- Email: [email protected]
Commercial immunoassays for human relaxin-2
Accepted Manuscript Commercial immunoassays for human Relaxin-2 Dennis R. Stewart PII:
S0303-7207(19)30004-8
DOI:
https://doi.org/10.1016/j.mce.2019.01.004
Reference:
MCE 10366
To appear in:
Molecular and Cellular Endocrinology
Received Date: 14 November 2018 Revised Date:
6 January 2019
Accepted Date: 7 January 2019
Please cite this article as: Stewart, D.R., Commercial immunoassays for human Relaxin-2, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.01.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Commercial Immunoassays for Human Relaxin-2
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[email protected]
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Dennis R. Stewart Molecular Medicine Research Institute 428 Oakmead Pkwy, Sunnyvale, CA 94085 USA
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Formerly: Novartis Pharmaceuticals, 1 Co-founder Corthera Inc., UC Davis School of Medicine
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Throughout the commercial development of relaxin/serelaxin, several different immunoassays have been used by the pharmaceutical company’s development programs. These assays have been validated for submission of GLP preclinical and clinical studies to the FDA and EU regulatory bodies. The requirements for these assays exceed that of most research assays commonly developed in academic research and have been made available directly from the pharmaceutical companies or indirectly through a third part vendor.
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In addition to these assays, there are also several commercial immunoassays for human relaxin that have appeared in many recent publications measuring relaxin2 in clinical studies. Typically, these commercial assays have not been validated by the vendor, nor any validation reported by the authors using them. In one article, authors cite prior validation of the Immundiagnostik assay which is actually nonexistent [1]. In other articles, authors did not even give the name of the assay they used [2] or simply refer to it as “commercially available ELISA Kit” [3]. In this latter case, concentrations reported for myocardial infarction were higher than seen in pregnancy, but these extraordinarily high concentrations were not discussed. The publication of these studies using questionable assays and results 1
Novartis was not affiliated and had no input into the design, implementation or the analysis of any of the subject matter of this presentation.
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indicates a lack of attention by authors, reviewers and editors which can lead to erroneous conclusions.
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The use of unknown and unvalidated assays presents a serious problem to the understanding of the role of relaxin-2 and potential treatment with serelaxin in serious medical conditions. This review examines some of the clinical articles that have been published and the assay used to determine relaxin-2.
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Forms and Sources of Human Relaxin Three forms of relaxin have been identified in the human and are commonly referred to as relaxin-1, relaxin-2 and relaxin-3 (often referred to as H1, H2, and H3. Relaxin-1 and relaxin-2 bind to the same receptor (RXFP1) which has wide distribution in the body while relaxin-3 has its own cognate receptor referred to as RXFP3 but distribution of the receptor is primarily localized in brain tissues with very low expression in peripheral tissues [4].
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The expression of relaxin-1 is in doubt but it may be expressed in decidua and placenta [4]. Relaxin-2 is produced in the corpus luteum, decidua, placenta, mammary glands, and prostate [4]. Relaxin-2 is found in circulation during the mid and late luteal phase of the menstrual cycle and peaks around 50 pg/ml [5-8]. It rises rapidly in pregnancy to peak in the first trimester at concentrations of 1.5 ng/ml and then falls gradually during the second and third trimesters to slightly less than 1 ng/ml [8-11]. In men, relaxin is found in seminal plasma at concentrations ranging from 6 - 140 ng/ml [12] but has not been definitively measured in plasma. Relaxin-3 is primarily a neuropeptide and not found in general circulation [4]
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Relaxin-2 is the primary focus of this article as it is the form found in circulation and also the focus of commercial development. Relaxin-2 is name of the endogenous form of the hormone while serelaxin is the International nonproprietary name for pharmaceutical relaxin but they are identical structures. Principles of Assay Validation There are many guides for proper validation of immunoassays and several excellent examples are DeSilva et al., [13] and Andreasson et al., [14]. In addition, the FDA has published guidance for industry in bioanalytical method validation [15]. These guides describe the same general principles for assay validation. These include specificity, selectivity, precision, accuracy, range of quantification, dilutional linearity, parallelism with standards, and recovery. The identity of the
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reference material must be fully understood and comparable with the target molecule.
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Additionally, in running an assay there needs to be quality control samples, criteria for assay acceptance, records and criteria for drift in assays, checks of sample stability in the matrix used, and other measures that are fundamental to assay quality.
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Commercial Relaxin-2 Assays A recent online search for commercial relaxin-2 assays has identified at least 9 kits available but only two of these have been most often cited. These are the R&D Systems Quantikine ELISA for Human Relaxin-2 and the Immundiagnostik Relaxin ELISA K9210. These assays will be further discussed.
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1. R&D Systems ELISA (DRL 200) This assay was co-developed by R&D Systems and Corthera, Inc. (prior to the acquisition by Novartis). This was done to provide the relaxin research community with access to a validated assay rather than through material transfer agreement programs as done earlier by Genentech and Connetics. The reference material used for this assay is recombinant serelaxin provided by Corthera, Inc. This assay uses a monoclonal capture antibody and a polyclonal labeled detection antibody. Its stated range is 7.8 to 500 pg / ml. Validation included within and between assay precision. The kit provides direct measurement in cell culture fluid, serum or plasma (heparin or EDTA as anticoagulant).
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Further validation included cross reactivity studies using several human peptides in the relaxin family which included IGF-I, IGF-II, insulin, proinsulin, insulin like 3, relaxin-1 and relaxin-3. Cross reactivity was not found at 50 ng/ml for any of these peptides. Relaxins from canine, mouse, porcine and rat did also not cross react at 50 ng/ml. Interference in the relaxin assay was also examined for all of these peptides and none was found. Dilutional linearity was determined for samples naturally containing relaxin or spiked samples and found to be acceptable over a range of 1:2 down to 1:16. 2. Immundiagnostik ELISA
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This assay utilized two different polyclonal antibodies for capture and detection. The reference material is not described and the required specificity, selectivity, interference, and full dilutional linearity studies were not reported. Partial dilutional linearity was listed for 1/5 – 1/7 dilutions, a very narrow and selected range. Only insulin was tested for cross reactivity. Cross reactivity of relaxin-1, relaxin-3 and other relaxin related peptides in this assay was not reported.
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This kit requires sample preparation prior to assay according the kit directions. Serum or plasma samples require at least a 1:3 dilution with assay buffer prior to analysis. Additionally, to avoid interference with heterophilic antibodies it is recommended that samples be treated twice with Anti Interference Reagent (5% v/v, shaken for 1 hour and centrifuged). Urine samples require a 1:4 dilution and seminal plasma samples a 1:10 dilution.
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3. Other commercial immunoassays Other commercial relaxin assays used in recent publications measuring serum relaxin in patient populations include kits from ADL Inc., Phoenix Pharmaceuticals, and ThermoFisher. The Phoenix kit recommends each plasma or tissue sample be extracted using C-18 SEP-Columns prior to assay. No validation data nor source of reference material could be found for these assays.
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Assays Used to Measure Relaxin-2 in Recent Clinically Oriented Articles There has been a general increased interest in relaxin-2 with the commercial development of serelaxin for treatment in acute heart failure. This has brought about many studies measuring relaxin-2/serelaxin in circulation in a number of clinical indications. This section reviews some of publications and which relaxin-2 assay was used in support of these studies. 1. R&D Systems Assay This immunoassay has been used by Corthera and Novartis for their clinical studies in the development of serelaxin in acute heart failure.2 This assay was also used in studies by Novartis looking at serelaxin pharmacokinetics
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This assay was used only for pharmacokinetics and not manufacturing or product released purposes.
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in patients with hepatic impairment [16], renal impairment [17] or ethnic sensitivity studies [18, 19].
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The R&D Systems assay was used to measure serum relaxin-2 in patients with ovarian cancer [20], aneurysm formation [21], shoulder instability [22], peripartum cardiomyopathy [23] and in female athletes [24-26].
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2. Immundiagnostik Assay The Immundiagnostik assay has been used in reports of acute heart failure [1, 27-31] as well as studies on chronic heart failure [32, 33]. It has also been used to examine acute heart failure patients and predict long term survival [31].
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The Immundiagnostik assay has also been used for reproductive studies including studies of women with premenstrual dysphoria [34] or prediction of preterm delivery in low risk pregnancies [35]. Additional indications studied are subjects with multiple sclerosis [36] or as a biomarker for prostate cancer [37]. This last article used a kit from ALPCO but it is believed to be the same as the Immundiagnostik assay.
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3. Other Commercial Relaxin Assays An assay from ADL Inc. was used in a report of relaxin in patients with congestive heart failure [38].
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A relaxin radioimmunoassay kit from Phoenix Pharmaceuticals was used in a study of relaxin concentrations in patients with diabetes [39].
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A relaxin assay from ThermoFisher was reportedly used to measure relaxin concentrations in a study of acute myocardial infarction [3]. However, the source of the assay was not reported in the original article and only revealed later in unpublished communications with the author.
Comparison of Assays There have been no published studies of head to head comparisons of any of the relaxin assays. However, some comparisons can be drawn from results of similar studies using different assays. Circulating relaxin has been reported by Dschietzig [40] and others [1] to be elevated in cases of acute heart failure patients utilizing the Immundiagnostik
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assay. Additionally, concentrations over 500 pg/ml have been reported in patients with acute heart failure [1, 27, 31] although this approaches the normal range for pregnancies. However, this observation of elevated serum relaxin concentrations was not confirmed when using the R&D System assay where patients admitted with acute heart failure had very low serum relaxin-2 concentrations [41].
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Serum relaxin concentrations were reported to be elevated and relatively stable throughout the menstrual cycle and averaged 427 pg/ml in the follicular phase and 466 pg/ml in the luteal phase [34] using the Immundiagnostik assay. Since it has been established that the source of serum relaxin-2 in the human female is the corpus luteum, the elevated serum relaxin during the follicular phase of the menstrual cycle in this article is physiologically unexplained. Additionally, the findings of this article contrasts markedly with the results from others who report serum relaxin concentrations only rise during the mid to late luteal phase and then only average about 50 pg/ml in non-conceptive cycles [5-8, 42]. Concentrations of serum relaxin above 200 pg/ml are considered to be an indication of an active pregnancy [7].
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It should be noted that these latter publications used the immunoassay provided by Genentech and later by Connetics to support their clinical programs. While an early version of this assay used an analog of relaxin as reference material [43] it was soon changed to natural sequence relaxin as reference material [44, 45]. The latter version was provided to many of the relaxin investigators at that time. It consisted of affinity purified goat polyclonal antibodies against the A chain (coat antibody) and affinity purified rabbit polyclonal antibodies against the B chain conjugated with horse radish peroxidase as the detection antibody [45]. Validation included precision, minimal detected dose and linearity of dilutions. Results from this assay were in good general agreement with research assays developed independently by Eddie [11, 46] and Goldsmith [47, 48] although validation details on cross-reactivity of peptides in the relaxin family or interference of these peptides were lacking for all of these assays. When we compared reference material from the R&D Systems assay with that of another commercial immunoassay it was found that they did not run in parallel and the competitor material showed about 85% potency (Figure 1).
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Figure 1 Comparison of reference material in R&D Systems immunoassay and reference material from a competitor assay.
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Further comparisons were made of relaxin-2 concentrations from serial dilutions of serum from pregnant women in both the R&D Systems immunoassay and that of a competitor (Figure 2).
Figure 2 Percent recovery of serial dilutions of pregnancy serum in the R&D Systems immunoassay compared with a competitor assay.
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Dilutional linearity was acceptable with the R&D assay but not that of the competitor assay. The data in Figures 1 and 2 serve to illustrate the need for authors to validate relaxin immunoassays in their own laboratories.
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Reliability of Results The conclusions reached in publications of relaxin in clinical settings hinges on the validity of assay used to measure relaxin. The better characterized and validated an assay, the greater the reliability one can place in the results obtained and make meaningful conclusions. There are clear differences in the extent of validation reported by the different vendors of commercial relaxin assays, ranging from very well validated and characterized to absolutely no information. The choice of an assay kit to use in measuring serum relaxin should be based in using the best quality assay available. It would also ideally be further validated by the end user.
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The fact that many articles focused on serum relaxin are being published using assays of unknown quality is disturbing and indicates a lack of due diligence by reviewers and weak editorial oversight. On occasion editors have allowed rebuttal to these conclusion [49, 50]. However, more disturbing is the trend to publish papers using assays with no validation and then reject letters to the editor questioning the use of these assays, as has occurred when this author responded to recent high profile articles [27-30].
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This lack of critical review undermines the integrity of the relaxin-2 field. As researchers, we must be sure of our measurement tools. As reviewers, we must demand the demonstration of assay validity. And editors must allow follow-up to articles to discuss weaknesses in methodology. We must each do our part in ensuring integrity of results and conclusions drawn from studies of relaxin-2. References 1. 2. 3. 4.
Solano, J.M., Mateo, J.J. S, Relaxin concentrations in acute heart failure patients: kinetics and clinical determinants. Rev Esp Cardiol (Engl Ed), 2016. 69(12): p. 1230-1232. Sanidas, E., et al., The impact of apelin and relaxin plasma levels in masked hypertension and white coat hypertension. J Clin Hypertens (Greenwich), 2018. Zhang, D., et al., Serum relaxin levels as a novel biomarker for detection of acute myocardial infarction. Int J Clin Exp Med, 2015. 8(9): p. 16937-40. Bathgate, R.A., et al., Relaxin family peptides and their receptors. Physiol Rev, 2013. 93(1): p. 405-80.
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Stewart, D.R., et al., Relaxin in the peri-implantation period. J Clin Endocrinol Metab, 1990. 70(6): p. 1771-3. Stewart, D.R., et al., Serum relaxin concentrations in patients with out-of-phase endometrial biopsies. Fertil Steril, 1992. 57(2): p. 453-5. Stewart, D.R., et al., Relaxin as a Biomarker for Human Pregnancy Detection, in Progress in Relaxin Research, A. MacLennan, G. Tregear, and G. Bryant-Greenwood, Editors. 1994, World Scientific Publishing Co: Singapore. p. 214-224. Stewart, D.R., et al., The relationship between hCG and relaxin secretion in normal pregnancies vs peri-implantation spontaneous abortions. Clin Endocrinol (Oxf), 1993. 38(4): p. 379-85. Johnson, M.R., et al., The effect of human chorionic gonadotropin and pregnancy on the circulating level of relaxin. J Clin Endocrinol Metab, 1991. 72(5): p. 1042-7. Witt, B.R., et al., Relaxin, CA-125, progesterone, estradiol, Schwangerschaft protein, and human chorionic gonadotropin as predictors of outcome in threatened and nonthreatened pregnancies. Fertil Steril, 1990. 53(6): p. 1029-36. Eddie, L.W., et al., Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin. Lancet, 1986. 1(8494): p. 1344-6. Colon, J.M., et al., Relaxin secretion into human semen independent of gonadotropin stimulation. Biol Reprod, 1994. 50(1): p. 187-92. DeSilva, B., et al., Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm Res, 2003. 20(11): p. 1885-900. Andreasson, U., et al., A Practical Guide to Immunoassay Method Validation. Front Neurol, 2015. 6: p. 179. FDA. Bioanalytical Method Validation Guidance for Industry. 2018; Available from: https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances /UCM070107.pdf. Kobalava, Z., et al., Pharmacokinetics of serelaxin in patients with hepatic impairment: A singledose, open-label, parallel-group study. Br J Clin Pharmacol, 2014. 79(6): p. 937-945. Dahlke, M., et al., Pharmacokinetics of serelaxin in patients with severe renal impairment or endstage renal disease requiring hemodialysis: A single-dose, open-label, parallel-group study. J Clin Pharmacol, 2016. 56(4): p. 474-83. Dahlke, M., et al., Safety and tolerability of serelaxin, a recombinant human relaxin-2 in development for the treatment of acute heart failure, in healthy Japanese volunteers and a comparison of pharmacokinetics and pharmacodynamics in healthy Japanese and Caucasian populations. J Clin Pharmacol, 2015. 56(4): p. 415-422. Sato, N., et al., Multicenter, Randomized, Double-Blinded, Placebo-Controlled Phase II Study of Serelaxin in Japanese Patients With Acute Heart Failure. Circ J, 2015. 79(6): p. 1237-47. Guo, X., et al., Serum relaxin as a diagnostic and prognostic marker in patients with epithelial ovarian cancer. Cancer Biomark, 2017. 21(1): p. 81-87. Papadopoulos, D.P., et al., Masked hypertension and atherogenesis: the impact of apelin and relaxin plasma levels. J Clin Hypertens (Greenwich), 2013. 15(5): p. 333-6. Owens, B.D., et al., Association Between Serum Relaxin and Subsequent Shoulder Instability. Orthopedics, 2016. 39(4): p. e724-8. Nonhoff, J., et al., Serelaxin treatment promotes adaptive hypertrophy but does not prevent heart failure in experimental peripartum cardiomyopathy. Cardiovasc Res, 2017. 113(6): p. 598608. Nose-Ogura, S., et al., Oral contraceptive therapy reduces serum relaxin-2 in elite female athletes. J Obstet Gynaecol Res, 2017. 43(3): p. 530-535.
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Dragoo, J.L., et al., Prospective Correlation Between Serum Relaxin Concentration and Anterior Cruciate Ligament Tears Among Elite Collegiate Female Athletes. Am J Sports Med, 2011. Dragoo, J.L., et al., Trends in serum relaxin concentration among elite collegiate female athletes. Int J Womens Health, 2011. 3: p. 19-24. Pintalhao, M., et al., Relaxin serum levels in acute heart failure are associated with pulmonary hypertension and right heart overload. Eur J Heart Fail, 2017. 19(2): p. 218-225. Herrero-Puente, P., et al., The relationship of circulating relaxin-2 concentrations with short-term prognosis in patients with acute heart failure: the RELAHF study. Eur J Heart Fail, 2017. 19(9): p. 1205-1209. Emmens, J.E., J.M. Ter Maaten, and A.A. Voors, Are circulating relaxin levels related to pulmonary hypertension in patients with heart failure? Eur J Heart Fail, 2017. Pintalhao, M., P. Castro-Chaves, and P. Bettencourt, Are circulating relaxin levels related to pulmonary hypertension in patients with heart failure? A reply. Eur J Heart Fail, 2017. Miro, O., et al., The subset of patients with acute heart failure able to secrete relaxin-2 at pregnancy concentrations could have a longer survival: a pilot study. Biomarkers, 2018: p. 1-7. Kruger, S., et al., Relaxin kinetics during dynamic exercise in patients with chronic heart failure. Eur J Intern Med, 2004. 15(1): p. 54-56. Fisher, C., et al., N-terminal pro B type natriuretic peptide, but not the new putative cardiac hormone relaxin, predicts prognosis in patients with chronic heart failure. Heart, 2003. 89(8): p. 879-81. Pehrsson, M., et al., Stable serum levels of relaxin throughout the menstrual cycle: a preliminary comparison of women with premenstrual dysphoria and controls. Arch Womens Ment Health, 2007. Vogel, I., et al., Prediction of preterm delivery using changes in serum relaxin in low risk pregnancies. Eur J Obstet Gynecol Reprod Biol, 2006. Garvin, R. and A. Burns, Serum relaxin levels in subjects with multiple sclerosis. Ital J Anat Embryol, 2016. 121(1): p. 51-59. Xu, Y., Q. Yu, and Y. Liu, Serum relaxin-2 as a novel biomarker for prostate cancer. Br J Biomed Sci, 2018: p. 1-4. Han, L., et al., Combined Assessment of Relaxin and B-Type Natriuretic Peptide Improves Diagnostic Value in Patients With Congestive Heart Failure. Am J Med Sci, 2017. 354(5): p. 480485. Zhang, X., et al., The plasma levels of relaxin-2 and relaxin-3 in patients with diabetes. Clin Biochem, 2013. 46(16-17): p. 1713-6. Dschietzig, T., et al., The pregnancy hormone relaxin is a player in human heart failure. Faseb J, 2001. 15(12): p. 2187-95. Mebazaa, A., et al., Imbalanced Angiogenesis in Peripartum Cardiomyopathy- Diagnostic Value of Placenta Growth Factor. Circ J, 2017. Stewart, D.R., et al., The role of relaxin in glycodelin secretion. J Clin Endocrinol Metab, 1997. 82(3): p. 839-46. Lucas, C., et al., An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys. J Endocrinol, 1989. 120(3): p. 449-57. Bethea, C.L., et al., The effect of relaxin infusion on prolactin and growth hormone secretion in monkeys. J Clin Endocrinol Metab, 1989. 69(5): p. 956-62. Ferraiolo, B.L., et al., The pharmacokinetics and metabolism of human relaxins in rhesus monkeys. Pharm Res, 1991. 8(8): p. 1032-8. Eddie, L.W., et al., Relaxin in paired samples of serum and milk from women after term and preterm delivery. Am J Obstet Gynecol, 1989. 161(4): p. 970-3.
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Iams, J.D., L.T. Goldsmith, and G. Weiss, The preterm prediction study: maternal serum relaxin, sonographic cervical length, and spontaneous preterm birth in twins. J Soc Gynecol Investig, 2001. 8(1): p. 39-42. Sandager, P., et al., Circulating relaxin and cervical length in midpregnancy are independently associated with spontaneous preterm birth. Am J Obstet Gynecol, 2009. 201(2): p. 169 e1-6. Stewart, D.R., Relaxin Concentrations in Acute Heart Failure Patients. Rev Esp Cardiol (Engl Ed), 2017. Stewart, D.R., Letter to Editor: Relaxin-acute myocardial infarction. Int J Exp Med, 2017. 10(2): p. 4243.
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Multiple commercial immunoassays for relaxin-2 are currently available but only two of these have been most widely used for clinically relevant publications. The amount of assay validation varies widely from well characterized to none and authors do not appear to have further characterized the assays they use. Direct comparisons of assays is lacking but similar studies using different assays have produced extremely different results. The use of unvalidated relaxin assays is a problem as it leads to unfounded conclusions regarding the role of relaxin in important clinical applications.
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S0303-7207(19)30004-8
DOI:
https://doi.org/10.1016/j.mce.2019.01.004
Reference:
MCE 10366
To appear in:
Molecular and Cellular Endocrinology
Received Date: 14 November 2018 Revised Date:
6 January 2019
Accepted Date: 7 January 2019
Please cite this article as: Stewart, D.R., Commercial immunoassays for human Relaxin-2, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.01.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Commercial Immunoassays for Human Relaxin-2
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[email protected]
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Dennis R. Stewart Molecular Medicine Research Institute 428 Oakmead Pkwy, Sunnyvale, CA 94085 USA
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Formerly: Novartis Pharmaceuticals, 1 Co-founder Corthera Inc., UC Davis School of Medicine
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Throughout the commercial development of relaxin/serelaxin, several different immunoassays have been used by the pharmaceutical company’s development programs. These assays have been validated for submission of GLP preclinical and clinical studies to the FDA and EU regulatory bodies. The requirements for these assays exceed that of most research assays commonly developed in academic research and have been made available directly from the pharmaceutical companies or indirectly through a third part vendor.
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In addition to these assays, there are also several commercial immunoassays for human relaxin that have appeared in many recent publications measuring relaxin2 in clinical studies. Typically, these commercial assays have not been validated by the vendor, nor any validation reported by the authors using them. In one article, authors cite prior validation of the Immundiagnostik assay which is actually nonexistent [1]. In other articles, authors did not even give the name of the assay they used [2] or simply refer to it as “commercially available ELISA Kit” [3]. In this latter case, concentrations reported for myocardial infarction were higher than seen in pregnancy, but these extraordinarily high concentrations were not discussed. The publication of these studies using questionable assays and results 1
Novartis was not affiliated and had no input into the design, implementation or the analysis of any of the subject matter of this presentation.
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indicates a lack of attention by authors, reviewers and editors which can lead to erroneous conclusions.
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The use of unknown and unvalidated assays presents a serious problem to the understanding of the role of relaxin-2 and potential treatment with serelaxin in serious medical conditions. This review examines some of the clinical articles that have been published and the assay used to determine relaxin-2.
M AN U
SC
Forms and Sources of Human Relaxin Three forms of relaxin have been identified in the human and are commonly referred to as relaxin-1, relaxin-2 and relaxin-3 (often referred to as H1, H2, and H3. Relaxin-1 and relaxin-2 bind to the same receptor (RXFP1) which has wide distribution in the body while relaxin-3 has its own cognate receptor referred to as RXFP3 but distribution of the receptor is primarily localized in brain tissues with very low expression in peripheral tissues [4].
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The expression of relaxin-1 is in doubt but it may be expressed in decidua and placenta [4]. Relaxin-2 is produced in the corpus luteum, decidua, placenta, mammary glands, and prostate [4]. Relaxin-2 is found in circulation during the mid and late luteal phase of the menstrual cycle and peaks around 50 pg/ml [5-8]. It rises rapidly in pregnancy to peak in the first trimester at concentrations of 1.5 ng/ml and then falls gradually during the second and third trimesters to slightly less than 1 ng/ml [8-11]. In men, relaxin is found in seminal plasma at concentrations ranging from 6 - 140 ng/ml [12] but has not been definitively measured in plasma. Relaxin-3 is primarily a neuropeptide and not found in general circulation [4]
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Relaxin-2 is the primary focus of this article as it is the form found in circulation and also the focus of commercial development. Relaxin-2 is name of the endogenous form of the hormone while serelaxin is the International nonproprietary name for pharmaceutical relaxin but they are identical structures. Principles of Assay Validation There are many guides for proper validation of immunoassays and several excellent examples are DeSilva et al., [13] and Andreasson et al., [14]. In addition, the FDA has published guidance for industry in bioanalytical method validation [15]. These guides describe the same general principles for assay validation. These include specificity, selectivity, precision, accuracy, range of quantification, dilutional linearity, parallelism with standards, and recovery. The identity of the
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reference material must be fully understood and comparable with the target molecule.
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Additionally, in running an assay there needs to be quality control samples, criteria for assay acceptance, records and criteria for drift in assays, checks of sample stability in the matrix used, and other measures that are fundamental to assay quality.
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Commercial Relaxin-2 Assays A recent online search for commercial relaxin-2 assays has identified at least 9 kits available but only two of these have been most often cited. These are the R&D Systems Quantikine ELISA for Human Relaxin-2 and the Immundiagnostik Relaxin ELISA K9210. These assays will be further discussed.
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1. R&D Systems ELISA (DRL 200) This assay was co-developed by R&D Systems and Corthera, Inc. (prior to the acquisition by Novartis). This was done to provide the relaxin research community with access to a validated assay rather than through material transfer agreement programs as done earlier by Genentech and Connetics. The reference material used for this assay is recombinant serelaxin provided by Corthera, Inc. This assay uses a monoclonal capture antibody and a polyclonal labeled detection antibody. Its stated range is 7.8 to 500 pg / ml. Validation included within and between assay precision. The kit provides direct measurement in cell culture fluid, serum or plasma (heparin or EDTA as anticoagulant).
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Further validation included cross reactivity studies using several human peptides in the relaxin family which included IGF-I, IGF-II, insulin, proinsulin, insulin like 3, relaxin-1 and relaxin-3. Cross reactivity was not found at 50 ng/ml for any of these peptides. Relaxins from canine, mouse, porcine and rat did also not cross react at 50 ng/ml. Interference in the relaxin assay was also examined for all of these peptides and none was found. Dilutional linearity was determined for samples naturally containing relaxin or spiked samples and found to be acceptable over a range of 1:2 down to 1:16. 2. Immundiagnostik ELISA
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This assay utilized two different polyclonal antibodies for capture and detection. The reference material is not described and the required specificity, selectivity, interference, and full dilutional linearity studies were not reported. Partial dilutional linearity was listed for 1/5 – 1/7 dilutions, a very narrow and selected range. Only insulin was tested for cross reactivity. Cross reactivity of relaxin-1, relaxin-3 and other relaxin related peptides in this assay was not reported.
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This kit requires sample preparation prior to assay according the kit directions. Serum or plasma samples require at least a 1:3 dilution with assay buffer prior to analysis. Additionally, to avoid interference with heterophilic antibodies it is recommended that samples be treated twice with Anti Interference Reagent (5% v/v, shaken for 1 hour and centrifuged). Urine samples require a 1:4 dilution and seminal plasma samples a 1:10 dilution.
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3. Other commercial immunoassays Other commercial relaxin assays used in recent publications measuring serum relaxin in patient populations include kits from ADL Inc., Phoenix Pharmaceuticals, and ThermoFisher. The Phoenix kit recommends each plasma or tissue sample be extracted using C-18 SEP-Columns prior to assay. No validation data nor source of reference material could be found for these assays.
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Assays Used to Measure Relaxin-2 in Recent Clinically Oriented Articles There has been a general increased interest in relaxin-2 with the commercial development of serelaxin for treatment in acute heart failure. This has brought about many studies measuring relaxin-2/serelaxin in circulation in a number of clinical indications. This section reviews some of publications and which relaxin-2 assay was used in support of these studies. 1. R&D Systems Assay This immunoassay has been used by Corthera and Novartis for their clinical studies in the development of serelaxin in acute heart failure.2 This assay was also used in studies by Novartis looking at serelaxin pharmacokinetics
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This assay was used only for pharmacokinetics and not manufacturing or product released purposes.
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in patients with hepatic impairment [16], renal impairment [17] or ethnic sensitivity studies [18, 19].
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The R&D Systems assay was used to measure serum relaxin-2 in patients with ovarian cancer [20], aneurysm formation [21], shoulder instability [22], peripartum cardiomyopathy [23] and in female athletes [24-26].
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2. Immundiagnostik Assay The Immundiagnostik assay has been used in reports of acute heart failure [1, 27-31] as well as studies on chronic heart failure [32, 33]. It has also been used to examine acute heart failure patients and predict long term survival [31].
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The Immundiagnostik assay has also been used for reproductive studies including studies of women with premenstrual dysphoria [34] or prediction of preterm delivery in low risk pregnancies [35]. Additional indications studied are subjects with multiple sclerosis [36] or as a biomarker for prostate cancer [37]. This last article used a kit from ALPCO but it is believed to be the same as the Immundiagnostik assay.
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3. Other Commercial Relaxin Assays An assay from ADL Inc. was used in a report of relaxin in patients with congestive heart failure [38].
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A relaxin radioimmunoassay kit from Phoenix Pharmaceuticals was used in a study of relaxin concentrations in patients with diabetes [39].
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A relaxin assay from ThermoFisher was reportedly used to measure relaxin concentrations in a study of acute myocardial infarction [3]. However, the source of the assay was not reported in the original article and only revealed later in unpublished communications with the author.
Comparison of Assays There have been no published studies of head to head comparisons of any of the relaxin assays. However, some comparisons can be drawn from results of similar studies using different assays. Circulating relaxin has been reported by Dschietzig [40] and others [1] to be elevated in cases of acute heart failure patients utilizing the Immundiagnostik
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assay. Additionally, concentrations over 500 pg/ml have been reported in patients with acute heart failure [1, 27, 31] although this approaches the normal range for pregnancies. However, this observation of elevated serum relaxin concentrations was not confirmed when using the R&D System assay where patients admitted with acute heart failure had very low serum relaxin-2 concentrations [41].
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Serum relaxin concentrations were reported to be elevated and relatively stable throughout the menstrual cycle and averaged 427 pg/ml in the follicular phase and 466 pg/ml in the luteal phase [34] using the Immundiagnostik assay. Since it has been established that the source of serum relaxin-2 in the human female is the corpus luteum, the elevated serum relaxin during the follicular phase of the menstrual cycle in this article is physiologically unexplained. Additionally, the findings of this article contrasts markedly with the results from others who report serum relaxin concentrations only rise during the mid to late luteal phase and then only average about 50 pg/ml in non-conceptive cycles [5-8, 42]. Concentrations of serum relaxin above 200 pg/ml are considered to be an indication of an active pregnancy [7].
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It should be noted that these latter publications used the immunoassay provided by Genentech and later by Connetics to support their clinical programs. While an early version of this assay used an analog of relaxin as reference material [43] it was soon changed to natural sequence relaxin as reference material [44, 45]. The latter version was provided to many of the relaxin investigators at that time. It consisted of affinity purified goat polyclonal antibodies against the A chain (coat antibody) and affinity purified rabbit polyclonal antibodies against the B chain conjugated with horse radish peroxidase as the detection antibody [45]. Validation included precision, minimal detected dose and linearity of dilutions. Results from this assay were in good general agreement with research assays developed independently by Eddie [11, 46] and Goldsmith [47, 48] although validation details on cross-reactivity of peptides in the relaxin family or interference of these peptides were lacking for all of these assays. When we compared reference material from the R&D Systems assay with that of another commercial immunoassay it was found that they did not run in parallel and the competitor material showed about 85% potency (Figure 1).
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Figure 1 Comparison of reference material in R&D Systems immunoassay and reference material from a competitor assay.
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Further comparisons were made of relaxin-2 concentrations from serial dilutions of serum from pregnant women in both the R&D Systems immunoassay and that of a competitor (Figure 2).
Figure 2 Percent recovery of serial dilutions of pregnancy serum in the R&D Systems immunoassay compared with a competitor assay.
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Dilutional linearity was acceptable with the R&D assay but not that of the competitor assay. The data in Figures 1 and 2 serve to illustrate the need for authors to validate relaxin immunoassays in their own laboratories.
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Reliability of Results The conclusions reached in publications of relaxin in clinical settings hinges on the validity of assay used to measure relaxin. The better characterized and validated an assay, the greater the reliability one can place in the results obtained and make meaningful conclusions. There are clear differences in the extent of validation reported by the different vendors of commercial relaxin assays, ranging from very well validated and characterized to absolutely no information. The choice of an assay kit to use in measuring serum relaxin should be based in using the best quality assay available. It would also ideally be further validated by the end user.
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The fact that many articles focused on serum relaxin are being published using assays of unknown quality is disturbing and indicates a lack of due diligence by reviewers and weak editorial oversight. On occasion editors have allowed rebuttal to these conclusion [49, 50]. However, more disturbing is the trend to publish papers using assays with no validation and then reject letters to the editor questioning the use of these assays, as has occurred when this author responded to recent high profile articles [27-30].
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This lack of critical review undermines the integrity of the relaxin-2 field. As researchers, we must be sure of our measurement tools. As reviewers, we must demand the demonstration of assay validity. And editors must allow follow-up to articles to discuss weaknesses in methodology. We must each do our part in ensuring integrity of results and conclusions drawn from studies of relaxin-2. References 1. 2. 3. 4.
Solano, J.M., Mateo, J.J. S, Relaxin concentrations in acute heart failure patients: kinetics and clinical determinants. Rev Esp Cardiol (Engl Ed), 2016. 69(12): p. 1230-1232. Sanidas, E., et al., The impact of apelin and relaxin plasma levels in masked hypertension and white coat hypertension. J Clin Hypertens (Greenwich), 2018. Zhang, D., et al., Serum relaxin levels as a novel biomarker for detection of acute myocardial infarction. Int J Clin Exp Med, 2015. 8(9): p. 16937-40. Bathgate, R.A., et al., Relaxin family peptides and their receptors. Physiol Rev, 2013. 93(1): p. 405-80.
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Stewart, D.R., et al., Relaxin in the peri-implantation period. J Clin Endocrinol Metab, 1990. 70(6): p. 1771-3. Stewart, D.R., et al., Serum relaxin concentrations in patients with out-of-phase endometrial biopsies. Fertil Steril, 1992. 57(2): p. 453-5. Stewart, D.R., et al., Relaxin as a Biomarker for Human Pregnancy Detection, in Progress in Relaxin Research, A. MacLennan, G. Tregear, and G. Bryant-Greenwood, Editors. 1994, World Scientific Publishing Co: Singapore. p. 214-224. Stewart, D.R., et al., The relationship between hCG and relaxin secretion in normal pregnancies vs peri-implantation spontaneous abortions. Clin Endocrinol (Oxf), 1993. 38(4): p. 379-85. Johnson, M.R., et al., The effect of human chorionic gonadotropin and pregnancy on the circulating level of relaxin. J Clin Endocrinol Metab, 1991. 72(5): p. 1042-7. Witt, B.R., et al., Relaxin, CA-125, progesterone, estradiol, Schwangerschaft protein, and human chorionic gonadotropin as predictors of outcome in threatened and nonthreatened pregnancies. Fertil Steril, 1990. 53(6): p. 1029-36. Eddie, L.W., et al., Radioimmunoassay of relaxin in pregnancy with an analogue of human relaxin. Lancet, 1986. 1(8494): p. 1344-6. Colon, J.M., et al., Relaxin secretion into human semen independent of gonadotropin stimulation. Biol Reprod, 1994. 50(1): p. 187-92. DeSilva, B., et al., Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macromolecules. Pharm Res, 2003. 20(11): p. 1885-900. Andreasson, U., et al., A Practical Guide to Immunoassay Method Validation. Front Neurol, 2015. 6: p. 179. FDA. Bioanalytical Method Validation Guidance for Industry. 2018; Available from: https://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances /UCM070107.pdf. Kobalava, Z., et al., Pharmacokinetics of serelaxin in patients with hepatic impairment: A singledose, open-label, parallel-group study. Br J Clin Pharmacol, 2014. 79(6): p. 937-945. Dahlke, M., et al., Pharmacokinetics of serelaxin in patients with severe renal impairment or endstage renal disease requiring hemodialysis: A single-dose, open-label, parallel-group study. J Clin Pharmacol, 2016. 56(4): p. 474-83. Dahlke, M., et al., Safety and tolerability of serelaxin, a recombinant human relaxin-2 in development for the treatment of acute heart failure, in healthy Japanese volunteers and a comparison of pharmacokinetics and pharmacodynamics in healthy Japanese and Caucasian populations. J Clin Pharmacol, 2015. 56(4): p. 415-422. Sato, N., et al., Multicenter, Randomized, Double-Blinded, Placebo-Controlled Phase II Study of Serelaxin in Japanese Patients With Acute Heart Failure. Circ J, 2015. 79(6): p. 1237-47. Guo, X., et al., Serum relaxin as a diagnostic and prognostic marker in patients with epithelial ovarian cancer. Cancer Biomark, 2017. 21(1): p. 81-87. Papadopoulos, D.P., et al., Masked hypertension and atherogenesis: the impact of apelin and relaxin plasma levels. J Clin Hypertens (Greenwich), 2013. 15(5): p. 333-6. Owens, B.D., et al., Association Between Serum Relaxin and Subsequent Shoulder Instability. Orthopedics, 2016. 39(4): p. e724-8. Nonhoff, J., et al., Serelaxin treatment promotes adaptive hypertrophy but does not prevent heart failure in experimental peripartum cardiomyopathy. Cardiovasc Res, 2017. 113(6): p. 598608. Nose-Ogura, S., et al., Oral contraceptive therapy reduces serum relaxin-2 in elite female athletes. J Obstet Gynaecol Res, 2017. 43(3): p. 530-535.
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Dragoo, J.L., et al., Prospective Correlation Between Serum Relaxin Concentration and Anterior Cruciate Ligament Tears Among Elite Collegiate Female Athletes. Am J Sports Med, 2011. Dragoo, J.L., et al., Trends in serum relaxin concentration among elite collegiate female athletes. Int J Womens Health, 2011. 3: p. 19-24. Pintalhao, M., et al., Relaxin serum levels in acute heart failure are associated with pulmonary hypertension and right heart overload. Eur J Heart Fail, 2017. 19(2): p. 218-225. Herrero-Puente, P., et al., The relationship of circulating relaxin-2 concentrations with short-term prognosis in patients with acute heart failure: the RELAHF study. Eur J Heart Fail, 2017. 19(9): p. 1205-1209. Emmens, J.E., J.M. Ter Maaten, and A.A. Voors, Are circulating relaxin levels related to pulmonary hypertension in patients with heart failure? Eur J Heart Fail, 2017. Pintalhao, M., P. Castro-Chaves, and P. Bettencourt, Are circulating relaxin levels related to pulmonary hypertension in patients with heart failure? A reply. Eur J Heart Fail, 2017. Miro, O., et al., The subset of patients with acute heart failure able to secrete relaxin-2 at pregnancy concentrations could have a longer survival: a pilot study. Biomarkers, 2018: p. 1-7. Kruger, S., et al., Relaxin kinetics during dynamic exercise in patients with chronic heart failure. Eur J Intern Med, 2004. 15(1): p. 54-56. Fisher, C., et al., N-terminal pro B type natriuretic peptide, but not the new putative cardiac hormone relaxin, predicts prognosis in patients with chronic heart failure. Heart, 2003. 89(8): p. 879-81. Pehrsson, M., et al., Stable serum levels of relaxin throughout the menstrual cycle: a preliminary comparison of women with premenstrual dysphoria and controls. Arch Womens Ment Health, 2007. Vogel, I., et al., Prediction of preterm delivery using changes in serum relaxin in low risk pregnancies. Eur J Obstet Gynecol Reprod Biol, 2006. Garvin, R. and A. Burns, Serum relaxin levels in subjects with multiple sclerosis. Ital J Anat Embryol, 2016. 121(1): p. 51-59. Xu, Y., Q. Yu, and Y. Liu, Serum relaxin-2 as a novel biomarker for prostate cancer. Br J Biomed Sci, 2018: p. 1-4. Han, L., et al., Combined Assessment of Relaxin and B-Type Natriuretic Peptide Improves Diagnostic Value in Patients With Congestive Heart Failure. Am J Med Sci, 2017. 354(5): p. 480485. Zhang, X., et al., The plasma levels of relaxin-2 and relaxin-3 in patients with diabetes. Clin Biochem, 2013. 46(16-17): p. 1713-6. Dschietzig, T., et al., The pregnancy hormone relaxin is a player in human heart failure. Faseb J, 2001. 15(12): p. 2187-95. Mebazaa, A., et al., Imbalanced Angiogenesis in Peripartum Cardiomyopathy- Diagnostic Value of Placenta Growth Factor. Circ J, 2017. Stewart, D.R., et al., The role of relaxin in glycodelin secretion. J Clin Endocrinol Metab, 1997. 82(3): p. 839-46. Lucas, C., et al., An enzyme-linked immunosorbent assay to study human relaxin in human pregnancy and in pregnant rhesus monkeys. J Endocrinol, 1989. 120(3): p. 449-57. Bethea, C.L., et al., The effect of relaxin infusion on prolactin and growth hormone secretion in monkeys. J Clin Endocrinol Metab, 1989. 69(5): p. 956-62. Ferraiolo, B.L., et al., The pharmacokinetics and metabolism of human relaxins in rhesus monkeys. Pharm Res, 1991. 8(8): p. 1032-8. Eddie, L.W., et al., Relaxin in paired samples of serum and milk from women after term and preterm delivery. Am J Obstet Gynecol, 1989. 161(4): p. 970-3.
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Iams, J.D., L.T. Goldsmith, and G. Weiss, The preterm prediction study: maternal serum relaxin, sonographic cervical length, and spontaneous preterm birth in twins. J Soc Gynecol Investig, 2001. 8(1): p. 39-42. Sandager, P., et al., Circulating relaxin and cervical length in midpregnancy are independently associated with spontaneous preterm birth. Am J Obstet Gynecol, 2009. 201(2): p. 169 e1-6. Stewart, D.R., Relaxin Concentrations in Acute Heart Failure Patients. Rev Esp Cardiol (Engl Ed), 2017. Stewart, D.R., Letter to Editor: Relaxin-acute myocardial infarction. Int J Exp Med, 2017. 10(2): p. 4243.
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Multiple commercial immunoassays for relaxin-2 are currently available but only two of these have been most widely used for clinically relevant publications. The amount of assay validation varies widely from well characterized to none and authors do not appear to have further characterized the assays they use. Direct comparisons of assays is lacking but similar studies using different assays have produced extremely different results. The use of unvalidated relaxin assays is a problem as it leads to unfounded conclusions regarding the role of relaxin in important clinical applications.
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