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Technical Briefs |
1 Laboratory of Radioimmunology and2 Department of Endocrinology, University Hospital, Vrije Universiteit, Brussels, Belgium;
aaddress correspondence to this author at: Laarbeeklaan 101, B-1090 Brussels, Belgium; fax 32-2-477-50-60, e-mail Johan.Schiettecatte{at}az.vub.ac.be
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 0.5 mL/min. The first 5 mL was discarded, and thirty-five 0.5-mL fractions were collected and analyzed for PRL by the automated assay on the Elecsys 2010 analyzer. Macroprolactin was identified as a peak of PRL immunoreactivity (above the Elecsys assay detection limit of 10 mIU/L), eluting between IgA and IgG (fractions 12–20) and before monomeric PRL (fractions 23–30). No distinct peak corresponded to the expected migration of big PRL, but small amounts may have occurred as shoulders of the monomeric PRL peak. Monomeric PRL and macroprolactin were quantified from relative areas under the PRL curve and total PRL results.
PRL was measured in 103 serum samples collected, in accordance with the 1975 Helsinki Declaration and subsequent amendments, from hyperprolactinemic individuals (PRL >600 mIU/L; 33 males, 5–81 years; 70 females, 10–70 years). PRL concentrations were 644–18 000 mIU/L, with PEG-precipitated PRL representing the entire range, particularly the gray zone.
The PEG precipitation test was performed as described previously (18). Serum samples were considered negative for macroprolactin when recovery was >50% and positive when it was 
40%. The 40%–50% range was defined as the gray zone (14)(18).
The serum sample IgG fraction was immunoprecipitated with ready-to-use PGA suspension (Roche Diagnostics) (20). After thorough mixing and incubation, samples were centrifuged for 2 min at 9500g and 20 °C. PRL concentrations in the serum (PRL before) and supernatant (PRL after) were immediately measured with the Elecsys assay. The percentage of PRL binding (%B) was calculated [100 x (PRL before – PRL after x 3)/PRL before]. PGA binding capacity was evaluated by assessment of the effects of serum volume, incubation time, and temperature on immunoprecipitation of PRL-IgG. %B decreased with increasing serum volume in a serum pool with macroprolactin. The highest binding was found with 150 µL of serum and 300 µL of PGA. Under these optimal conditions, serum IgG binding was >90%. Binding after immunoprecipitation was independent of incubation time (15 min, 30 min, 1 h, and 3 h) and temperature (2–8 °C and room temperature). PGA (300 µL) was added to 150 µL serum in conical tubes and incubated at room temperature for 15 min with rotation (10 rpm).
The real PRL dilution was determined in 12 serum samples without macroprolactin (shown by GFC) before and after treatment with PGA. The dilution factor (PRL before/PRL after) approached 3 [mean (SD), 2.90 (0.16)], showing small matrix effects from treatment; because the PRL concentration in the supernatant was slightly overestimated [recovery, 103.7 (5.1)%], some samples containing only monomeric PRL showed negative binding.
Reproducibility of the immunoprecipitation was evaluated for 2 months by repeated analyses (n = 15) of 2 serum pools containing macroprolactin. Between-run CVs for PRL binding were 1.2% (binding, 62%; PRL before, 3256 mIU/L) and 0.45% (binding, 84%; PRL before, 1995 mIU/L). Reproducibility for macroprolactin-containing serum was confirmed in a second experiment (n = 10) with a between-run CV of 1.2% (binding, 73.9%; PRL before, 2643 mIU/L). The between-run CV for immunoprecipitation in a pool containing only monomeric PRL was 66% (binding, –4.3%; PRL before, 1811 mIU/L). Immunoprecipitation showed better precision than PEG precipitation in samples with macroprolactin (between-run CVs for recovery after PGA treatment, 2.0%, 2.3%, and 3.3% vs 8.4% for recovery after PEG treatment) and comparable precision in samples containing only monomeric PRL (2.8% vs 3.3%).
Binding to PGA was evaluated in hyperprolactinemic samples with no evidence of macroprolactin (n = 28) and in serum samples containing macroprolactin (n = 23; confirmed with GFC). Mean %B in 28 samples containing monomeric PRL was –4.4% (–9.2% to 9.7%) and in 23 samples containing macroprolactin (percentage macroprolactin after GFC, 12.8%–92%) was 54% (–9.0% to 89%). In 21 of 23 samples from the macroprolactin-positive group (13%–89%), %B was clearly increased, whereas 2 samples that may have contained forms of macroprolactin other than PRL-IgG complexes (5)(20) showed no PRL binding (percentage macroprolactin after GFC, 19.5% and 15.5%; binding, –5.1% and –9.0%).
The %B correlated positively with the percentage of macroprolactin as measured with GFC (r = 0.971; P <0.0001). Because macroprolactin may be present with increased monomeric PRL (21)(22), we compared the GFC-determined monomeric PRL concentration with the residual PRL concentration after immunoprecipitation with PGA and found very good agreement [Passing–Bablok regression: monomeric PRL PGA = 1.031 monomeric PRL GFC + 40 mIU/L (r = 0.998; P <0.0001)].
PRL binding to PGA compared with recovery after PEG precipitation in 103 hyperprolactinemic serum samples is summarized in Fig. 1
. In 37 hyperprolactinemic sera with PEG recovery >50%, the binding was always <10% (–10.3% to 9.7%). In 37 of 38 samples with PEG recovery 40%, binding to PGA was >10% (27.1%–89.1%). One sample with a PEG recovery of 37.1% showed no PRL binding in the immunoprecipitation test but contained macroprolactin with a markedly heterogeneous chromatographic profile, with higher and lower molecular-mass forms than the 150- to 170-kDa macroprolactin. We tested for PRL-IgA or PRL-IgM complexes with goat anti-human IgA or IgM bound covalently to agarose (Sigma). Serum (100 µL) was incubated with 400 µL of anti-IgA- or anti-IgM-agarose for 3 h at 4 °C with rotation (10 rpm). After centrifugation for 2 min at 9500g and 20 °C, PRL amounts in the supernatant were compared with those from a similar dilution of unprecipitated serum in PBS, and %B was calculated: [PRL in diluted serum – PRL in supernatant)/PRL in diluted serum x 100]. Mean binding for anti-IgA- and anti-IgM-agarose was 0.8% (–10.3% to 5.3%) and –0.4% (–13.4% to 4.5%), respectively, in hyperprolactinemic samples (n = 20), with no evidence of macroprolactin. For 2 of 3 macroprolactin-positive samples (no serum was available for 1 sample) after GFC without the IgG component, binding to anti-IgA-agarose was 90% and 18% and binding to anti-IgM-agarose was 18% and 15%, indicating the presence of PRL-IgA and PRL-IgM complexes.
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A variable percentage of PRL bound to PGA in 28 samples in the gray zone of the PEG test (recovery >40% and 50%; see Table 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue9/). Fifteen were >10% (15%–42%), and 13 were <10% (–11% to 8.5%). Six samples with increased protein G-precipitated PRL tested with GFC contained substantial amounts of macroprolactin (13%–42%). Twelve of 13 samples with <10% PRL-IgG binding were tested with GFC (no serum was available for 1 sample). Four samples were negative by GFC and showed no PRL binding to anti-IgA- and anti-IgM-agarose. Of 8 GFC-positive samples, 1 had increased PRL binding to anti-IgA- and anti-IgM-agarose (58.8% and 5.0%) and 3 showed increased PRL binding to anti-IgA-agarose (6.2%, 58.8%, and 60.8%) but were negative for anti-IgM-agarose. Three samples with slightly increased PRL-IgG binding of 8.5%, 7.6%, and 3.1% were negative for anti-IgA- and anti-IgM-agarose. The residual PRL concentration after immunoprecipitation of the gray-zone samples was consistent with the concentration of monomeric PRL after GFC.
Unlike the PEG test, immunoprecipitation showed negligible nonspecific PRL fixation and less chance of interference in PRL immunoassays because a PBS suspension is used instead of a viscous solution. Although costlier than the PEG test, immunoprecipitation tests are analytically more accurate and can be used with immunoassays in which PEG interferes.
In conclusion, immunoprecipitation is reliable for routine serum screening and helps resolve cases in the gray zone of the PEG test. We report for the first time PRL-IgA and PRL-IgM complexes in macroprolactin-positive samples without PRL-IgG complexes, underlining the heterogeneity of macroprolactin.
Acknowledgments
This work was supported by a research grant from the Belgian Study Group for Pediatric Endocrinology. We thank Dr. Erich Schneider, Roche Germany, for performing the gel-filtration chromatography.
References
The following articles in journals at HighWire Press have cited this article:
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  L. Kavanagh, T. J. McKenna, M. N. Fahie-Wilson, J. Gibney, and T. P. Smith Specificity and Clinical Utility of Methods for the Detection of Macroprolactin Clin. Chem., July 1, 2006; 52(7): 1366 - 1372. [Abstract] [Full Text] [PDF]
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