate-buffered saline (PBS; 0.05 mol/L, pH 7.0) at a flow rate of …