A rapid and sensitive bioassay for the simultaneous measurement of multiple bone morphogenetic proteins. Identification and quantification of BMP4, BMP6 and BMP9 in bovine and human serum.
BACKGROUND
Bone morphogenetic proteins (BMPs) are pleiotropic members of the TGF-beta superfamily which regulate many biological processes during development and adult tissue homeostasis and are implicated in the pathogenesis of a number of human diseases. Their involvement in both normal and aberrant physiology creates a need for rapid, sensitive and methodologically simple assays to evaluate their activity from a variety of biological samples. Previously alkaline phosphatase based assays, ELISA and luciferase based bioassays have been developed to evaluate either individual or total BMP activity. In this paper, we describe a highly sensitive, rapid and specific cell based assay for the simultaneous quantification of total and isoform specific BMP activity from biological samples.


RESULTS
A C2C12 cell line stably transfected with a reporter plasmid consisting of the BMP response element (BRE) from the Id1 promoter fused to a luciferase reporter gene was generated. Exposure of this cell line to human recombinant BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10 induced the expression of luciferase which was quantified using a luminometer. This assay was specific for BMP activity as the other TGF-beta superfamily members TGF-beta 1, Nodal and Mullerian Inhibiting Substance (MIS) did not induce the reporter. Pretreatment of samples with isoform specific BMP blocking antibodies coupled with isoform specific titration analysis allowed the simultaneous identification and quantification of BMP4, BMP6 and BMP9 in serum samples.


CONCLUSIONS
The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.
Herrera
Blanca
B
Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK. b.herrera@beatson.gla.ac.uk
Inman
Gareth J
GJ
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D001681:Biological Assay / Q000379:methods*; D055415:Bone Morphogenetic Protein 4 / Q000097:blood; D055418:Bone Morphogenetic Protein 6 / Q000097:blood; D019485:Bone Morphogenetic Proteins / Q000097:blood*; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D017930:Genes, Reporter; D055427:Growth Differentiation Factor 2 / Q000097:blood; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D012015:Reference Standards
D019138:Antibodies, Blocking; D055415:Bone Morphogenetic Protein 4; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; D055427:Growth Differentiation Factor 2; D008156:Luciferases