Bioactivity assay of porcine relaxin based on cAMP accumulation in THP-1 cells quantified by LC-MS/MS.
This study describes a reliable bioactivity assay of porcine relaxin (pRLX) based on cyclic adenosine 3',5'-monophosphate (cAMP) accumulation in the human monocyte cell-line quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). As a result, the LC-MS/MS was based on a positive selected reaction monitoring of cAMP with a stable internal standard, 8-Br-cAMP and a protein precipitation procedure by HClO4. The standard curve of cAMP was linear from 5.0 ng mL(-1) to 992.0 ng mL(-1), with lower limits of detection and quantification of 0.5 ng mL(-1) and 5.0 ng mL(-1), respectively. The satisfactory validation data including stability assay were obtained. When measured by the LC-MS/MS, the pRLX sample showed a time- and dose-dependent stimulation of cAMP with the concentration for 50% of the maximal effect (EC50) of 40.6 ng mL(-1). The developed method indicated higher precision and selectivity than the commercial enzyme linked immunosorbent assay kits, which showed EC50 of 66.6 ng mL(-1).
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China; Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Xiaohong
X
Ren
Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Caifen
C
Wang
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China; Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Li
L
Wu
Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Haiyan
H
Li
Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Liandi
L
Chi
School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China.
Lixin
L
Sun
Rizhao Lanshan Biochemical Products Co. Ltd., Rizhao 276800, China.
Bin
B
Wang
Rizhao Lanshan Biochemical Products Co. Ltd., Rizhao 276800, China.
Hu
H
Cui
IBSA Institut Biochimique S.A., 6574 Lugano, Switzerland.
Luca
L
Angiolini
IBSA Institut Biochimique S.A., 6574 Lugano, Switzerland.
Alessandro
A
Bianchi
IBSA Institut Biochimique S.A., 6574 Lugano, Switzerland.
Paolo
P
Caccia
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China. Electronic address: xingshu@jlu.edu.cn.
Shu
S
Xing
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China. Electronic address: fxq@jlu.edu.cn.
Xueqi
X
Fu
Edmond H. Fischer Signal Transduction Laboratory, College of Life Sciences, Jilin University, Changchun 130012, China; Center for Drug Delivery Systems, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. Electronic address: jwzhang@simm.ac.cn.
Jiwen
J
Zhang
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D057230:Limit of Detection; D009000:Monocytes / Q000378:metabolism*; D012065:Relaxin / Q000737:chemistry* / Q000378:metabolism*; D015203:Reproducibility of Results; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods
D012065:Relaxin; D000242:Cyclic AMP
Bioactivity; ELISA; LC–MS/MS; Porcine relaxin; cAMP