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Point of care testing of phospholipase A2 group IIA for serological diagnosis of rheumatoid arthritis Received 00th January 20xx, Accepted 00th January 20xx
a,b
a
a
b
c
Nathan Liu, Robert Chapman, Yiyang Lin, Jonas Mmesi, Andrew Bentham, Matthew c b,* a,* Tyreman, Sonya Abraham, Molly M. Stevens.
DOI: 10.1039/x0xx00000x www.rsc.org/
Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care. Outcomes in arthritic conditions are complex, and prognosis requires combinations of indicators to assess disease activity including joint swelling, joint pain, functional status, and patient perception of pain and fatigue. It is widely acknowledged that a current lack of reliable markers exists for improving patient management for rheumatoid and psoriatic arthritis. While biomarkers have been reported for diagnosis of rheumatoid arthritis (RA), these markers have been demonstrated to be poor predictors of disease activity and prognosis.1 In addition, psoriatic arthritis (PsA) lacks specific biomarkers for monitoring disease activity and treatment response.2 Consequently, there remains a significant clinical need for new biomarkers to guide management of rheumatoid and psoriatic arthritis. Secretory phospholipase A2 (sPLA2) was first explored as an inflammatory biomarker in rheumatoid arthritis over two decades ago by Pruzanski et al.,3 who noted levels of phospholipase activity correlated with disease indices including tender and swollen joint count, Landsbury index, functional class, and erythrocyte sedimentation rate (ESR). The group IIA form of sPLA2 (sPLA2-IIA) was further explored as a drug target but failed to produce additional benefit adjunct to
treatment with disease modifying anti-rheumatic drugs 4 (DMARDs) in rheumatoid arthritis cohorts. Attempts to reproduce the prognostic capabilities using sPLA2 have led to 5,6 varied and inconclusive results. It is possible that these previous measurements of sPLA2 were not specific, as it is now known that as many as eleven subgroups of sPLA2 exist, of which only a few have been conclusively implicated in 7 inflammation and rheumatic disease, notably sPLA2-IIA. While sPLA2 has not been studied recently, interest remains in its 8 utility as a biomarker in arthritic diseases. Recently, we described a point of care lateral flow device (LFD) assay utilizing sPLA2-mediated cleavage of a liposomal substrate for ultrasensitive measurement of sPLA2-IB enzyme activity in 9 serum/blood within 15 minutes. In this report, we expand on this work to explore the use of sPLA2-IIA activity as a point of care prognostic biomarker for patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA). To this end 30 patients diagnosed with RA and 25 patients with PsA, were recruited from a biobank at the Imperial College Healthcare NHS Trust. Patients had received varied treatments with DMARDs such as methotrexate and TNF inhibitors such as etanercept as outlined in Table 1. Disease activity for each patient was determined using the Nijmegen 28-joint Disease Activity Score (DAS28) for RA patients, and both the psoriatic arthritis response criteria (PsARC) and the minimal disease activity (MDA) criteria for PsA patients. Point of care devices were fabricated as in our previous work,9 and used to measure the sPLA2-IIA activity in 5 µL of serum relative to a standard curve generated from recombinant PLA2-IIA (R&D systems). The liposomes used in this assay were prepared by extrusion and purification over Sephadex gel, and showed good uniformity by transmission electron microscopy and dynamic light scattering (see SI, Figure S1). Images of the lateral flow strips, which were measured in duplicate and read using a Forsite LFD reader, are shown in the supporting information (Figure S2). The sPLA2-IIA activity was compared to the concentration ([sPLA2-IIA]), which was measured by enzyme linked immunosorbent assays (ELISA) using a novel antibody pairing prepared in house. The ELISA showed good standard
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Figure 1. a) Schematic of the point of care lateral flow device (LFD) assay. Activity of the enzyme cleaves the liposome, releasing linker (4-arm PEG-Biotin), which adheres polystreptavidin-coated gold nanoparticles to a streptavidin test line giving a red signal proportional to the sPLA2 activity. b-c) Scatter plots relating the concentrations and activity of sPLA2-IIA (measured by ELISA and LFD respectively) (b); the concentrations of sPLA2-IIA and CRP (c); as well as the activity of sPLA2-IIA and concentration of CRP (d). Each point represents a pair of values from one of 55 total patients with rheumatoid (red) or psoriatic (blue) arthritis.
Table 1. Patient baseline characteristics and clinical performance of markers
Patients, n Age, yrs Male/Female, n (%) DAS28-ESR Biologics, n (%) - Etanercept - Adalimumab - Golimumab DMARDS, n (%) - Methotrexate - Sulfasalazine - Hydroxychloroquine - Azathioprine Active/Inactive (n) [CRP], mg/L - Active - Inactive - Difference - p-value [sPLA2-IIA], ng/mL - Active - Inactive - Difference - p-value sPLA2-IIA activity, units - Active - Inactive - Difference - p-value
Rheumatoid Arthritis (RA) 30 58.6 ± 14.1 5 (17%)/25 4.49 ± 1.99 25 (83%) 23 (77%) 2 (7%) 0 (0%) 24 (80%) 19 (63%) 11 (37%) 10 (33%) 1 (3%) 20/10
Psoriatic Arthritis (PsA) 25 44.8 ± 15.8 15 (60%)/9 N/A 24 (96%) 8 (32%) 12 (48%) 4 (16%) 14 (56%) 7 (28%) 10 (40%) 0 (0%) 0 (0%) 18/7
14.7 ± 15.7 1.6 ± 1.3 13.07 0.0015
9.8 ± 17.9 11.6 ± 18.8 -1.86 0.80
191.0 ± 167.4 57.8 ± 33.2 133.2 0.0024
95.2 ± 60.3 85.5 ± 97.6 9.680 0.79
3010 ± 1800 959 ± 956 2050 0.0068
1780 ± 1720 1510 ± 1740 271.0 0.70
Continuous variables are expressed as means ± SD
curves with a broad linear range and sensitivity at < 1 ng/mL (see SI, Figure S3), and the LFD assay performed well across the full range of clinical samples. The activity of sPLA2-IIA was found to be strongly correlated with its concentration, with a Spearman correlation coefficient of 0.932 and a Pearson correlation coefficient of 0.836 (Figure 1b, Table S1). Both sPLA2-IIA concentration and activity were found to correlate with C-reactive protein (CRP) concentration, an inflammatory biomarker commonly used in RA diagnosis, with Spearman correlation coefficients of 0.651 and 0.521, respectively (Figure 1b-c, Table S1). Using the MDA criteria for differentiating between active and inactive disease within psoriatic arthritis, mean CRP concentrations were slightly decreased in the active disease cohort (9.8 ± 17.9 mg/L) relative to the inactive disease cohort (11.6 ± 18.8), but the difference was not statistically significant (Table 1, Figure 2a). In line with previous studies,10,11 CRP was found to discriminate significantly (p < 0.05) between the active and inactive disease cohorts in rheumatoid arthritis, with mean levels of 14.7 ± 15.7 mg/L in the active cohort and 1.6 ± 1.3 mg/L in the inactive (Table 1, Figure 2d). Given the speed and ease of the sPLA2-IIA LFD assay (15 min) compared to the CRP ELISA, and the strong correlations found between the markers, we expected sPLA2-IIA to prove an attractive analogue to CRP in RA diagnosis. Estimates of background levels of sPLA2-IIA in healthy serum vary from 2.1 65 ng/mL depending on the method used.12,13 Using our novel antibody pairing we measured the concentration of sPLA2-IIA in a set of 10 serum samples from healthy patients obtained from SeraLabs to be 21.9 ± 23.9 ng/mL, in line with these estimates. The sPLA2-IIA concentration and activity were found to be not significantly different between the inactive and active PsA cohorts (85.5 ± 97.6 ng/mL vs. 95.2 ± 60.3 ng/mL respectively for concentration and 1780 ± 1720 LFD units vs 1510 ± 1740 LFD units respectively for activity) (Table 1, Figure 2b-c). However, sPLA2-IIA concentration and activity was significantly (p < 0.05) elevated in the active disease relative to the inactive RA cohort, with measured levels of 191.0 ± 167.4 ng/mL (3010 ± 1800 LFD units) and 57.8 ± 33.2 ng/mL (959 ± 956 LFD units), respectively (Table 1, Figure 2e-f). Both active and inactive RA cohorts showed elevated sPLA2-IIA levels relative to the healthy control set (see SI, Figure S4). These results confirm the value of sPLA2 in diagnosis of RA first suggested over two decades ago,3 but specifically highlight the role of the sPLA2-IIA form of the enzyme, through the use of novel antibodies specific to this isoform. Both the concentration and activity of sPLA2-IIA appear to be comparable to CRP as a biomarker for predicting disease activity in RA, as evidenced by the strong correlations between these markers (Figure 1) and their similar diagnostic accuracy in RA (Figure 2c-f), reported for the first time in this work. This correlation supports the suggested role of sPLA2-IIA as a mediator of inflammation through the arachidonate 7,14 pathway. While sPLA2 has been previously found in synovial 15 fluid of PsA, this is the first report investigating the prognostic capability of sPLA2-IIA as a biomarker in psoriatic arthritis. However, neither sPLA2 nor CRP could distinguish
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Figure 2. Beeswarm and box plots depicting the distribution of CRP concentration (a), sPLA2-IIA concentration (b) and sPLA2-IIA activity (c) in psoriatic arthritis patient cohorts; and the CRP concentration (d), sPLA2-IIA concentration (e) and sPLA2-IIA activity (f) in rheumatoid arthritis patient cohorts. *Significant (p 2.6, and inactive disease was characterized by DAS28-ESR < 2.6. Psoriatic arthritis disease activity was assessed utilizing both the psoriatic arthritis response criteria (PsARC) and the minimal disease activity (MDA) criteria. Minimally active disease activity in PsA was defined by tender joint count (TJC) ≤ 1, swollen joint count (SJC) ≤ 1, and a patient global assessment (PTGA) ≤ 20%. Active disease in PsA was characterized by TJC > 3, SJC > 3, or PTGA >20%. All sera and blood were stored at -80 °C, and thawed to room temperature 30 minutes prior to use. Biomarker assays: sPLA2-IIA and C-reactive protein (CRP) concentration was measured using enzyme-linked immunosorbent assays (ELISA). Biotinylated and unconjugated mouse monoclonal IgG antibodies for human C-reactive protein (CRP) were obtained from R&D systems (Abingdon, U.K.), and bovine serum albumin (BSA) conjugated fragment antibodies (fAb) for capture and detection of sPLA2-IIA were obtained from the Mologic BITs PLA2G2A ELISA (Bedford, UK). In each ELISA, the capture antibody was incubated (50 µL per well, 2 µg/mL) in PBS overnight onto high-binding 96-well plates (Corning, NY, USA). Plates were washed (50 mM TRIS + 0.02% w/v Tween 20 x 3, PBS x 3), and blocked (10 mg/mL BSA in PBS), washed as before, and then incubated for 2h with the serum diluted to the appropriate concentration in PBS + 1 mg/mL BSA / high spec diluent (1:1 v/v). After washing, the plates were incubated with the detection antibody (200 ng/mL, PBS + 0.1% w/v BSA) for 1h, washed and then incubated with streptavidin conjugated HRP (160 ng/mL) for 30 min. Plates were developed with TMB (0.5 mM 3,3′,5,5′tetramethylbenzidine + 0.5 mM H2O2 in sodium acetate buffer, 50 µl) for 30 min before addition of H2SO4 (10 µL) to stop the reaction. sPLA2-IIA activity was measured utilizing an LFD assay previously described, omitting the use of a sPLA2 type IIA 9 inhibitor. Briefly, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphorylglycerol (POPG, Sigma-Aldrich) liposomes containing 4-arm PEG-biotin (2 kDa, 0.1 mM) were prepared by extruding multilamellar liposomal suspensions through a
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Nanoscale Tissue Bank for facilitating the transfer of the clinical samples from the Charing Cross Hospital Biobank. References 1
J. D. Isaacs, Nat. Rev. Immunol., 2010, 10, 605–611.
2
D. Gladman, J Rheumatol Suppl, 2012, Jul, 106–10.
3 W. Pruzanski, E. C. Keystone, B. Sternby, C. Bombardier, K. M. Snow and P. Vadas, J Rheumatol, 1988, 15, 1351–1355. 4 J. D. Bradley, A. A. Dmitrienko, A. J. Kivitz, O. S. Gluck, A. L. Weaver, G. Wiesenhutter, S. L. Myers and G. D. Sides, J Rheumatol, 2005, 32, 417–423. 5 M. K. S. Lin, V. Farewell, P. Vadas, A. A. M. Bookman, E. C. Keystone and W. Pruzanski, J Rheumatol, 1996, 23, 1162–1166.
Statistical Analysis: Statistical analyses were performed using R (3.1.3). All retrieved clinical and measured data for each subject was recorded and tabulated. All correlations between markers were performed using the Pearson and nonparametric Spearman rank correlation test. Group differences were assessed using Welch’s t-test. All significance tests were two-sided with a significance level of α = 0.05.
6 R. M. Michaels, J. C. Reading, D. H. Beezhold and J. R. Ward, J Rheumatol, 1996, 23, 226–229.
Notes and References
10 M. J. Plant, A. L. Williams, M. M. O’Sullivan, P. a Lewis, E. C. Coles and J. D. Jessop, Arthritis Rheum., 2000, 43, 1473–1477.
Acknowledgements The authors are grateful for funding from the Technology Strategy Board (TSB), the UK Engineering and Physical Sciences Research Council (EPSRC) (EP/K502352/1), and the NIHR and BRC at the Imperial College Healthcare NHS Trust. NJL was supported by the US-UK Fulbright Commission and the Whitaker Foundation. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The authors also thank Sandra Hemmington, Mark Burnapp and Josh Kirby at Mologic for fruitful discussions throughout the study as well as Sarah Chilcott-Burns and the Imperial College Healthcare NHS Trust
7 D. Six and E. Dennis, Biochim. Biophys. Acta, 2000, 1488, 1– 19. 8
W. Pruzanski, J Rheumatol, 2005, 32, 400–402.
9 R. Chapman, Y. Lin, M. Burnapp, A. Bentham, D. Hillier, A. Zabron, S. Khan, M. Tyreman and M. M. Stevens, ACS Nano, 2015, 9, 2567–2573.
11 G. Wells, J.-C. Becker, J. Teng, M. Dougados, M. Schiff, J. Smolen, D. Aletaha and P. L. C. M. van Riel, Ann. Rheum. Dis., 2009, 68, 954–960. 12 T. J. Nevalainen, Clin. Chem., 1993, 39, 2453–2459. 13 T. J. Nevalainen, J. M. Gronroos and P. T. Kortesuo, Gut, 1993, 34, 1133–1136. 14 S. Masuda, M. Murakami, K. Komiyama, M. Ishihara, Y. Ishikawa, T. Ishii and I. Kudo, FEBS J., 2005, 272, 655–72. 15 J. Seilhamer, E. Stefanski, P. Vadas, L. K. Johnson, D. T. Nterminal and J. Biochem, J. Biochem., 1989, 106, 38–42.
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TM
polycarbonate membrane with Nucleopore 200 nm pores (Whatman, Maidstone, UK), followed by purification by gel filtration through a Sephadex G-100 column. The liposome fractions were collected and diluted to a lipid concentration of 1 mg/mL in HEPES buffer (50 mM, pH 7.0) containing 150 mM NaCl. These liposomes (2 μL) were incubated with serum (5 μL) diluted in assay buffer (15 μL, 1% w/v BSA + HEPES (50 mM) + NaCl (150 mM) + CaCl2 (20 mM)) for 5 min before addition of polystreptavidin-functionalized gold nanoparticles (2 μL, 0.05 mg/mL streptavidin + 40 nM gold colloid, BBI) (Figure 1a). The solution was allowed to flow up the LFD via capillary action, and the LFDs were washed with assay buffer (15 μL). Upon drying, the signal on the LFD was quantified using a lateral flow device reader (Forsite Diagnostics, Abingdon, UK).
Nanoscale Accepted Manuscript
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This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Point of care testing of phospholipase A2 group IIA for serological diagnosis of rheumatoid arthritis Received 00th January 20xx, Accepted 00th January 20xx
a,b
a
a
b
c
Nathan Liu, Robert Chapman, Yiyang Lin, Jonas Mmesi, Andrew Bentham, Matthew c b,* a,* Tyreman, Sonya Abraham, Molly M. Stevens.
DOI: 10.1039/x0xx00000x www.rsc.org/
Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care. Outcomes in arthritic conditions are complex, and prognosis requires combinations of indicators to assess disease activity including joint swelling, joint pain, functional status, and patient perception of pain and fatigue. It is widely acknowledged that a current lack of reliable markers exists for improving patient management for rheumatoid and psoriatic arthritis. While biomarkers have been reported for diagnosis of rheumatoid arthritis (RA), these markers have been demonstrated to be poor predictors of disease activity and prognosis.1 In addition, psoriatic arthritis (PsA) lacks specific biomarkers for monitoring disease activity and treatment response.2 Consequently, there remains a significant clinical need for new biomarkers to guide management of rheumatoid and psoriatic arthritis. Secretory phospholipase A2 (sPLA2) was first explored as an inflammatory biomarker in rheumatoid arthritis over two decades ago by Pruzanski et al.,3 who noted levels of phospholipase activity correlated with disease indices including tender and swollen joint count, Landsbury index, functional class, and erythrocyte sedimentation rate (ESR). The group IIA form of sPLA2 (sPLA2-IIA) was further explored as a drug target but failed to produce additional benefit adjunct to
treatment with disease modifying anti-rheumatic drugs 4 (DMARDs) in rheumatoid arthritis cohorts. Attempts to reproduce the prognostic capabilities using sPLA2 have led to 5,6 varied and inconclusive results. It is possible that these previous measurements of sPLA2 were not specific, as it is now known that as many as eleven subgroups of sPLA2 exist, of which only a few have been conclusively implicated in 7 inflammation and rheumatic disease, notably sPLA2-IIA. While sPLA2 has not been studied recently, interest remains in its 8 utility as a biomarker in arthritic diseases. Recently, we described a point of care lateral flow device (LFD) assay utilizing sPLA2-mediated cleavage of a liposomal substrate for ultrasensitive measurement of sPLA2-IB enzyme activity in 9 serum/blood within 15 minutes. In this report, we expand on this work to explore the use of sPLA2-IIA activity as a point of care prognostic biomarker for patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA). To this end 30 patients diagnosed with RA and 25 patients with PsA, were recruited from a biobank at the Imperial College Healthcare NHS Trust. Patients had received varied treatments with DMARDs such as methotrexate and TNF inhibitors such as etanercept as outlined in Table 1. Disease activity for each patient was determined using the Nijmegen 28-joint Disease Activity Score (DAS28) for RA patients, and both the psoriatic arthritis response criteria (PsARC) and the minimal disease activity (MDA) criteria for PsA patients. Point of care devices were fabricated as in our previous work,9 and used to measure the sPLA2-IIA activity in 5 µL of serum relative to a standard curve generated from recombinant PLA2-IIA (R&D systems). The liposomes used in this assay were prepared by extrusion and purification over Sephadex gel, and showed good uniformity by transmission electron microscopy and dynamic light scattering (see SI, Figure S1). Images of the lateral flow strips, which were measured in duplicate and read using a Forsite LFD reader, are shown in the supporting information (Figure S2). The sPLA2-IIA activity was compared to the concentration ([sPLA2-IIA]), which was measured by enzyme linked immunosorbent assays (ELISA) using a novel antibody pairing prepared in house. The ELISA showed good standard
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Figure 1. a) Schematic of the point of care lateral flow device (LFD) assay. Activity of the enzyme cleaves the liposome, releasing linker (4-arm PEG-Biotin), which adheres polystreptavidin-coated gold nanoparticles to a streptavidin test line giving a red signal proportional to the sPLA2 activity. b-c) Scatter plots relating the concentrations and activity of sPLA2-IIA (measured by ELISA and LFD respectively) (b); the concentrations of sPLA2-IIA and CRP (c); as well as the activity of sPLA2-IIA and concentration of CRP (d). Each point represents a pair of values from one of 55 total patients with rheumatoid (red) or psoriatic (blue) arthritis.
Table 1. Patient baseline characteristics and clinical performance of markers
Patients, n Age, yrs Male/Female, n (%) DAS28-ESR Biologics, n (%) - Etanercept - Adalimumab - Golimumab DMARDS, n (%) - Methotrexate - Sulfasalazine - Hydroxychloroquine - Azathioprine Active/Inactive (n) [CRP], mg/L - Active - Inactive - Difference - p-value [sPLA2-IIA], ng/mL - Active - Inactive - Difference - p-value sPLA2-IIA activity, units - Active - Inactive - Difference - p-value
Rheumatoid Arthritis (RA) 30 58.6 ± 14.1 5 (17%)/25 4.49 ± 1.99 25 (83%) 23 (77%) 2 (7%) 0 (0%) 24 (80%) 19 (63%) 11 (37%) 10 (33%) 1 (3%) 20/10
Psoriatic Arthritis (PsA) 25 44.8 ± 15.8 15 (60%)/9 N/A 24 (96%) 8 (32%) 12 (48%) 4 (16%) 14 (56%) 7 (28%) 10 (40%) 0 (0%) 0 (0%) 18/7
14.7 ± 15.7 1.6 ± 1.3 13.07 0.0015
9.8 ± 17.9 11.6 ± 18.8 -1.86 0.80
191.0 ± 167.4 57.8 ± 33.2 133.2 0.0024
95.2 ± 60.3 85.5 ± 97.6 9.680 0.79
3010 ± 1800 959 ± 956 2050 0.0068
1780 ± 1720 1510 ± 1740 271.0 0.70
Continuous variables are expressed as means ± SD
curves with a broad linear range and sensitivity at < 1 ng/mL (see SI, Figure S3), and the LFD assay performed well across the full range of clinical samples. The activity of sPLA2-IIA was found to be strongly correlated with its concentration, with a Spearman correlation coefficient of 0.932 and a Pearson correlation coefficient of 0.836 (Figure 1b, Table S1). Both sPLA2-IIA concentration and activity were found to correlate with C-reactive protein (CRP) concentration, an inflammatory biomarker commonly used in RA diagnosis, with Spearman correlation coefficients of 0.651 and 0.521, respectively (Figure 1b-c, Table S1). Using the MDA criteria for differentiating between active and inactive disease within psoriatic arthritis, mean CRP concentrations were slightly decreased in the active disease cohort (9.8 ± 17.9 mg/L) relative to the inactive disease cohort (11.6 ± 18.8), but the difference was not statistically significant (Table 1, Figure 2a). In line with previous studies,10,11 CRP was found to discriminate significantly (p < 0.05) between the active and inactive disease cohorts in rheumatoid arthritis, with mean levels of 14.7 ± 15.7 mg/L in the active cohort and 1.6 ± 1.3 mg/L in the inactive (Table 1, Figure 2d). Given the speed and ease of the sPLA2-IIA LFD assay (15 min) compared to the CRP ELISA, and the strong correlations found between the markers, we expected sPLA2-IIA to prove an attractive analogue to CRP in RA diagnosis. Estimates of background levels of sPLA2-IIA in healthy serum vary from 2.1 65 ng/mL depending on the method used.12,13 Using our novel antibody pairing we measured the concentration of sPLA2-IIA in a set of 10 serum samples from healthy patients obtained from SeraLabs to be 21.9 ± 23.9 ng/mL, in line with these estimates. The sPLA2-IIA concentration and activity were found to be not significantly different between the inactive and active PsA cohorts (85.5 ± 97.6 ng/mL vs. 95.2 ± 60.3 ng/mL respectively for concentration and 1780 ± 1720 LFD units vs 1510 ± 1740 LFD units respectively for activity) (Table 1, Figure 2b-c). However, sPLA2-IIA concentration and activity was significantly (p < 0.05) elevated in the active disease relative to the inactive RA cohort, with measured levels of 191.0 ± 167.4 ng/mL (3010 ± 1800 LFD units) and 57.8 ± 33.2 ng/mL (959 ± 956 LFD units), respectively (Table 1, Figure 2e-f). Both active and inactive RA cohorts showed elevated sPLA2-IIA levels relative to the healthy control set (see SI, Figure S4). These results confirm the value of sPLA2 in diagnosis of RA first suggested over two decades ago,3 but specifically highlight the role of the sPLA2-IIA form of the enzyme, through the use of novel antibodies specific to this isoform. Both the concentration and activity of sPLA2-IIA appear to be comparable to CRP as a biomarker for predicting disease activity in RA, as evidenced by the strong correlations between these markers (Figure 1) and their similar diagnostic accuracy in RA (Figure 2c-f), reported for the first time in this work. This correlation supports the suggested role of sPLA2-IIA as a mediator of inflammation through the arachidonate 7,14 pathway. While sPLA2 has been previously found in synovial 15 fluid of PsA, this is the first report investigating the prognostic capability of sPLA2-IIA as a biomarker in psoriatic arthritis. However, neither sPLA2 nor CRP could distinguish
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COMMUNICATION measurement of this marker may be undertaken within 15 minutes and without any equipment for the reading of the signal, this technology has significant advantages over traditional “blood tests” which need to be analysed in a laboratory setting.
Figure 2. Beeswarm and box plots depicting the distribution of CRP concentration (a), sPLA2-IIA concentration (b) and sPLA2-IIA activity (c) in psoriatic arthritis patient cohorts; and the CRP concentration (d), sPLA2-IIA concentration (e) and sPLA2-IIA activity (f) in rheumatoid arthritis patient cohorts. *Significant (p 2.6, and inactive disease was characterized by DAS28-ESR < 2.6. Psoriatic arthritis disease activity was assessed utilizing both the psoriatic arthritis response criteria (PsARC) and the minimal disease activity (MDA) criteria. Minimally active disease activity in PsA was defined by tender joint count (TJC) ≤ 1, swollen joint count (SJC) ≤ 1, and a patient global assessment (PTGA) ≤ 20%. Active disease in PsA was characterized by TJC > 3, SJC > 3, or PTGA >20%. All sera and blood were stored at -80 °C, and thawed to room temperature 30 minutes prior to use. Biomarker assays: sPLA2-IIA and C-reactive protein (CRP) concentration was measured using enzyme-linked immunosorbent assays (ELISA). Biotinylated and unconjugated mouse monoclonal IgG antibodies for human C-reactive protein (CRP) were obtained from R&D systems (Abingdon, U.K.), and bovine serum albumin (BSA) conjugated fragment antibodies (fAb) for capture and detection of sPLA2-IIA were obtained from the Mologic BITs PLA2G2A ELISA (Bedford, UK). In each ELISA, the capture antibody was incubated (50 µL per well, 2 µg/mL) in PBS overnight onto high-binding 96-well plates (Corning, NY, USA). Plates were washed (50 mM TRIS + 0.02% w/v Tween 20 x 3, PBS x 3), and blocked (10 mg/mL BSA in PBS), washed as before, and then incubated for 2h with the serum diluted to the appropriate concentration in PBS + 1 mg/mL BSA / high spec diluent (1:1 v/v). After washing, the plates were incubated with the detection antibody (200 ng/mL, PBS + 0.1% w/v BSA) for 1h, washed and then incubated with streptavidin conjugated HRP (160 ng/mL) for 30 min. Plates were developed with TMB (0.5 mM 3,3′,5,5′tetramethylbenzidine + 0.5 mM H2O2 in sodium acetate buffer, 50 µl) for 30 min before addition of H2SO4 (10 µL) to stop the reaction. sPLA2-IIA activity was measured utilizing an LFD assay previously described, omitting the use of a sPLA2 type IIA 9 inhibitor. Briefly, 1-palmitoyl-2-oleoyl-sn-glycero-3phosphorylglycerol (POPG, Sigma-Aldrich) liposomes containing 4-arm PEG-biotin (2 kDa, 0.1 mM) were prepared by extruding multilamellar liposomal suspensions through a
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DOI: 10.1039/C5NR08423G
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Nanoscale Tissue Bank for facilitating the transfer of the clinical samples from the Charing Cross Hospital Biobank. References 1
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2
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Statistical Analysis: Statistical analyses were performed using R (3.1.3). All retrieved clinical and measured data for each subject was recorded and tabulated. All correlations between markers were performed using the Pearson and nonparametric Spearman rank correlation test. Group differences were assessed using Welch’s t-test. All significance tests were two-sided with a significance level of α = 0.05.
6 R. M. Michaels, J. C. Reading, D. H. Beezhold and J. R. Ward, J Rheumatol, 1996, 23, 226–229.
Notes and References
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Acknowledgements The authors are grateful for funding from the Technology Strategy Board (TSB), the UK Engineering and Physical Sciences Research Council (EPSRC) (EP/K502352/1), and the NIHR and BRC at the Imperial College Healthcare NHS Trust. NJL was supported by the US-UK Fulbright Commission and the Whitaker Foundation. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The authors also thank Sandra Hemmington, Mark Burnapp and Josh Kirby at Mologic for fruitful discussions throughout the study as well as Sarah Chilcott-Burns and the Imperial College Healthcare NHS Trust
7 D. Six and E. Dennis, Biochim. Biophys. Acta, 2000, 1488, 1– 19. 8
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9 R. Chapman, Y. Lin, M. Burnapp, A. Bentham, D. Hillier, A. Zabron, S. Khan, M. Tyreman and M. M. Stevens, ACS Nano, 2015, 9, 2567–2573.
11 G. Wells, J.-C. Becker, J. Teng, M. Dougados, M. Schiff, J. Smolen, D. Aletaha and P. L. C. M. van Riel, Ann. Rheum. Dis., 2009, 68, 954–960. 12 T. J. Nevalainen, Clin. Chem., 1993, 39, 2453–2459. 13 T. J. Nevalainen, J. M. Gronroos and P. T. Kortesuo, Gut, 1993, 34, 1133–1136. 14 S. Masuda, M. Murakami, K. Komiyama, M. Ishihara, Y. Ishikawa, T. Ishii and I. Kudo, FEBS J., 2005, 272, 655–72. 15 J. Seilhamer, E. Stefanski, P. Vadas, L. K. Johnson, D. T. Nterminal and J. Biochem, J. Biochem., 1989, 106, 38–42.
4 | J. Name., 2012, 00, 1-3
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polycarbonate membrane with Nucleopore 200 nm pores (Whatman, Maidstone, UK), followed by purification by gel filtration through a Sephadex G-100 column. The liposome fractions were collected and diluted to a lipid concentration of 1 mg/mL in HEPES buffer (50 mM, pH 7.0) containing 150 mM NaCl. These liposomes (2 μL) were incubated with serum (5 μL) diluted in assay buffer (15 μL, 1% w/v BSA + HEPES (50 mM) + NaCl (150 mM) + CaCl2 (20 mM)) for 5 min before addition of polystreptavidin-functionalized gold nanoparticles (2 μL, 0.05 mg/mL streptavidin + 40 nM gold colloid, BBI) (Figure 1a). The solution was allowed to flow up the LFD via capillary action, and the LFDs were washed with assay buffer (15 μL). Upon drying, the signal on the LFD was quantified using a lateral flow device reader (Forsite Diagnostics, Abingdon, UK).
