Insulin-like growth factor 1 (IGF1) is an important biomarker for the treatment of growth hormone disorders. Recently, there has been increased interest in using mass spectrometric (MS) detection methods to measure IGF1. However, widespread clinical deployment of an MS-based IGF1 assay requires increased throughput and speed to justify analysis costs, as well as robust industrial platforms that are reproducible across laboratories.
Presented here is an MS-based quantitative IGF1 assay with a performance evaluation of >1,000 samples/day and an ability to quantify IGF1 point mutations and post-translational modifications. The throughput of the IGF1Mass spectrometry immunoassays (MSIA) benefited from a simplified sample preparation step, IGF1 immunocapture in a cutting-edge format, and high-throughput MALDI-TOF-MS analysis. The resulting assay had a limit of detection and limit of quantification of 1.5 μg/L and 5 μg/L, respectively, with intra- and inter-assay precision CVs of less than 10% and good linearity and recovery. The IGF1-MSIA was compared to the commercially available IGF1-ELISA via the Bland-Altman method comparison test, resulting in a slight positive bias of 16%.
The IGF1 MSIA was deployed in an optimized parallel workflow using two pipetting robots and MALDI-TOF MS instruments synchronized into 1-hour phases of sample preparation, extraction and MSIA pipette tip elution, MS data acquisition and data processing. With this workflow, high-throughput IGF1 quantification of 1,054 human samples was achieved in approximately 9 hours. This testing rate is a significant improvement over existing MS-based IGF1 assays. and is on par with enzyme-based immunoassays. In addition, a mutation was detected in ~1% of the samples (SNP: rs17884626,creating an A→T substitution at position 67 of IGF1), demonstrating the ability of IGF1-MSIA to detect point mutations and post-translational modifications .
introduction
Insulin-like growth factor 1 (IGF1) is an important biomarker for the treatment of growth hormone disorders. IGF1 is produced by the IGF1 gene, located on chromosome 12 in humans, and is a critical mediator involved in cell growth, differentiation and transformation. Human IGF1 is a 70 amino acid protein containing three intra-disulfide bonds with a mass of 7648.7 Da.
- Serum IGF1 reference values in healthy people are 20–600 μg/L. Most of the IGF1 produced acts as an endocrine hormone via secretion from the liver, but the molecule can also serve as a paracrine hormone in certain tissues, including cartilage cells, and even as an oncogene in autocrine mode.
- IGF1 exerts its effects by binding to the IGF1 receptor on target tissues. In the blood, 99% of IGF1 is bound to IGFBPs (Insulin-like Growth Factor Binding Proteins), with 80% of IGF1 circulating in a ternary complex consisting of one molecule of IGF1, one molecule of IGFBP3, and one molecule of an acid labile subunit . Therefore, the presentation of circulating IGF1 has created the need for methods to disrupt these complexes for accurate IGF1 quantification.
- In the last thirty years, IGF1 has been generally quantified using radioimmunoassay (RIA), immunoradiometric assay (IRMA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence. These methods use various sample preparation steps to break down and remove IGFBPs (thus providing free IGF1 for testing), including low pH, size exclusion chromatography, and acid-ethanol extraction. Some methods also involve the addition of an IGFBP blocker, typically IGF2 .
- Because of these variability, commercial assays have not provided comparable measurements of serum IGF1.
- Since 2001 there has been increasing interest in the use of mass spectrometric detection methods to measure IGF1. Such methods can be categorized as either bottom-up (enzymatic digestion followed by analysis of surrogate peptides representative of IGF1) or top-down (no digestion – the intact IGF1 is analyzed directly) and in some cases use immunoprecipitation as a separation step before detection.
- In 2004, our group used a top-down mass spectrometry immunoassay (MSIA) to quantify IGF1 in human samples. The approach used a novel sodium dodecyl sulfate (SDS) treatment to disrupt IGFBPs prior to immunoaffinity capture of IGF1, followed by direct detection of intact IGF1 using MALDI-TOF mass spectrometry.
Endogenous IGF1 was quantitated by introducing a mass-shifted IGF1 analog (internal standard) into the samples prior to any processing.
The internal standard was present throughout the workflow/analysis and was finally registered as a mass-resolved signal in the mass spectra used for normalization and quantification of the IGF1 signal (via a working curve method). In 2008, Thevis et. Al. used immunoprecipitation coupled to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/MS) to quantify IGF1 and related analogues. This approach monitored productions arising from dissociations of terminal IGF1 peptide fragments (the molecule was fragmented from its intact form, limiting sequence coverage due to the three remaining intact disulfide bonds). Recently, MS-based non-immunoprecipitation methods for IGF1 quantification have gained traction. In 2009, Kay et. al implemented an acetonitrile precipitation extraction using a bottom-up LC/MS /MS-SRM approach . This approach monitored MS 3 ions of the N-terminal IGF1 fragment (residues 1-21), provided a dynamic range of 16-2000 μg/L and had CVs <13%. Solid phase extraction methods have also been found to work for IGF1 quantification. Bystrom et. Al. demonstrated top-down quantification of IGF1 via solid-phase extraction coupled with narrow mass extraction (via Q-TOF) of a single IGF1 isotope in the 7 + charge state (m/z 1093.5209) yielded excellent analytical metrics (CVs <5.2%, LOD 3.7). μg/l) . Also Kay et. al . further refined their approach by now introducing solid phase extraction (SPE) at the front end of the workflow and extended the method to monitor two IGF1 tryptic fragments (1-21 and 38-50). Each of these approaches has its own merits and taken together they clearly demonstrate the value of IGF1 quantification with MS detection.
However, apart from providing an unequivocal detection of IGF1 with high analytical performance, the widespread clinical adoption of an MS-based IGF1 assay requires increased throughput and speed to justify the cost of the analyzes (mainly due to time unit and consumables) , robust industrial platforms that are reproducible across laboratories (turnkey systems), and the long-term sustainability of the supply chains associated with the IGF1 assay (as well as any additional assays). In addition, and considering the ability of mass spectrometry to easily detect molecular variants of a target protein, the current status of IGF1 mass spectrometry assays couldalso benefit from improved configurations that account for protein microheterogeneity across populations. Such heterogeneity could potentially cause currently used top-down and bottom-up MS approaches to fail when a patient is phenotypically heterozygous via a single nucleotide polymorphism or when N- or C-terminal truncations affect the monitored m/z signals change in LC/MS. In addition, such protein heterogeneity may have undiscovered pathophysiological implications and potential clinical utility.
Human Insulin Like Growth Factor 1 (IGF1) siRNA
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Human Insulin Like Growth Factor 1 (IGF1) Protein
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abx260489-2g
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2 µg
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abx060289-100g
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Ask for price
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Recombinant Human Insulin Like Growth Factor 1 (IGF1)
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EKN46078-48T
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EKN46078-5x96T
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EKN46078-96T
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385.3 EUR
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Biomatik Corporation
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386.7 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS4500091-10x96Tests
MyBiosource
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3145 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS4500091-48Tests
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300 EUR
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MBS4500091-5x96Tests
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1615 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS4500091-96Test
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365 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2024324-10x96StripWells
MyBiosource
10x96-Strip-Wells
4070 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2024324-24StripWells
MyBiosource
24-Strip-Wells
295 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2024324-48StripWells
MyBiosource
48-Strip-Wells
420 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2024324-5x96StripWells
MyBiosource
5x96-Strip-Wells
2225 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2024324-96StripWells
MyBiosource
96-Strip-Wells
550 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS451804-10x96StripWells
MyBiosource
10x96-Strip-Wells
3070 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS451804-48StripWells
MyBiosource
48-Strip-Wells
295 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS451804-5x96StripWells
MyBiosource
5x96-Strip-Wells
1575 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS451804-96StripWells
MyBiosource
96-Strip-Wells
355 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2700905-10x96StripWells
MyBiosource
10x96-Strip-Wells
2275 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2700905-24StripWells
MyBiosource
24-Strip-Wells
225 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2700905-48StripWells
MyBiosource
48-Strip-Wells
280 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2700905-5x96StripWells
MyBiosource
5x96-Strip-Wells
1190 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
MBS2700905-96StripWells
MyBiosource
96-Strip-Wells
335 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RDR-IGF1-Hu-48T
Reddot Biotech
48T
259 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RDR-IGF1-Hu-48Tests
Reddot Biotech
48 Tests
368.4 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RDR-IGF1-Hu-96T
Reddot Biotech
96T
371 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RDR-IGF1-Hu-96Tests
Reddot Biotech
96 Tests
501.6 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
SEA050Hu-10x96wellstestplate
Cloud-Clone
10x96-wells test plate
4091.29 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
SEA050Hu-1x48wellstestplate
Cloud-Clone
1x48-wells test plate
442.1 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
SEA050Hu-1x96wellstestplate
Cloud-Clone
1x96-wells test plate
580.15 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
SEA050Hu-5x96wellstestplate
Cloud-Clone
5x96-wells test plate
2250.68 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
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Cloud-Clone
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RD-IGF1-Hu-48T
Reddot Biotech
48T
247 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RD-IGF1-Hu-48Tests
Reddot Biotech
48 Tests
354 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RD-IGF1-Hu-96T
Reddot Biotech
96T
353 EUR
Human Insulin Like Growth Factor 1 (IGF1) ELISA Kit
RD-IGF1-Hu-96Tests
Reddot Biotech
96 Tests
480 EUR
Human Insulin Like Growth Factor 1 (IGF1) Peptide (OVA)
20-abx651781
Abbexa
Human Insulin Like Growth Factor 1 (IGF1) Peptide (OVA)
abx651781-5mg
Abbexa
5 mg
562.5 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx652297-100g
Abbexa
100 µg
237.5 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
20-abx652297
Abbexa
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx652297-10g
Abbexa
10 µg
175 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx652297-50g
Abbexa
50 µg
212.5 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx651354-100g
Abbexa
100 µg
575 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
20-abx651354
Abbexa
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx651354-10g
Abbexa
10 µg
337.5 EUR
Human Insulin Like Growth Factor 1 (IGF1) Protein (Active)
abx651354-50g
Abbexa
50 µg
475 EUR
Human IGF1 (Insulin Like Growth Factor 1) ELISA Kit
MBS8800136-10x96StripWells
MyBiosource
10x96-Strip-Wells
3130 EUR
Human IGF1 (Insulin Like Growth Factor 1) ELISA Kit
MBS8800136-48StripWells
MyBiosource
48-Strip-Wells
315 EUR
Human IGF1 (Insulin Like Growth Factor 1) ELISA Kit
MBS8800136-5x96StripWells
MyBiosource
5x96-Strip-Wells
1710 EUR
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385 EUR
Active Insulin Like Growth Factor 1 (IGF1)
APA050Hu01
Cloud-Clone
10ug
39 EUR
Active Insulin Like Growth Factor 1 (IGF1)
APA050Hu61
Cloud-Clone
10ug
140 EUR
Active Insulin Like Growth Factor 1 (IGF1)
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Active Insulin Like Growth Factor 1 (IGF1)
APA050Hu62
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144 EUR
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Active Insulin Like Growth Factor 1 (IGF1)
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122 EUR
Active Insulin Like Growth Factor 1 (IGF1)
APA050Mu03
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124 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2105063-001mg
MyBiosource
0.01mg
115 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2105063-005mg
MyBiosource
0.05mg
170 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2105063-01mg
MyBiosource
0.1mg
225 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2105063-02mg
MyBiosource
0.2mg
260 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2105063-05mg
MyBiosource
0.5mg
460 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122262-001mg
MyBiosource
0.01mg
100 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122262-005mg
MyBiosource
0.05mg
135 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122262-01mg
MyBiosource
0.1mg
160 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122262-02mg
MyBiosource
0.2mg
180 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122262-05mg
MyBiosource
0.5mg
280 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122263-001mg
MyBiosource
0.01mg
145 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122263-005mg
MyBiosource
0.05mg
235 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122263-01mg
MyBiosource
0.1mg
330 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122263-02mg
MyBiosource
0.2mg
405 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2122263-05mg
MyBiosource
0.5mg
755 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2086061-001mg
MyBiosource
0.01mg
155 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2086061-005mg
MyBiosource
0.05mg
260 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2086061-01mg
MyBiosource
0.1mg
375 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2086061-02mg
MyBiosource
0.2mg
460 EUR
Active Insulin Like Growth Factor 1 (IGF1)
MBS2086061-05mg
MyBiosource
0.5mg
860 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU54794-100ug
Biomatik Corporation
100ug
554.4 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU54794-1mg
Biomatik Corporation
1mg
2184 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU54794-50ug
Biomatik Corporation
50ug
336 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU55460-100ug
Biomatik Corporation
100ug
235.2 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU55460-1mg
Biomatik Corporation
1mg
1092 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU55460-50ug
Biomatik Corporation
50ug
168 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56075-100ug
Biomatik Corporation
100ug
219 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56075-1mg
Biomatik Corporation
1mg
605.3 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56075-50ug
Biomatik Corporation
50ug
93.2 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56102-100ug
Biomatik Corporation
100ug
374 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56102-1mg
Biomatik Corporation
1mg
1665.3 EUR
Active Insulin Like Growth Factor 1 (IGF1)
RPU56102-50ug
Biomatik Corporation
50ug
302.5 EUR
Insulin Like Growth Factor 1 (IGF1) Protein
20-abx263464
Abbexa
Insulin Like Growth Factor 1 (IGF1) Peptide
20-abx266444
Abbexa
Insulin Like Growth Factor 1 (IGF1) Peptide
abx266444-1ml
Abbexa
1 ml
525 EUR
Insulin Like Growth Factor 1 (IGF1) Peptide
abx266444-200l
Abbexa
200 µl
350 EUR
Human Insulin Like Growth Factor I (IGF1) ELISA Kit
abx573274-96tests
Abbexa
96 tests
468.75 EUR
Insulin Like Growth Factor 1 (IGF1) Monoclonal Antibody (Human)
4-MAA050Hu22
Cloud-Clone
Insulin Like Growth Factor 1 (IGF1) Polyclonal Antibody (Human)
4-PAA050Hu06
Cloud-Clone
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102882-100l
Abbexa
100 µl
200 EUR
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20-abx102882
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102882-1ml
Abbexa
1 ml
462.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102882-200l
Abbexa
200 µl
250 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102883-100l
Abbexa
100 µl
212.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx102883
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102883-1ml
Abbexa
1 ml
475 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102883-200l
Abbexa
200 µl
250 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102884-100l
Abbexa
100 µl
262.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx102884
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102884-1ml
Abbexa
1 ml
687.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102884-200l
Abbexa
200 µl
325 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102885-100l
Abbexa
100 µl
287.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx102885
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102885-1ml
Abbexa
1 ml
812.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102885-200l
Abbexa
200 µl
362.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102886-100l
Abbexa
100 µl
287.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx102886
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102886-1ml
Abbexa
1 ml
812.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx102886-200l
Abbexa
200 µl
362.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx109912-100l
Abbexa
100 µl
162.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx109912
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx125982-100l
Abbexa
100 µl
175 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx125982
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx125982-1ml
Abbexa
1 ml
400 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx125982-200l
Abbexa
200 µl
275 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx125984
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131192-100l
Abbexa
100 µl
462.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx131192
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131192-20l
Abbexa
20 µl
200 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131192-50l
Abbexa
50 µl
250 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131938-100g
Abbexa
100 µg
712.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx131938
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131938-20g
Abbexa
20 µg
325 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx131938-5g
Abbexa
5 µg
262.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx137012-01mg
Abbexa
0.1 mg
487.5 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx137012
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx137628-100tests
Abbexa
100 tests
225 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
abx128905-100l
Abbexa
100 µl
200 EUR
Insulin Like Growth Factor 1 (IGF1) Antibody
20-abx128905
Abbexa
Insulin Like Growth Factor 1 (IGF1) Antibody
abx128905-1ml
Abbexa
1 ml
462.5 EUR
In this work, we present an MS-based quantitative IGF1 assay that meets all of the above requirements by achieving these two goals: 1) To rigorously quantify IGF1 in human plasma samples at a rate of >1,000 samples/day over time and to objectively assess economic considerations associated with the transfer of such targeted mass spectrometric assays from research laboratories to clinical use, and 2) to consider the IGF1 heterogeneity discovered during the analysis of large populations in order to intelligently design IGF1 mass spectrometric assays that avoid errors avoid (or allow for the study of variants in alternative disease situations) due to structural variants.To the best of our knowledge, none of these aspects of assay/biomarker translation have previously been tested and reported to any significant extent.