Limits...
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.

Herrera B, Inman GJ - BMC Cell Biol. (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK. b.herrera@beatson.gla.ac.uk

ABSTRACT

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.

Conclusion: The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.

Show MeSH

Related in: MedlinePlus

Determination of the linear range of activation of BRE-Luc by BMPs. C2C12BRE cells were treated as in Figure 1 with increasing concentrations of (A) BMP4, (B) BMP6, (C) BMP7, (D) BMP9 and (E) BMP10. Luciferase activity was normalised to protein content. Relative luciferase units (RLU) following background subtraction are shown. Data shown is a representative experiment performed in quadruplicate ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2663541&req=5

Figure 2: Determination of the linear range of activation of BRE-Luc by BMPs. C2C12BRE cells were treated as in Figure 1 with increasing concentrations of (A) BMP4, (B) BMP6, (C) BMP7, (D) BMP9 and (E) BMP10. Luciferase activity was normalised to protein content. Relative luciferase units (RLU) following background subtraction are shown. Data shown is a representative experiment performed in quadruplicate ± SD.

Mentions: Having established that the C2C12BRE cells were sensitive to BMP9 treatment we investigated the dose dependent effects of multiple recombinant BMPs on this cell line. Cells were incubated with increasing concentrations of recombinant human BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10. All 6 BMPs tested stimulated luciferase activity in a dose dependent manner, with maximal stimulation reached with >50 ng/ml (>2 nM) BMP2, 2 ng/ml (77 pM) BMP4, >50 ng/ml (>1.66 nM) BMP6, 10 ng/ml (427 pM) BMP7, 5 ng/ml (205 pM) BMP9 and 20 ng/ml (820 pM) BMP10 (Figure 1A–F and data not shown). We next assessed the specificity of the assay for BMPs by treating the cells with other members of the TGF-β superfamily. TGF-β1, Nodal and Mullerian Inhibiting Substance (MIS) failed to increase luciferase activity, even at concentrations as high as 10 ng/ml (400 pM) TGF-β1, 200 ng/ml (7.75 nM) Nodal or 1640 ng/ml (70 nM) MIS (Figure 1G–I). Having observed that BMPs stimulate luciferase production in C2C12BRE cells over a range of concentrations in an isoform specific fashion, we next determined the linear range of the assay with each BMP tested. We found that the assay maintained linearity up to 1 ng/ml (40 pM) BMP4, 10 ng/ml (333 pM) BMP6, 10 ng/ml (427 pM) BMP7, 1 ng/ml (40 pM) BMP9 and 5 ng/ml (205 pM) BMP10 (Figure 2A–E and data not shown). Using these titration analyses we also determined the lower sensitivity limits for each BMP. We could reproducibly measure as little as 0.05 ng/ml (2 pM) BMP4, 0.5 ng/ml (16.5 pM) BMP6, 1 ng/ml (42 pM) BMP7, 0.1 ng/ml (4.1 pM) BMP9 and 1 ng/ml (41 pM) BMP10 (Figure 2 and data not shown). Thus, this assay is sensitive and robust enough to measure 0.05–1 ng/ml (2–40 pM) BMP4, 0.1–1 ng/ml (4–40 pM) BMP9, 0.5–10 ng/ml (16.5–333 pM) BMP6, 1–10 ng/ml (42–427 pM) BMP7 and 1–5 ng/ml (40–205 pM) BMP10.


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.

Herrera B, Inman GJ - BMC Cell Biol. (2009)

Determination of the linear range of activation of BRE-Luc by BMPs. C2C12BRE cells were treated as in Figure 1 with increasing concentrations of (A) BMP4, (B) BMP6, (C) BMP7, (D) BMP9 and (E) BMP10. Luciferase activity was normalised to protein content. Relative luciferase units (RLU) following background subtraction are shown. Data shown is a representative experiment performed in quadruplicate ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2663541&req=5

Figure 2: Determination of the linear range of activation of BRE-Luc by BMPs. C2C12BRE cells were treated as in Figure 1 with increasing concentrations of (A) BMP4, (B) BMP6, (C) BMP7, (D) BMP9 and (E) BMP10. Luciferase activity was normalised to protein content. Relative luciferase units (RLU) following background subtraction are shown. Data shown is a representative experiment performed in quadruplicate ± SD.
Mentions: Having established that the C2C12BRE cells were sensitive to BMP9 treatment we investigated the dose dependent effects of multiple recombinant BMPs on this cell line. Cells were incubated with increasing concentrations of recombinant human BMP2, BMP4, BMP6, BMP7, BMP9 and BMP10. All 6 BMPs tested stimulated luciferase activity in a dose dependent manner, with maximal stimulation reached with >50 ng/ml (>2 nM) BMP2, 2 ng/ml (77 pM) BMP4, >50 ng/ml (>1.66 nM) BMP6, 10 ng/ml (427 pM) BMP7, 5 ng/ml (205 pM) BMP9 and 20 ng/ml (820 pM) BMP10 (Figure 1A–F and data not shown). We next assessed the specificity of the assay for BMPs by treating the cells with other members of the TGF-β superfamily. TGF-β1, Nodal and Mullerian Inhibiting Substance (MIS) failed to increase luciferase activity, even at concentrations as high as 10 ng/ml (400 pM) TGF-β1, 200 ng/ml (7.75 nM) Nodal or 1640 ng/ml (70 nM) MIS (Figure 1G–I). Having observed that BMPs stimulate luciferase production in C2C12BRE cells over a range of concentrations in an isoform specific fashion, we next determined the linear range of the assay with each BMP tested. We found that the assay maintained linearity up to 1 ng/ml (40 pM) BMP4, 10 ng/ml (333 pM) BMP6, 10 ng/ml (427 pM) BMP7, 1 ng/ml (40 pM) BMP9 and 5 ng/ml (205 pM) BMP10 (Figure 2A–E and data not shown). Using these titration analyses we also determined the lower sensitivity limits for each BMP. We could reproducibly measure as little as 0.05 ng/ml (2 pM) BMP4, 0.5 ng/ml (16.5 pM) BMP6, 1 ng/ml (42 pM) BMP7, 0.1 ng/ml (4.1 pM) BMP9 and 1 ng/ml (41 pM) BMP10 (Figure 2 and data not shown). Thus, this assay is sensitive and robust enough to measure 0.05–1 ng/ml (2–40 pM) BMP4, 0.1–1 ng/ml (4–40 pM) BMP9, 0.5–10 ng/ml (16.5–333 pM) BMP6, 1–10 ng/ml (42–427 pM) BMP7 and 1–5 ng/ml (40–205 pM) BMP10.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK. b.herrera@beatson.gla.ac.uk

ABSTRACT

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.

Conclusion: The assay is rapid (<48 hours) and can be used to simultaneously measure isoform specific and total BMP activity in complex solutions.

Show MeSH
Related in: MedlinePlus