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. 2017 Apr 5;12(4):e0174447.
doi: 10.1371/journal.pone.0174447. eCollection 2017.

A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies

Daniel I Bromage  1 , Stasa Taferner  1 , Mahesh Pillai  1 , Derek M Yellon  1 , Sean M Davidson  1
Affiliations

A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies

Daniel I Bromage et al. PLoS One. .

Abstract

Background: Stromal derived factor-1α (SDF-1α/CXCL12) is a chemokine that is up-regulated in diseases characterised by tissue hypoxia, including myocardial infarction, ischaemic cardiomyopathy and remote ischaemic conditioning (RIC), a technique of cyclical, non-injurious ischaemia applied remote from the heart that protects the heat from lethal ischaemia-reperfusion injury. Accordingly, there is considerable interest in SDF-1α as a potential biomarker of such conditions. However, SDF-1α is rapidly degraded and inactivated by dipeptidyl peptidase 4 and other peptidases, and the kinetics of intact SDF-1α remain unknown.

Methods & results: To facilitate investigation of full-length SDF-1α we established an ELISA using a novel recombinant human antibody we developed called HCI.SDF1. HCI.SDF1 is specific to the N-terminal sequence of all isoforms of SDF-1 and has a comparable KD to commercially available antibodies. Together with a detection antibody specific to the α-isoform, HCI.SDF1 was used to specifically quantify full-length SDF-1α in blood for the first time. Using RIC applied to the hind limb of Sprague-Dawley rats or the arms of healthy human volunteers, we demonstrate an increase in SDF-1α using a commercially available antibody, as previously reported, but an unexpected decrease in full-length SDF-1α after RIC in both species.

Conclusions: We report for the first time the development of a novel recombinant antibody specific to full-length SDF-1. Applied to RIC, we demonstrate a significant decrease in SDF-1α that is at odds with the literature and suggests a need to investigate the kinetics of full-length SDF-1α in conditions characterised by tissue hypoxia.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Representation of binding sites of HCI.SDF1α and commercial antibodies on full-length SDF-1α and SDF-1α after cleavage of its N-terminal di-peptide by DPP4.
Fig 2
Fig 2. SDF-1α sequence according to species.
Alignment of SDF-1α from humans, rats and mice demonstrates the N-terminal sequence is highly conserved between species. Accession numbers NP_001171605.1, NP_001029055.1 and NP_001012495.1 were used, respectively. Sequences in red represent the sequence of interest used to generate HCI.SDF1. Blue residues represent differences in the primary sequence between species; note that none of these are at the N-terminal end.
Fig 3
Fig 3. In vivo rat protocols.
Remote ischaemic conditioning (RIC) was applied to the rat hind limb in the following groups: A) Control: 35 min stabilisation; B) RIC: 5 min stabilisation, 3 cycles of 5 min ischemia and 5 min reperfusion. Blood samples were taken either immediately after RIC (sample 1) or 60 min thereafter (sample 2).
Fig 4
Fig 4. Laser Doppler blood flow assessment in the subject hind limb during RIC and reperfusion.
Panel A shows a colour photograph of the subject hind limb. Panel B demonstrates complete cessation of blood flow during RIC ischaemia (C) and restoration after reperfusion (D).
Fig 5
Fig 5. Representative HCI.SDF1 and MAB350 binding kinetics data.
Representative data obtained from label-free sensor evaluation of HCI.SDF1 and MAB350 with Amine Reactive Second-Generation (AR2G) Biosensors.
Fig 6
Fig 6. ELISA for intact and cleaved SDF-1α using HCI.SDF1 and MAB350.
An ELISA assay demonstrates that HCI.SDF1 is specific for active SDF-1α, with very low signal in the absence of intact SDF-1α, whereas there is substantial residual signal in the absence of intact SDF-1α when using the commercial antibody MAB350.
Fig 7
Fig 7. HCI.SDF1 used for Western blotting.
Western blots demonstrated a concentration-signal relationship when HCI.SDF1 was used to detect varying concentrations of rhSDF-1α.
Fig 8
Fig 8. The effect of sample type on SDF-1α concentration.
Serum samples, collected by allowing whole blood to clot for 10 min prior to centrifuging to remove the clot, had significantly more active SDF-1α than either platelet-free (PFP) or unfractionated plasma samples that were collected into a citrated tube and prepared by differential centrifugation, when measured using either capture antibody as indicated.
Fig 9
Fig 9. Effect of RIC on intact and cleaved SDF-1α.
Following RIC, ELISA using commercial antibodies detects an increase in plasma SDF-1α levels whereas HCI.SDF1α, which is more specific for full-length SDF-1α, measures a decrease immediately after RIC that is restored to baseline by 1 h.
Fig 10
Fig 10. Effect of RIC on intact SDF-1α in humans.
Plasma levels of full-length SDF-1α (quantified using HCI.SDF1α ELISA), decreased significantly in healthy male volunteers subjected to RIC.
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