Development and validation of a multiplex bead assay for measuring

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Development and validation of a multiplex bead assay for measuring growth mediators in wound fluid† Thanasak Rakmanee,ab Irwin Olsen,*a Gareth S. Griffithsc and Nikolaos Donosa Received 17th June 2009, Accepted 23rd September 2009 First published as an Advance Article on the web 17th November 2009 DOI: 10.1039/b911863b Large amounts of biological samples are usually required to measure multiple components by the enzyme-linked immunosorbant assay. However, the amounts of many tissue extracts and fluids, including gingival crevicular fluid (GCF), are generally extremely small. The aim of this study was, therefore, to develop and validate a novel multiplex bead assay (MBA) to simultaneously measure a profile of healing-related mediators in the GCF of treated periodontal wounds. An MBA was developed and validated by assessment of assay selectivity, recovery, precision and sensitivity, using eight recombinant human growth mediators as assay standards. GCF samples were collected on paper strips from healing wound (test) and healthy unaffected (control) sites of 15 patients with periodontitis, seven days post-periodontal surgery. Each GCF sample was eluted and the levels of the mediators measured using the MBA and antibody pairs specific for angiopoietin-1, vascular endothelial growth-factor, bone morphogenetic protein-2, osteoprotegerin, tissue inhibitor of metalloprotease-1 (TIMP-1), basic fibroblast growth-factor, keratinocyte growth-factor, and platelet derived growth-factor. Less than 1.8% of cross-reactivity was observed between antibodies and the eight different analytes, for which the recovery was more than 85%. Mean intra- and inter-assay precision were within the acceptance criteria of 20% and 25%, respectively. Detection of all mediators was highly sensitive (#70 ng/L) except for TIMP-1 (215 ng/L). Angiogenic factors were the most highly secreted in the GCF seven days post-surgery. This new MBA can simultaneously measure small amounts of eight different growth mediators in the GCF of healing periodontal wounds. It might also be a valuable tool for evaluating the components of wound fluids as a prognostic indicator of the success of therapeutic intervention.

Introduction Wound healing involves several interrelated stages of coagulation, inflammation, angiogenesis, re-epithelialisation (in some tissues) and remodelling. Many cytokines and growth factors (GF) have been identified and their functions in tissue destruction and in wound healing studied in vitro and in vivo.1–3 These polypeptide mediators are secreted from cells and exert their biological activity by binding to their corresponding transmembrane receptors and initiating an intracellular cascade of molecular signalling events which ultimately activate specific target genes.4 Moreover, the presence of such mediators in body fluids is generally considered to reflect the activity of the underlying tissue, a number of studies reporting the close association between GF in skin drainage fluid,5 nasal fluid6 and periodontal fluid (gingival crevicular fluid; GCF)7 and the wound healing process.3

a Eastman Dental Institute for Oral Health Care Sciences, Division of Biomaterials and Tissue Engineering, UCL-Eastman Dental Institute, 256 Gray’s Inn Road, London, UK WC1X 8LD. E-mail: i.olsen@ eastman.ucl.ac.uk; Fax: +44(0)207975 1254; Tel: +44(0)207915 1254 b Faculty of Dentistry, Thammasat University, Patumthani, Thailand c School of Clinical Dentistry, University of Sheffield, Sheffield, UK † Electronic supplementary information (ESI) available: Growth mediators and antibodies, and schematic illustration of the multiplex bead assay. See DOI: 10.1039/b911863b

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GF are pivotal in mediating periodontal wound healing and regeneration and their effects on periodontal cells in vitro and in vivo have been extensively investigated. For example, platelet derived growth-factor (PDGF) was found to promote regeneration of both hard and soft periodontal tissues,8 while keratinocyte growth-factor (KGF) has a major role in re-epithelialisation,9 including the formation of the long-junctional gingival epithelium.10 Basic fibroblast growth-factor (bFGF), vascular endothelial growth-factor (VEGF) and angiopoietin-1 (Ang-1) play important parts in vascularisation/angiogenesis during the early phase of wound healing,11–13 while bone morphogenetic protein-2 (BMP-2) has been shown to recruit undifferentiated mesenchymal cells and induce bone formation14 and cementogenesis in vivo.15 Osteoprotegerin (OPG) has a major role in bone deposition and bone remodelling,16 and tissue inhibitor of metalloprotease-1 (TIMP-1) is associated with the turnover of extracellular matrix components as well as with cell migration and angiogenesis.17–19 Studies of the molecular events which accompany the healing of damaged tissue in vivo have, however, been limited by the very small amounts of wound fluid which can generally be obtained. Thus, the majority of such studies have examined only one mediator in each situation, usually by enzyme-linked immunosorbant assay (ELISA). Although this technique is relatively specific and sensitive, it precludes the systematic examination of the profile of multiple factors in a small sample of biological This journal is ª The Royal Society of Chemistry 2010

material. While a ‘sequential’ ELISA could possibly overcome some of these constraints,20 it is time-consuming, impractical for routine analysis and not cost-effective, therefore there is a need for a procedure which is able to rapidly and accurately determine the profile of multiple components in small amounts of biological fluid. Multiplex bead technology utilising flow cytometry (FCM) enables several analytes to be measured simultaneously in a small volume of biological material.21 This technique has been used for the assay of cytokines, chemokines and other inflammatory markers, and a number of multiplex panels are now commercially available. However, a multiplex bead assay (MBA) has hitherto not been applied to the measurement of multiple GF in wound fluids. Additionally, the MBA has usually utilised duallaser FCM, which is not readily available. The present study reports, for the first time, an MBA for simultaneously measuring growth-associated mediators in wound fluids using single-laser FCM (BD FACScan, BD Biosciences). A Tyramide Signal Amplification (TSA) procedure22 has also been utilised which, in conjunction with the MBA, significantly increases the assay sensitivity for measuring the following eight mediators in the GCF of healing periodontal wounds: Ang-1, VEGF, BMP-2, OPG, TIMP-1, bFGF, KGF, and PDGF.

ethanesulfonic acid (MES), phosphate-buffered saline (PBS), BSA, Tween20 and sodium azide (NaN3) from Sigma-Aldrich. Collection and elution of GCF samples GCF samples were collected from 15 subjects (age 32  7; 9 males) seven days after periodontal surgery. The subjects were diagnosed with advance periodontitis23 and periodontal surgery was scheduled as part of their corrective treatment. Informed consent was obtained from all participants and the collection of the GCF was reviewed and approved by the Eastman/UCLH Joint Research Ethics Committee, London, UK (study reference: 04/Q0512/93). For each subject, the test GCF was collected from the sites undergoing periodontal surgery (four samples) while the control GCF was collected from healthy unaffected sites (four samples), using an intracrevicular method with pre-cut filter strips as previously described.24 The volume of GCF collected was measured, the strips pooled according to the group category, transferred to plastic micro-centrifuge tubes (0.4 mL) (Alpha Laboratories) and stored at 70 C until elution using a previously described technique.25 Antibody-bead conjugation

Materials and methods Reagents The recombinant human (rh) proteins and antibodies (Ab) used in the present study are shown in Supplemental Data Table 1.† Carboxylated fluorescent beads (7.6 mm) (excitation 488 nm; emission 635 nm), which comprise a set of eight fluorescent beads each with a different intensity of the same colour fluorescence, and non-fluorescent beads pre-coated with bovine serum albumin (BSA) (0.8 mm, ‘carrier beads’) were obtained from Spherotech Inc. Streptavidin-AlexaFluor488 was used as ‘reporter’ fluorescence (excitation 495 nm; emission 519 nm) (Molecular Probes). The TSA Biotin System kit was obtained from PerkinElmer, Streptavidin-HRP from R&D Systems, 1-Ethyl-3-(3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfoNHS) from Pierce Biotechnology, 2-(N-morpholino)

‘Capture’ Ab corresponding to each of the eight mediators were conjugated to the beads using a modified two-step protocol,26 with all incubation processes carried out at room temperature (RT) in the dark. 2  106 Beads were washed by centrifugation (MicroCentaur centrifuge) at 13000 rpm for 4 min, the supernatant removed and the beads resuspended in 0.2 mL of distilled water, centrifuged again and resuspended in 40 ml of 50 mmol/L MES buffer (pH 5.5). Freshly prepared sulfo-NHS in sterile water (30 ml; 100 g/L) was added, then fresh EDC in sterile water (30 ml; 100 g/L). The suspension was mixed by vortex (IKA Works), gently shaken for 20 min, washed 3 times with 0.1 mL of the MES buffer and centrifuged again. To determine the optimal conjugation efficacy, 10, 20, 50 and 100 mg of the ‘capture’ Ab in 0.2 mL of MES buffer were incubated with the beads for either 2 h or overnight, with end-to-end rotation (Jencons Scientific). After incubation the beads were washed 3 times with wash buffer (PBS containing 0.5 mL/L Tween 20) and resuspended in 0.2 mL of PBS buffer containing 10 g/L BSA, 0.5 mL/L Tween 20 and

Table 1 Selectivity of the MBA Selectivity (%)a Corresponding Ab Target antigens

Concentration (ng/L)

Ang-1

VEGF

BMP-2

OPG

TIMP-1

bFGF

KGF

PDGF

Ang-1 VEGF BMP-2 OPG TIMP-1 bFGF KGF PDGF-AB

5000 5000 5000 5000 10000 8000 5000 5000

100.0 1.3 1.2 1.7 0.6 0.8 1.7 0.8

0.3 100.0 1.8 1.8 0.3 0.8 0.9 0.5

0.1 0.4 100.0 0.5 0.2 0.1 0.3 0.1

0.0 0.2 0.3 100.0 0.1 0.1 0.3 0.1

0.2 1.0 1.0 1.4 100.0 0.6 1.0 0.7

0.2 1.0 0.8 1.0 0.4 100.0 0.2 0.5

0.3 0.6 0.8 0.8 0.9 0.6 100.0 0.7

0.0 0.2 0.2 0.2 0.1 0.1 0.2 100.0

a

Selectivity of 100% is defined as the median fluorescence intensity (MFI) of each target antigen with its own specific corresponding Ab.

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0.5 g/L NaN3, then stored in the dark at 4  C. The relative level of ‘capture’ Ab binding to the beads was assessed by incubating for 1 h with goat anti-mouse Ab conjugated with AlexaFluor488 (1:500 dilution in PBS containing 10 g/L BSA). The beads were then washed and analysed by FCM.

A schematic illustration of the MBA of the eight mediators is shown in the Supplemental Data.† The MBA was carried out in 0.2 mL polypropylene microcentrifuge tubes (Alpha Laboratories) in the dark at RT, with end-to-end rotation. Following all incubation processes, the beads were washed three times by the addition of 0.1 mL of wash buffer and centrifuged at 13000 rpm for 4 min. Vortex shaking and 1 min of sonication (Elma GmbH & Co.) were used to re-suspend the pellets following centrifugation. Mixtures of the Ab-coated beads were incubated overnight with 0.1 mL of the eluted GCF samples and also with 0.1 mL of a mixture of known concentrations of the eight mediators used as standard samples for calibration (2-fold dilution series of six concentrations of the rh antigens, excluding zero, starting with 10 mg/L of TIMP-1, 4 mg/L of bFGF and 2 mg/L of each of the other mediators). After incubation, carrier beads were added to each sample and washed as above. The beads were re-suspended and incubated for 30 min in 0.5% TSA blocking buffer, centrifuged, washed and incubated for 1.5 h with a mixture of the eight (biotinylated) detection Ab (at the pre-determined optimal concentrations ranging from 45–250 mg/L). To evaluate the use of TSA in the MBA, dilutions (0, 125, 250, 500, 1000 ng/L) of a mixture of the 8 rh antigens were prepared in diluent buffer, in duplicate, and the procedure carried out with and without the addition of TSA. In the MBA without TSA, the beads were incubated for 45 min with 0.1 mL of Streptavidin-AlexaFluor488 (1:1000 dilution in diluent buffer), washed and analysed by FCM. In the MBA with the addition of TSA, the beads were resuspended in 0.1 mL of Streptavidin-HRP (1:50 dilution in the TSA blocking buffer) and incubated for 45 min, washed again as above and incubated for 10 min in 0.05 mL of TSA reagent (1:50 dilution in the amplification buffer). After washing, the beads were incubated for 45 min in 0.1 mL of Streptavidin-AlexaFluor488 (1:1000 dilution in diluent buffer). Finally, the beads were washed and resuspended in 0.1 mL of distilled water and analysed by FCM. CellQuest (v 3.3) and BD Cytometric Bead Array (CBA) software (BD Biosciences) were used for data acquisition and data analysis, respectively. The absolute amount of the analytes (ng) was determined using the standard curve obtained for each antigen, carried out at the same time. A 4-parameter logistic model of the concentration-response relationship was used for fitting the curve, as previously recommended.27 Because each GCF was eluted in a total volume of 0.1 mL, the absolute amount was calculated by dividing the outcome from the standard curve (ng/L) by 10 4 and expressed as ng/site.

assessment of assay selectivity, precision, recovery and sensitivity, as follows. Selectivity is the ability of the assay to distinguish a specific analyte in the presence of other components in the samples.28 To assess selectivity, a mixture of eight Ab-coated beads was added to 0.1 mL of a high concentration of each of the eight mediators individually (10 mg/L of TIMP-1, 8 mg/L of bFGF and 5 mg/L of each of the other mediators, in diluent buffer) and in duplicate, and the MBA carried out as above. The control was the diluent buffer alone. Selectivity of individual mediators was also evaluated by diluting each of the eight mediators at the above concentration in 10% normal human serum (NHS) (pooled from six healthy donors), which is equivalent to the final 10% concentration of the eluted GCF samples.29 Precision is the closeness of measurements when the procedure is repeated.28 To evaluate precision, the eight mediators were mixed at three concentrations (five replicates each), including the concentrations at the lower limit of quantification (LLOQ), the approximate midrange of the calibration curve and the upper limit of quantification (ULOQ). To be accepted as the lowest and highest concentrations on the standard curve, the LLOQ and ULOQ need to meet the following criteria: (1) the response (median fluorescence intensity, MFI) at the anticipated LLOQ is $5 times higher than the MFI of the blank (diluent buffer alone); and (2) the response should be reproducible with a mean coefficient of variation (CV) of