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A dual-monoclonal sandwich ELISA specific for hepcidin-25.

Recent reports indicate that hepcidin plays a key role in the regulation ofiron metabolism inhumans (1). Hepcidin exerts its effects by binding to the iron transporter ferroportin, which is present in macrophages and on the basolateral side of intestinal enterocytes (2-6). Through an incompletely understood mechanism, binding of hepcidin to ferroportin causes internalization and degradation of ferroportin, resulting in cells being unable to export iron across the plasma membrane (2-6).

The recent discovery of hepcidin and elucidation of its unique properties have added important information to the field of iron metabolism (7-10). Evidence has emerged that in certain types of hereditary hemochromatosis, the cause of the disease is a deficiency of hepcidin, resulting in unregulated uptake of iron and subsequent iron overload (11-13). In contrast, in anemia of chronic disease and anemia of cancer, data suggest that hepcidin concentrations are increased, causing decreased absorption of iron and increased sequestration of iron in the reticulo-endothelial system, which together account for the observed anemia (6, 14-18). It is also suggested that patients with chronic kidney disease may have increased hepcidin, which could contribute to their observed renal anemia that is treated with erythropoietin and oral iron (19,20).

One of the major issues in further exploring the role of hepcidin in disorders of iron metabolism is the reported difficulty in developing a relatively simple and robust immunoassay for measuring hepcidin-25 in human serum (21). Because of the small size of the molecule (25 amino acids) and the fact that it contains 4 disulfide bonds (22, 23), it has been difficult to raise suitable antibodies. In addition, because of its small size and compact structure, hepcidin is not well suited to analysis via Western blotting.

Further complicating matters, the active 25-amino acid protein is derived from preprohepcidin, an 84-amino acid precursor that is first cleaved at the N-terminus to yield the inactive 64-amino acid pro-hepcidin (24). Prohepcidin is further processed via removal of its N-terminal 39 amino acids to yield hepcidin-25 (the active form of the hormone), which can then undergo further N-terminal processing to yield hepcidin-20 and hepcidin-22, both of which are inactive (24). Hence, early competitive immunoassays struggled to differentiate physiologically relevant hepcidin-25 from the inactive, less relevant hepcidin-22, hepcidin-20, and prohepcidin species (25, 26).

As a result, many of the described assays specific for hepcidin-25 have been LC-MS-type assays (27-29). Although such assays are accurate and precise, their complexity, expense, and high level of operator expertise prevent their implementation into most routine clinical laboratories. To address the need for a highly specific and robust immunoassay, we have developed a sandwich ELISA by using 2 monoclonal antibodies for the measurement of hepcidin-25 in human serum. In this study, we show that our sandwich ELISA specifically measures hepcidin-25 and has a high correlation with LC-MS (28). In addition, we demonstrate that hepcidin-25 concentrations are increased in patients with cancer and rheumatoid arthritis compared with healthy individuals.

Materials and Methods

HUMAN SPECIMENS

One hundred serum samples from healthy volunteers (ages 18-64 years, mean age 37 years) were purchased from Valley Biomedical. The samples spanned a broad ethnic distribution (24 African American, 24 Hispanic, and 52 Caucasian), with each group consisting of 50% women and 50% men. Among women, 40 of 50 individuals were age [less than or equal to] 45 years. We also obtained 34 serum samples from cancer patients. These specimens included 17 samples from patients with hematological malignancies (8 acute myelocytic leukemia, 3 lymphoma, 2 multiple myeloma, 2 myelodysplastic syndrome, 1 chronic lymphocytic leukemia, and 1 chronic myelocytic leukemia) and 17 samples from patients with nonhematological tumors (3 renal, 2 head and neck, 2 melanoma, 2 lung, 2 squamous cell carcinoma, 1 ameloblastoma, 1 prostate, 1 thyroid, 1 colon, 1 hepatic, and 1 urethral). Samples from 76 patients with rheumatoid arthritis were also obtained from patients who gave their permission for serum samples to be banked for future exploratory analysis. After obtaining protocol approval from an institutional review board and proper informed consent, all samples were collected, stored, and deidentified to protect patient privacy. Samples were received on dry ice and stored at -70[degrees]C before analysis of hepcidin levels. Ferritin analysis was performed by using a Beckman DxI 800 chemiluminescent immunoassay.

HEPCIDIN-25 GENERATION, PRODUCTION OF ANTIHEPCIDIN ANTIBODIES, AND LABELING OF ANTIBODIES

Synthesized human hepcidin-25, hepcidin-22, and hepcidin-20 proteins were obtained from Peptides International or produced recombinantly. Proteins were divided into aliquots and stored at -70[degrees]C before use. Two different monoclonal antihepcidin antibodies (antihepcidin-1 and antihepcidin-2) were produced by immunizing mice with either human hepcidin-25 protein or an N-terminal hepcidin peptide (amino acids 1-7) conjugated with a 5-amino acid peptide linker to an ovalbumin peptide (amino acids 232-336) sequence. Of each antibody, 1 mg was biotinylated by using a Pierce kit, and 1 mg was labeled with ruthenium by using a MesoScale Discovery (MSD)2 kit for electro-chemiluminescent detection. Afterward, the labeled antibodies were diluted in 50% glycerol and stored at -20[degrees]C.

HEPCIDIN ELISA

A human hepcidin-25 MSD ELISA was constructed by using the reagents described above. Streptavidin-coated and blocked wells were incubated for 1 h with biotinylated antihepcidin-2 antibody (2 mg/L). Afterward, the wells were aspirated and washed 3 times with Tris-buffered saline with Tween (TBST) containing 10 mmol/L Tris, pH 7.40, 150 mmol/L NaCl, with 1 mL Tween 20 per liter. Next, 100/[micro]L hepcidin standards (varying concentrations of hepcidin-25 protein in assay buffer consisting of 50 mmol/L HEPES, pH 7.40, 150 mmol/L NaCl, 10 mL/L Triton X-100, 5 mmol/L EDTA, and 5 mmol/L EGTA) was added to the wells to generate a calibration curve. Serum samples were diluted 1:20 in an assay buffer and added to their respective wells, and the ELISA plate was incubated for 1 h at room temperature. After aspiration, the wells were washed 3 times with TBST, and 100/[micro]L of a 1:1000 dilution of conjugate antibody (ruthenium-labeled antihepcidin-1 antibody, 1 mg/L) was added to the wells for a 1-h incubation at room temperature. After aspiration, the wells were washed 3 times with TBST, and the plate was developed by using an MSD reader, which recorded ruthenium electrochemiluminescence.

MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT (MALDI-TOF) ANALYSIS

Antibodies were evaluated for their ability to immuno-precipitate endogenous hepcidin from human serum via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer on antibody bound hepcidin performed after sample reduction. The different antihepcidin antibodies were coated onto wells of a 96-well Nunc standard ELISA plate in carbonate-bicarbonate (pH 9.4) buffer for 1 h at room temperature at a concentration of 2 mg/L. The wells were aspirated and washed 3 times with TBST. Human serum samples containing a known amount of hepcidin (diluted in assay buffer) were added at 100 [micro]/well for1hat room temperature. The wells were aspirated and washed 3 times with TBST. Captured hepcidin was eluted by adding 40 [micro]L/well of 0.1% formic acid for 5 min at room temperature. Eluted samples were collected and concentrated with a C4 ZipTip (Millipore). A 0.5-[micro]L volume of sample was spotted onto a MALDI target, and an equal volume of matrix solution was added (50% acetonitrile, 0.1% trifluoroacetic acid saturated with [alpha]-cyan-4-hydroxycinnamic acid). The sample was dried and analyzed with a 4700 TOF-TOF Mass Spectrometer (Applied Biosystems) operated in linear mode.

DATA ANALYSIS

MSD software and SigmaPlot version 8.0 were used for fitting ELISA calibration curves. Data were plotted by using version 2.98 of the program FigP (Biosoft). For each group of individuals or patients studied, the median, 25th percentile, 75th percentile, and interquartile range were determined. Comparisons of hepcidin-25 concentrations between respective groups were performed by using the Wilcoxon nonparametric rank sum test. In each case, a P value of <0.05 was considered to indicate statistical significance.

Results

In the first series of experiments, MALDI-TOF spectrometry was used to determine which hepcidin species were bound to antihepcidin antibodies in human serum. Antihepcidin-1 antibody bound mainly to hepcidin-25 (Fig. 1, A and B) and to a lesser extent to hepcidin-20. There was no indication that antihepcidin-1 antibody bound to prohepcidin (predicted molecular weight of 7.7 kDa).

Similar experiments were performed to determine which hepcidin species were bound by antihepcidin-2 antibody. We observed that the antihepcidin-2 antibody was only bound to hepcidin-25 in human serum (Fig. 1, C and D). No hepcidin-20, hepcidin-22, prohepcidin, or other hepcidin species were bound, indicating that the antihepcidin-2 antibody was highly specific for hepcidin-25.

The antibodies were then investigated for pairing in a sandwich ELISA. The optimal pairing was found to be antihepcidin-2 antibody as the capture antibody and antihepcidin-1 antibody as the conjugate antibody. Fig. 2A shows a typical calibration curve obtained with the final ELISA orientation described above, in which hepcidin-25 protein was prepared at a concentration of 10 [micro]g/L and serially diluted to create a calibration curve. Based on a 3-SD evaluation from the zero calibrator, the limit of quantification of the ELISA was determined to be 0.01 [micro]g/L and therefore indicated that the ELISA was adequate for measuring serum hepcidin-25 concentrations, on the basis of previous estimates ofhuman serum hepcidin-25 concentrations measured by LC-MS assays (28). The sandwich ELISA was confirmed to be specific for hepcidin-25 (Fig. 2A) and did not recognize hepcidin-20 or hepcidin-22. To ensure that hepcidin-20 and hepcidin-22 were not being generated from hepcidin-25 during the activation of proteolytic enzymes associated with the clotting process, we collected matching serum and EDTA plasma samples from 6 healthy donors at 2 different time points (12 serum samples and 12 matching EDTA plasma samples). We compared hepcidin-25 results obtained from each pair of matched samples by using the ELISA. All serum results were within 15% of the EDTA plasma values, suggesting that hepcidin-20 and hepcidin-22 were not generated to any appreciable degree during the clotting process. Finally, ELISA dilution curves for the recombinant standard and actual human serum samples were determined to be parallel, and ELISA demonstrated dilutional linearity.

On the basis of these results, we compared the sandwich ELISA method to a previously described gold standard LC-MS assay shown to be specific for hepcidin-25 (28) by using 52 human serum samples from a mixture of healthy individuals and cancer patients. The hepcidin-25 ELISA values showed a correlation of r = 0.98 (P < 0.001) with the LC-MS values, confirming that the sandwich ELISA specifically measured hepcidin-25.

Freeze-thaw stability was evaluated by testing 4 different serum samples. The results showed a consistent 80%-120% recovery, even after 5 freeze-thaw cycles (sample A: 0.16, 0.16, 0.17, 0.17, 0.17, and 0.17 [micro]g/L, respectively; sample B: 4.5, 4.4, 4.6, 4.6, 4.6, and 4.4 [micro]g/L respectively; sample C: 8.9, 9.6, 9.7, 9.7, 9.9, and 9.6 [micro]g/L, respectively; sample D: 15.1, 15.1, 15.4, 15.7, 15.4, and 15.4 [micro]g/L, respectively). The imprecision of the ELISA was assessed by using human serum samples containing 0.16, 4.5, and 15.1 [micro]g/L of endogenous hepcidin-25. Intraassay (n = 20) imprecision results (CVs) were 3.4%, 4.5%, and 3.5%, respectively.

To assess the recovery of hepcidin-25 added into human serum, hepcidin-25 protein was added to 3 different human serum samples, each containing verylow concentrations of endogenous hepcidin-25, at concentrations of 250, 25, 2.5, and 0.25 [micro]g/L. These samples were analyzed by using the sandwich ELISA. Mean (SD) results were 287 (6) [micro]g/L, 24.2 (0.2) [micro]g/L, 2.0 (0.1) [micro]g/L, and 0.23 (0.01) [micro]g/L, respectively.

[FIGURE 1 OMITTED]

Next, 100 serum samples from healthy individuals (50 men and 50 women) were analyzed by ELISA. The concentrations of hepcidin-25 ranged from <0.02 to 25 [micro]g/L (median 1.20 [micro]g/L, 25%-75% range 0.42-3.07 [micro]g/L) (Fig. 2A), consistent with the concentrations previously reported in healthy individuals by using an LC-MS assay (28). Hepcidin-25 concentrations in healthy individuals were lower in women (median 0.84 [micro]g/L, 25%-75% range 0.42-2.35 [micro]g/L) (Fig. 3B) than in men (median 1.70 [micro]g/L, 25%-75% range 0.57-4.76 [micro]g/L). Hepcidin-25 concentrations in these 100 individuals were also compared with serum ferritin concentrations (Fig. 3C) and were found to correlate with serum ferritin (r = 0.71, P < 0.001). Not surprisingly, hepcidin-25 concentrations were only modestly correlated with hemoglobin concentrations (r = 0.22, P = 0.03). We observed that 4 of 50 men and 27 of 50 women had ferritin concentrations <10 [micro]g/L (Fig. 3C), suggesting that many individuals were iron deficient. Iron-deficient individuals would be expected to have very low hepcidin concentrations, which is what we observed. All but 4 of these individuals had a hepcidin-25 concentration of <1 [micro]g/L, and the remaining 4 individuals had hepcidin-25 concentrations between 1 and 2 [micro]g/L. Because the data in Fig. 3A did not appear to be evenly distributed, we also plotted the data as a frequency distribution of log hepcidin. A much more even distribution of data was observed (Fig. 3D), with a log hepcidin mean of 0.01 and a 95% CI of -0.14-0.16.

Finally, we compared sandwich ELISA hepcidin-25 results in patients with cancer (n = 34) and rheumatoid arthritis (n = 76) to results of healthy individuals (n = 100). These comparisons (Fig. 4) demonstrated that hepcidin-25 concentrations were increased in patients with cancer (median 54.8 [micro]g/L, 25%-75% range 23.2-93.5 [micro]g/L) and rheumatoid arthritis (median 10.6 [micro]g/L, 25%-75% range 5.9-18.4 [micro]g/L) compared with healthy individuals (median 1.20 [micro]g/L, 25%-75% range 0.42-3.07 [micro]g/L). In patients with rheumatoid arthritis, hepcidin-25 concentrations were higher in men (median 21.3 [micro]g/L, 25%-75% range 15.2-25.1 [micro]g/L, n = 9) than women (median 9.7 [micro]g/L, 25%-75% range 5.7-18.3 [micro]g/L, n = 67). These data further underscored the large increase in hepcidin-25 concentrations in women with rheumatoid arthritis (median 9.7 [micro]g/L, 25%-75% range 5.7-18.3 [micro]g/L, n = 67) compared with healthy women (median 0.84 [micro]g/L, 25%-75% range 0.42-2.35 [micro]g/L, n = 50). There was no appreciable difference in hepcidin-25 concentrations between men (median 44.2 [micro]g/L, 25%-75% range 23.2-85.8 [micro]g/L, n = 21) and women (median 54.8 [micro]g/L, 25%75% range 22.1-93.5 [micro]g/L, n = 13) who had cancer. Interestingly, cancer patients with hematological (median 76.3 [micro]g/L, 25%-75% range 54.8-112.0 [micro]g/L, n = 17) and nonhematological malignancies (median 38.9 [micro]g/L, 25%-75% range 16.2-44.2 [micro]g/L, n = 17) each demonstrated markedly increased hepcidin-25 concentrations compared with healthy individuals.

[FIGURE 2 OMITTED]

Because of the log-normal distribution of hepcidin-25 concentrations for healthy individuals (Fig. 3D), group distributions were not assumed to be normal. Therefore, statistical analyses were performed that did not assume normality. For each group of individuals or patients studied, the median, 25th percentile, 75th percentile, and interquartile range were summarized (Table 1). Next, comparisons were performed between respective groups by using the Wilcoxon nonparametric rank sum test. The results from these comparisons (Table 2) indicated that for each comparison performed, the differences in hepcidin-25 concentrations between respective groups were statistically significant.

Discussion

Our results demonstrate that the sandwich ELISA described here is capable of measuring hepcidin-25 concentrations in human serum. This work builds upon previously reported assays for hepcidin (25,26, 30-33). Compared with previous reported immunoassays, however, which are single antibody and competitive (25, 26, 30 -33), this assay uses 2 independent monoclonal antibodies in a sandwich format to specifically measure hepcidin-25.

Compared with existing assays that use a competitive format (either ELISA or RIA), this method has advantages inherent in the sandwich assay format. In particular, responses are directly correlated with increasing hepcidin concentrations. In contrast, in competitive ELISA methods, absorbance values are inversely correlated with hepcidin concentrations (25, 26, 30 -33). Also, because competitive ELISA methods rely on a single antibody, specificity may be an issue, and indeed, some competitive assays may cross-react with prohepcidin or other inactive species, making results difficult to interpret (25, 26). Another advantage of our assay is its improved limit of quantification of 0.01 [micro]g/L compared with existing assays (25, 26, 30 -33). This may be especially important, since we observed that 44% of healthy volunteers had serum hepcidin-25 concentrations of <0.9 [micro]g/L. Interestingly, 31 of 100 healthy individuals (4 men and 27 women) also had very low ferritin concentrations (< 10 [micro]g/L), suggesting that some healthy donors, particularly premenopausal women, may be iron deficient, perhaps because of menstrual blood loss and/or frequent blood donations. As expected, all individuals with ferritin concentrations <10 [micro]g/L had very low hepcidin-25 concentrations.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

As a result of the 2 antibodies used, our sandwich ELISA is specific for hepcidin-25 and correlates extremely well with a previously described gold standard method LC-MS assay for hepcidin-25 (28). From a practical standpoint, the advantage of an ELISA over an LC-MS is that the ELISA can be implemented in clinical laboratories that do not have the complex equipment or the highly specialized operator expertise required to perform LC-MS type assays. In addition, ELISA has the potential for higher throughput than an LC-MS assay and therefore provides the basis for first dual monoclonal sandwich immunoassay, which can be used to measure hepcidin-25.

This higher throughput is particularly relevant for a clinical assay that measures human serum hepcidin-25 concentrations (1,21). The sandwich ELISA developed and described here can be used clinically to further increase our understanding of the role of hepcidin-25 in regulating iron metabolism. Using ELISA, we were able to demonstrate that circulating hepcidin-25 concentrations were increased in patients with cancer and rheumatoid arthritis (34) compared with healthy individuals. Data obtained from cancer patients, however, must be interpreted with caution. We studied a limited number of patients, and some of the most common cancer types were relatively underrepresented. It will be important to investigate additional factors such as the effect of radiation (which may induce cytokines) and coexisting infections on hepcidin-25 concentrations in patients with different tumor types.

In addition to these observations, there are several other uses for the sandwich ELISA that we developed. In cases of suspected hereditary hemochromatosis, the low limit of quantification of ELISA should be able to indicate

whether abnormally low concentrations of hepcidin are responsible for the disease, thus obviating the need for a complex genetic workup. In suspected cases ofsecondary iron overload, measurement of serum hepcidin-25 concentrations would provide useful information as well (35, 36). In patients with anemia of chronic disease that is unresponsive to erythropoietin, this hepcidin ELISA may be used to verify that increased serum hepcidin is at least partly responsible for the erythropoietin-resistant anemia. Likewise, in the setting ofchronic renal disease, this ELISA could be used to determine serum hepcidin concentrations to better direct therapy.

This sandwich ELISA could also be used to help diagnose iron deficiency anemia in difficult cases in which it may coexist with anemia of chronic disease. In uncomplicated iron deficiency, hepcidin concentrations would be expected to be quite low, whereas in anemia of chronic disease without coexisting iron deficiency, hepcidin concentrations would be expected to be increased (1, 7-10). Thus, in patients with anemia of chronic disease, a relatively low serum hepcidin concentration might also indicate the presence of coexisting iron deficiency.

In summary, our hepcidin sandwich ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism. Unlike recently described immunoassays (25,26, 30-33), the use of the 2 antibodies in the sandwich format provides specificity for the active form of the protein, a limit of quantification of 0.01 [micro] g/L, and a broad dynamic range. It is thus possible that this assay or one similar to it could become part of the routine iron profile panel performed on automated clinical instruments. As a result, patient workups that currently include serum iron, ferritin, and percent transferrin saturation may someday also include hepcidin-25.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data, (b) drafting or revising the article for intellectual content, and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: A.M. Butterfield, Eli Lilly; V.J. Wroblewski, Eli Lilly.

Consultant or Advisory Role: None declared.

Stock Ownership: A.M. Butterfield, Eli Lilly; P. Luan, Eli Lilly; D.R. Witcher, Eli Lilly; J. Manetta, Eli Lilly; A.T. Murphy, Eli Lilly; V.J. Wroblewski, Eli Lilly; R.J. Konrad, Eli Lilly.

Honoraria: None declared.

Research Funding: P. Luan, Eli Lilly; R. Witcher, Eli Lilly; J. Manetta, Eli Lilly; A.T. Murphy, Eli Lilly; R.J. Konrad, Eli Lilly.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played a direct role in the design and choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: We thank Paula Santa and Jayne Talbot for support and Michael Knierman, Bill Alborn, Patrick Haslett, Guoqing Cao, and Ming-Dauh Wang for technical assistance. This work was supported entirely by Eli Lilly and Company.

References

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Anthony M. Butterfield, [1] Peng Luan, [1] Derrick R. Witcher, [1] Joseph Manetta, [1] Anthony T. Murphy, [1] Victor J. Wroblewski, [1] and Robert J. Konrad [1] *

[1] Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN.

* Address correspondence to this author at: Eli Lilly and Company, Indianapolis, IN 46285. Fax 317-276-5281; e-mail konrad_robert@lilly.com.

Received June 3, 2010; accepted August 12, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.151522

[2] Nonstandard abbreviations: MSD, MesoScale Discovery; TBST, Tris-buffered saline with Tween.
Table 1. Hepcidin-25 concentrations for groups of
healthy individuals or patients. (a)

 25th
 n percentile Median

Healthy men 50 0.57 1.70
Healthy women 50 0.42 0.84
All healthy individuals 100 0.42 1.20
Nonhematological cancer patients 17 16.2 38.9
Hematological cancer patients 17 54.8 76.3
Rheumatoid arthritis patients 76 5.9 10.6

 75th Interquartile
 percentile range

Healthy men 4.76 4.19
Healthy women 2.35 1.93
All healthy individuals 3.07 2.65
Nonhematological cancer patients 44.2 28.0
Hematological cancer patients 112.0 57.2
Rheumatoid arthritis patients 18.4 12.5

(a) For each group of individuals or patients studied, the
median, 25th percentile, 75th percentile, and interquartile range
were determined.

Table 2. Comparisons of hepcidin-25 concentrations
between respective groups of healthy individuals
and patients. (a)

 P

Healthy men (n = 50) vs healthy women (n = 50) 0.034

Non-hematological cancer patients (n = 17) vs healthy <0.001
 individuals (n = 100)

Hematological cancer patients (n = 17) vs healthy <0.001
individuals (n = 100)

Rheumatoid arthritis patients (n = 76) vs healthy <0.001
individuals (n = 100)

(a) Comparisons of hepcidin-25 concentrations between healthy men and
women and between healthy individuals and patients with cancer or
rheumatoid arthritis were performed by using a Wilcoxon nonparametric
rank sum test.
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Title Annotation:Proteomics and Protein Markers
Author:Butterfield, Anthony M.; Luan, Peng; Witcher, Derrick R.; Manetta, Joseph; Murphy, Anthony T.; Wrobl
Publication:Clinical Chemistry
Date:Nov 1, 2010
Words:5031
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