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Endocrinology Vol. 148, No. 5 2465-2470
Copyright © 2007 by The Endocrine Society

Development of Anti-PRL (Prolactin) Autoantibodies by Homologous PRL in Rats: A Model for Macroprolactinemia

Naoki Hattori, Yasuhisa Nakayama, Kaori Kitagawa, Tiesong Li and Chiyoko Inagaki

Department of Pharmacology, Kansai Medical University, Osaka 570-8506, Japan

Address all correspondence and requests for reprints to: Naoki Hattori, M.D., Ph.D., Department of Pharmacology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi-City, Osaka 570-8506, Japan. E-mail: hattorin{at}takii.kmu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macroprolactinemia is hyperprolactinemia in humans mainly due to anti-PRL (prolactin) autoantibodies and is a pitfall for the differential diagnosis of hyperprolactinemia. Despite its high prevalence, the pathogenesis remains unclear. In this study, we examined whether anti-PRL autoantibodies develop via immunization with homologous rat pituitary PRL in rats to elucidate what mechanisms are involved and whether they cause hyperprolactinemia with low PRL bioactivity, as seen in human macroprolactinemia. Anti-PRL antibodies were developed in 19 of 20 rats immunized with homologous rat pituitary PRL and 29 of 30 rats with heterogeneous bovine or porcine pituitary PRL but did not develop in 25 control rats. In rats with anti-PRL antibodies, the basal serum PRL levels were elevated, and a provocative test for PRL secretion using dopamine D2 receptor antagonist (metoclopramide) showed a normal rising response with a slower clearance of PRL because of the accumulation of macroprolactin in blood. Antibodies developed by porcine or rat pituitary PRL reduced the bioactivity of rat serum PRL, and gonadal functions in these rats were normal despite hyperprolactinemia. Anti-PRL antibodies were stable and persisted for at least 5 wk after the final injection of PRL. These findings suggest that pituitary PRL, even if homologous, has antigenicity, leading to the development of anti-PRL autoantibodies. We successfully produced an animal model of human macroprolactinemia, with which we can explain the mechanisms of its clinical characteristics, i.e. asymptomatic hyperprolactinemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SERUM CONCENTRATIONS of prolactin (PRL) increase physiologically in pregnancy and during lactation. Pathological hyperprolactinemia occurs due to prolactinoma, hypothalamic or pituitary tumors compressing the pituitary stalk, drugs such as dopamine receptor D2 antagonists, hypothyroidism, or hepatorenal disorders (1). The clinical characteristics of hyperprolactinemia include amenorrhea or oligomenorrhea, galactorrhea, and infertility in women and impotence and galactorrhea in men.

Recently it has been shown that there are quite a number of hyperprolactinemic patients who are not included in any of the above etiologies. Serum PRL in those patients mainly consists of big-big PRL (molecular mass greater than 100 kDa), whereas that in normal subjects and hyperprolactinemic patients with other etiologies is composed of monomeric PRL (23 kDa) as a predominant form (2). The condition was termed macroprolactinemia (3, 4) and reportedly accounts for 10–46% of hyperprolactinemia (5, 6, 7, 8, 9). Patients with macroprolactinemia usually lack clinical symptoms of hyperprolactinemia, and pregnancy is possible without medication in these patients (8, 9, 10). Although the frequency of macroprolactinemia is high, this etiology still remains a diagnostic pitfall. Unnecessary examination such as computerized axial tomography or magnetic resonance imaging of the pituitary gland have been performed and unnecessary medication prescribed until the diagnosis of macroprolactinemia is finally made.

Several groups as well as our own have demonstrated that anti-PRL autoantibody is a major player in the pathogenesis of macroprolactinemia (11, 12, 13, 14, 15, 16), although other forms such as oligomeric forms of monomeric PRL (17), covalently or noncovalently bound glycosylated PRL (18), and stable PRL-IgG complexes not displaceable by the human PRL standard (19, 20) have also been reported. Despite many clinical studies, much remains unknown about how anti-PRL autoantibodies naturally arise so frequently and how they cause asymptomatic hyperprolactinemia. We recently found that human PRL is phosphorylated in the pituitary gland and partially dephosphorylated in the serum (21). This prompted us to postulate that immunization with pituitary PRL, which is phosphorylated, even if of the same species (homologous), could produce autoantibodies. In this study we examined whether injections of homologous and heterogeneous pituitary PRL into rats could lead to the production of antibodies against rat PRL and whether these antibodies cause hyperprolactinemia with similarity explicable for clinical and laboratory characteristics of macroprolactinemia in humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wistar rats (54 males and 21 females, body weight 180–200 g) were used in three series of experiments. Bovine, porcine, or rat highly purified pituitary PRL (National Hormone and Peptide Program, Torrance, CA) was emulsified at a ratio of 1:1 in complete Freund’s adjuvant (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and 50 µg peptide in 200 µl emulsion was injected ip into rats. The same dose of peptides emulsified in incomplete Freund’s adjuvant (Wako) was injected every 2 wk four times in the first and second series of experiments and twice in the third series. Control rats were injected with 200 µl emulsion consisting of adjuvant and 0. 01 mol/liter NaHCO3.

In the first series of immunizations using 25 male rats (five for bovine PRL, five for porcine PRL, five for rat PRL, and 10 controls), blood (3–5 ml) was taken from the heart by needle puncture under urethane anesthesia (500 mg/kg) a week after the final immunization.

In the second series of immunizations using 29 male rats (five for bovine PRL, 10 for porcine PRL, five for rat PRL, and nine controls), a provocative test for PRL secretion was performed a week after the final immunization. The jugular veins on both sides were exposed under urethane anesthesia, and then a dopamine D2 receptor antagonist, metoclopramide (0.25 mg/kg), was administered through the jugular vein. The blood samples (100–200 µl) were collected at 0, 15, 30, 60, 90, and 120 min from the contralateral jugular vein. The needle (23 gauge) was inserted into the jugular vein through the muscle flap so that repeated blood samplings were possible without bleeding. Blood (3–5 ml) was taken from the heart at 180 min by needle puncture.

In the third series of immunizations using 21 female rats (five for porcine PRL, 10 for rat PRL, and six controls), blood samples (300–400 µl) were collected from the tail veins on d 7, 21, and 35 after the final immunization. In addition, blood samples were collected from 14 female rats in the morning for 4 consecutive days between 1 and 3 wk after the final immunization to examine the estrous cycles. In every series, the sera were separated immediately by centrifugation and stored at –40 C until the assay. The treatment of animals and experimental procedures were based on the Guidelines for Animal Care and Use Committee of Kansai Medical University.

Enzyme immunoassay (EIA) for rat PRL
An EIA for rat PRL was set up according to an EIA for human GH (22). Rat PRL (rPRL-B-9) and antiserum to rat PRL (anti-rPRL A.S.) were kindly provided from Dr. A. F. Parlow (National Hormone and Peptide Program). PRL concentrations were measured in duplicate in serum samples diluted 200 times with 0.01 mol/liter phosphate buffer containing 0.1% BSA, and column eluates with rPRL-B-9 (National Hormone and Peptide Program) as a reference. The minimum detectable quantity of rat PRL was 0.01 µg/liter using 100 µl samples, and the intra- and interassay coefficients of variation were 7.2 and 9.0%, respectively. The developed antibodies were found not to interfere with this EIA.

Bioassay for rat PRL
Nb2 rat lymphoma cells were kindly provided from Dr. T. Tanaka (National Children’s Medical Research Center, Tokyo, Japan), and bioassay for rat PRL was performed according to his original report (23) except that the cell proliferation was measured by Cell Counting Kit-8 (Dojindo, Tokyo, Japan). The minimal detectable quantity of rat PRL was 4 µg/liter using 10 µl of samples, and the intra- and interassay coefficients of variation were 10 and 12%, respectively. Bioactive PRL concentrations were measured at least in three series of diluted samples, and the bioactivity was determined at the linear part of the dilution curve using the same rat PRL standard (rPRL-B-9; National Hormone and Peptide Program) as EIA. Rat GH up to 10 mg/liter was found not to cross-react to this bioassay as originally reported (23).

EIA for rat testosterone and estradiol
Serum testosterone and estradiol concentrations were measured using commercially available kits (a testosterone kit from Assay Designs Inc., Ann Arbor, MI, and an estradiol kit from Cayman Chemical Co., Ann Arbor, MI). The minimal detectable quantities of rat testosterone and estradiol were 5.7 and 7.8 ng/liter, respectively.

Detection of antibodies against rat PRL
A protein G column method.
To detect IgG-bound PRL, a protein G column was used. Protein G agarose of 1 ml (Roche Diagnostics GmbH, Mannheim, Germany) was packed into a 2.5-ml syringe and equilibrated with 0.02 mol/liter sodium phosphate buffer (pH 7.0). Serum samples (100 µl) were applied on the column and the unbound proteins were washed away with 7 ml of the same buffer. Then the bound PRL-IgG complex was eluted with 0.1 mol/liter glycine-HCl buffer (pH 2.7) every 1 ml and neutralized immediately with 40 µl of 1 mol/liter Tris-HCl buffer (pH 9.0). IgG was mainly eluted from the second to the fourth tube. The amounts of PRL bound to IgG were determined by EIA and expressed as the ratio to the total PRL (percent) (antibody titers). When the PRL standard was applied on the column, no PRL was bound to protein G.

Polyethylene glycol (PEG) method.
PEG has been widely used for the screening of macroprolactinemia. The sera were mixed with cold PEG (final 12.5%) and centrifuged at 15,000 rpm for 10 min to remove precipitated antibody-bound PRL. The supernatant was collected for the determination of free PRL. Recovery was calculated by free PRL in the supernatant/total PRL x 100%.

Gel filtration chromatography.
Gel filtration chromatography was performed at 4 C using a 1- x 70-cm column of Ultrogel AcA 44 (IBF, La Garenne, France) and equilibrated with 0.01 mol/liter sodium phosphate buffer (pH 7.0) containing 0.1 mol/liter NaCl, 0.1% BSA, and 0.01% NaN3. Samples were applied on the column, and fractions of 1 ml were collected for PRL determination. The column was calibrated with various molecular weight markers (Sigma, St. Louis, MO).

Statistical analyses
Statistical analyses were performed using unpaired Student’s t tests and differences were considered to be statistically significant at P < 0.05. All values are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of antibodies against rat PRL
In the first series of immunizations using 25 male rats, we examined whether antirat PRL antibodies were developed by injecting rat PRL or heterologous PRL into rats. As shown in Fig. 1AGo, injection of bovine or porcine PRL expectedly increased the ratio of protein G-bound serum PRL, i.e. IgG-bound PRL, in four of five rats for bovine PRL (19.5 ± 6.6%, P = 0.001) and all five rats for porcine PRL (23.3 ± 2.0%, P < 0.0001) but not in control rats (0.1 ± 0.1%, n = 10). Injection of rat PRL into rats also led to the production of antirat PRL autoantibodies in four of five rats (41.0 ± 14.4%, P = 0.001).


Figure 1
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  		FIG. 1. Development of antirat PRL autoantibodies. A, The ratio of protein G-bound rat PRL in sera from control male rats (n = 10) and male rats immunized with bovine PRL (n = 5), porcine PRL (n = 5), and rat PRL (n = 5). Closed circles show the sera with anti-PRL antibodies and bars indicate the mean values. The asterisk indicates a significant difference between control rats and bovine PRL (P = 0.001), porcine PRL (P < 0.0001), or rat PRL (P = 0.001). B, The recovery ratio of rat PRL in the supernatant after precipitating PRL-IgG complexes with 12.5% PEG in sera from control rats (n = 10) and rats immunized with bovine PRL (n = 5), porcine PRL (n = 5), and rat PRL (n = 5). Closed circles show the sera with anti-PRL antibodies determined by the protein G method and bars indicate the mean values. The asterisk indicates a significant difference between control rats and bovine PRL (P = 0.0001), porcine PRL (P < 0.0001), and rat PRL (P = 0.002). C, Representative gel filtration profiles of rat pituitary PRL (upper panel), serum PRL without anti-PRL antibodies (middle panel), and serum PRL with anti-PRL antibodies (lower panel) on Ultrogel AcA 44 column (1 x 70 cm).

 
Then we compared the results of the protein G method with those of the PEG method, which is widely used for the screening of macroprolactinemia in humans, to examine the validity of the PEG method. The recovery of PRL after precipitating IgG-bound PRL in all of bovine, porcine, and rat PRL-injected rats significantly decreased, compared with that in control rats (18.6 ± 6.0%, P = 0.0001 for bovine PRL, 10.0 ± 1.9%, P < 0.0001 for porcine PRL, and 24.0 ± 12.5%, P = 0.002 for rat PRL vs. 71.7 ± 6.2% for control rats) (Fig. 1BGo).

The presence of antibody-bound rat PRL was further examined by gel chromatography, which is used to confirm macroprolactinemia (Fig. 1CGo). Rat pituitary PRL was mainly eluted at 23 kDa, and rat serum PRL without antirat PRL antibody was predominantly eluted at 23 kDa with a small amount of large molecular mass PRL, probably an aggregated form of PRL. In contrast, the main peak of serum PRL in 13 rats with antirat PRL antibodies was observed at 150 kDa, reflecting a complex of rat PRL and the antibody.

Serum PRL concentrations and the response to metoclopramide
In the second series of immunizations using 29 male rats, a dopamine D2 receptor antagonist, metoclopramide, was administered to rats that had been immunized with bovine PRL (n = 5), porcine PRL (n = 10), and rat PRL (n = 5), and control rats (n = 9). This time, antibodies against rat PRL were produced in all rats that had received PRL of any species, as judged by the PEG method (recovery less than 40%, n = 20). Basal serum PRL levels were compared among the four groups (total of 54 male rats including those in the first and second series of immunizations) (Fig. 2Go). The basal serum PRL concentrations were significantly higher in rats immunized with bovine PRL (46.5 ± 7.7 µg/liter, P = 0.0009), porcine PRL (81.8 ± 16.7 µg/liter, P = 0.0002), or rat PRL (124 ± 46.8 µg/liter, P = 0.0035), compared with that in control rats (15.8 ± 4.4 µg/liter). The rats that did not develop antibodies in the first immunization with bovine and rat PRL showed lower serum PRL concentrations.


Figure 2
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  		FIG. 2. Serum PRL levels in male rats with and without anti-PRL antibodies. Basal serum PRL concentrations in control male rats (n = 19) and those immunized with bovine PRL (n = 10), porcine PRL (n = 15), and rat PRL (n = 10). Closed circles show the sera with anti-PRL antibodies and bars indicate the mean values. The asterisk indicates a significant difference between control rats and those immunized with bovine PRL (P = 0.0009), porcine PRL (P = 0.0002), or rat PRL (P = 0.0035).

 
Then we compared the hemodynamics of serum PRL after metoclopramide injection in rats with and without antibodies against rat PRL. Serum PRL concentrations rapidly increased by the iv administration of metoclopramide with peaks around 30 min in both antibody-negative and -positive groups, followed by a gradual decline (Fig. 3AGo). The decreasing rate of serum PRL, which corresponds to the clearance of PRL from the circulation, seemed to be slow in rats with antibodies. As summarized in Fig. 3BGo, the time taken for serum PRL levels to decrease to 50% of the peak values was significantly (P = 0.0017) longer in anti-PRL antibody-positive rats (64.8 ± 7.9 min) than that in negative ones (22.6 ± 2.9 min).


Figure 3
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  		FIG. 3. PRL response to metoclopramide in male rats with [Ab (+)] and without [Ab (–)] anti-PRL antibodies. A, Dopamine D2 antagonist, metoclopramide (0.25 mg/kg), was administered iv via jugular veins in urethane anesthetized male rats with (n = 20) and without (n = 9) anti-PRL antibodies. Blood was taken from the opposite jugular veins at 0, 15, 30, 60, 90, and 120 min to determine serum rat PRL concentrations. B, The time taken for serum PRL levels to decrease to 50% of the peak values (T1/2) in rats with (closed circles) and without (open circles) anti-PRL antibodies. Bars indicate the mean values. The asterisk indicates a significant difference (P = 0.0017) between antibody-negative and -positive groups.

 
Changes in the gel filtration profiles of serum PRL were examined after metoclopramide injection in antirat PRL antibody-positive rats (Fig. 4Go). Monomeric and dimeric forms of PRL rapidly increased and returned to the basal levels within 180 min, whereas PRL bound to antirat PRL antibody (macroprolactin) kept increasing gradually during this period.


Figure 4
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  		FIG. 4. Gel filtration profiles of rat serum PRL during metoclopramide loading test. Representative gel filtration profiles of serum PRL after metoclopramide injection in rats with anti-PRL antibodies. Shaded areas indicate macroprolactin.

 
Bioactivity of antibody-bound PRL
The effects of anti-PRL antibodies on bioactivity were examined using serum samples in the first and second immunizations by the Nb2 rat lymphoma proliferation assay (Fig. 5Go). Bioactive PRL/immunoreactive PRL in sera of control rats varied from 1.5 to 3.5 (2.4 ± 0.3). The ratio was not significantly different in rats immunized with bovine PRL (1.8 ± 0.2, P = 0.14), even though anti-PRL antibodies were developed in these rats. In contrast, the ratio was significantly decreased in rats immunized with porcine PRL (0.8 ± 0.1, P < 0.0001) and rat PRL (1.2 ± 0.2, P = 0.0049), with considerable individual variations.


Figure 5
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  		FIG. 5. Effects of anti-PRL antibodies on the biological activity of rat PRL. The ratio of bioactive PRL to immunoreactive PRL in sera from control male rats (n = 8) and those immunized with bovine PRL (n = 10), porcine PRL (n = 15), and rat PRL (n = 10). Bioactive PRL was determined by the Nb2 lymphoma cell count 3 d after the application of serum samples referring the cell count when rat pituitary PRL preparations (rPRL-B-9) were applied. Immunoreactive PRL was determined by EIA for rat PRL referring the same rat pituitary PRL preparations as in bioassay. Among sera from 19 control rats, the bioactivities of PRL in 11 serum samples were below the detection limit of this bioassay system. Closed circles show the sera with anti-PRL antibodies and bars indicate the mean values. The asterisk indicates a significant difference between control rats and porcine (P < 0.0001) or rat (P = 0.0049) PRL.

 
Gonadal functions
Serum concentrations of testosterone were not significantly different between male rats with anti-PRL antibodies (3.86 ± 0.35 µg/liter, n = 33) and those without them (3.46 ± 0.47 µg/liter, n = 21), as shown in Fig. 6AGo. Serum estradiol concentrations were measured in 14 of 21 female rats in the third series of experiment for 4 consecutive days to examine estrous cycles. Estrous cycles were observed in all 14 female rats except one with antibodies, which showed persistently high estradiol levels. Peak serum concentrations of estradiol, probably reflecting preovulatory rises on proestrous phase, were not significantly different between female rats with anti-PRL antibodies (44.4 ± 6.0 ng/liter, n = 7) and those without them (46.3 ± 7.5 ng/liter, n = 6), as shown in Fig. 6BGo.


Figure 6
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  		FIG. 6. Gonadal functions in rats with (+) and without (–) anti-PRL antibodies (Ab). A, Serum testosterone concentrations in male rats with (n = 33) and without (n = 21) anti-PRL antibodies. B, The highest concentrations of estradiol in serum samples taken in the morning during 4 consecutive days in female rats with (n = 7) and without (n = 6) anti-PRL antibodies. The serum sample from a female rat with anti-PRL antibody was excluded because the serum estradiol levels were persistently high with unknown reason.

 
Changes in antibody titers
Changes in the titers of antibodies after the final immunization were examined in the third series of experiments using 21 female rats. Antibodies against rat PRL were produced in all rats immunized with porcine PRL (five of five) and rat PRL (10 of 10), but did not develop in controls (none of six), as judged by the protein G column method. As shown in Fig. 7AGo, the titers of anti-PRL antibodies did not show significant changes during 5 wk after the final immunizations. Serum PRL levels in female rats with antibodies were significantly (P < 0.05) higher than those without them throughout this period, as shown in Fig. 7BGo.


Figure 7
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  		FIG. 7. Changes in antibody titers (Ab) and serum PRL concentrations in female rats. A, The percentages of protein G bound PRL (antibody titers) in 15 female rats immunized with porcine PRL (n = 5) and rat PRL (n = 10) 1, 3, and 5 wk after the last immunization. B, Basal serum PRL concentrations in control female rats (n = 6) and those with anti-PRL antibodies (n = 15). The asterisk indicates a significant difference between control rats and those with anti-PRL antibodies 1 wk (P = 0.004), 3 wk (P = 0.033), and 5 wk (P = 0.037) after the last immunization.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data support the hypothesis that pituitary PRL, even if homologous, can elicit an immune response to produce anti-PRL autoantibodies. Regarding heterogeneous bovine and porcine PRL, having 60 and 64% homology with rat PRL, respectively (17), it is most probable that antibodies raised against different amino acids with shared protein sequences cross-reacted to rat PRL. Such a cross-reactivity of antibodies against peptides of different species or altered antigens has been previously demonstrated (24, 25). Regarding autoantibodies against homologous peptide, however, another explanation for the development is required. The break of tolerance by homologous proteins has been partly explained by the use of adjuvant, which stimulates the immune system (26). However, in this study, adjuvant alone did not stimulate the production of autoantibodies to endogenous rat serum PRL. Several posttranslational modifications of PRL have been reported (17): glycosylation in most species; phosphorylation in rats, bovines and birds; deamidation in rats, mice, sheep, and humans; and sulfation in ovines, sheep, and buffalo. We have recently shown that human pituitary PRL is phosphorylated at serine residues and partially dephosphorylated in blood (21). If pituitary forms of PRL such as phosphorylated ones are intolerant to the immune system, leakage of such forms of PRL due to hypophysitis or lack of dephosphorylation may cause an autoimmune response. There are several reports that posttranslational modifications such as glycosylation and phosphorylation of some proteins create neoepitopes for autoantibody production (27, 28).

This animal model with anti-PRL antibodies can explain several clinical characteristics and the underlying mechanisms of macroprolactinemia in humans. First, this model clearly demonstrates how macroprolactinemia is associated with hyperprolactinemia. The clearance of PRL was shown to be slower in the presence of anti-PRL antibodies, suggesting that antibody-bound PRL is big enough to be confined to vascular spaces, and hyperprolactinemia develops due to the delayed clearance of PRL rather than increased production. Such an idea is supported by gel filtration profiles during the metoclopramide test, whereby dimeric and monomeric PRL levels increased and rapidly returned to the basal levels, whereas macroprolactin kept increasing. Normal response of PRL to metoclopramide despite hyperprolactinemia implicates a lack of negative feedback (29), probably because PRL-autoantibody complex cannot freely access to the hypothalamus, and this may also contribute to the pathogenesis of hyperprolactinemia. Second, this model demonstrates why patients with macroprolactinemia are usually asymptomatic. Anti-PRL antibodies developed by porcine and rat PRL had reduced PRL bioactivity, although such reduced bioactivity was not observed in rats immunized with bovine PRL. Because PRL molecule has several epitopes, it is possible that several kinds of anti-PRL antibodies develop in rats immunized with PRL of different species. Some antibodies may bind to the epitopes important for the binding to PRL receptors and reduce bioactivity; others may bind to the epitopes unrelated to receptor binding. We have recently reported that epitopes of anti-PRL autoantibodies in patients with macroprolactinemia were located near binding site 1 to human PRL receptors (30), raising a possibility that anti-PRL autoantibodies may compete with the PRL molecule for binding to its receptors, resulting in reduced bioactivity in vivo. Normal levels of sex hormones despite hyperprolactinemia, as shown in this study, may support this possibility because hyperprolactinemia reportedly causes gonadal dysfunction (31, 32). Lastly, this model demonstrates that anti-PRL autoantibodies are stable with time, for at least 5 wk, and supports the idea that macroprolactinemia is a chronic condition in humans.

In conclusion, rat pituitary PRL has antigenicity, leading to the production of anti-PRL autoantibodies, and these autoantibodies reduce PRL bioactivity and delay PRL clearance. This animal model can explain asymptomatic hyperprolactinemia in human macroprolactinemia.


   Acknowledgments
 
We thank Dr. A. F. Parlow (a scientific director of the National Hormone and Pituitary Program, Torrance, CA) for supplying PRL and the antisera and his scientific advice. We also thank Kansai Medical University Laboratory Center for providing experimental instruments and assistance.


   Footnotes
 
This work was supported in part by the Japanese Ministry of Education, Science Sports, and Culture and the Japanese Private School Promotion Foundation.

First Published Online February 15, 2007

Abbreviations: EIA, Enzyme immunoassay; PEG, polyethylene glycol; PRL, prolactin.

Received September 5, 2006.

Accepted for publication February 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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