Immunospecific analysis of in vitro and ex vivo surface-immobilized protein complex

A. Quantification of ATH immobilized on polyurethane catheters

Central venous catheters (CVCs) are used for the long-term collection of blood samples and delivery of medication. Our previous studies have demonstrated that the modification of the CVCs with ATH can be successful in reducing plasma protein binding on indwelling catheters and in increasing potential anticoagulant properties.10,15,3110. Y. J. Du, J. L. Brash, G. McClung, L. R. Berry, P. Klement, and A. K. C. Chan, J. Biomed. Mater. Res. A 80A, 216 (2007). https://doi.org/10.1002/jbm.a.3097715. K. N. Sask, L. R. Berry, A. K. C. Chan, and J. L. Brash, J. Biomed. Mater. Res. A 100A, 2821 (2012). https://doi.org/10.1002/jbm.a.3421831. K. N. Sask, L. R. Berry, A. K. C. Chan, and J. L. Brash, Langmuir 28, 2099 (2012). https://doi.org/10.1021/la203821g Six PU catheters coated with covalently bound ATH conjugate were examined. An EIA method was used to evaluate the variations in the density of ATH immobilized onto the catheter surface, as outlined in the workflow in Fig. 1. The method is based on a chromogenic reaction with HRP-conjugated antibodies and TMB/H2O2 substrates. The HRP enzyme combines with H2O2 to oxidize TMB and produce a blue color.3232. E. S. Bos, A. A. van der Doelen, N. van Rooy, and A. H. W. M. Schuurs, J. Immunoassay 2, 187 (1981). https://doi.org/10.1080/15321818108056977 With the addition of a sulfuric acid stop solution, the color changes to yellow and colorimetric detection can be used to compare samples.The surface area of the catheters was 0.0332 cm2/mg, which was calculated from the geometric characteristics of the catheter. Based on the OD readings of the wells containing catheter segments, the total amount of ATH on the modified catheter was calculated by extrapolating from the OD readings of the wells containing known concentrations of ATH standards. A representative standard curve used for quantification of surface ATH is provided in Fig. 2.

The standard curve includes six data points in the dilution series starting from 12.5 down to 0.39 ng/ml with replicates for each point. The coefficient of determination for this standard curve was 0.993 with all other curve values 0.99 or greater. A standard curve was repeated for each assay to ensure the greatest accuracy in determining protein values. For all standard curves completed, the coefficient of variation for each point was less than 10%, indicating low variation among assays when measuring ATH immobilized on a reference surface.

As seen in Fig. 3, on the surfaces of six PU catheters, the measured ATH density ranged from 0.09 to 0.19 ng/cm2 with a mean density of 0.13 ± 0.03 ng/cm2 and a median density of 0.13 ng/cm2.Three sets of PU catheters were modified by incubation with three different concentrations of ATH. There was no significant difference (p > 0.1) in the surface density of ATH when a higher concentration (4 mg/ml) or a lower concentration (1 mg/ml) of ATH incubation solution was compared with the usual 2 mg/ml of ATH used for modification (Fig. 4).

B. Qualitative immunohistochemical determination of the spatial distribution of surface-immobilized ATH

After quantitative evaluation of the ATH density on catheter surfaces following immobilization, the catheter segments were examined and stained with TrueBlue peroxidase substrates. As can be seen in the images, ATH modified catheters were significantly more stained than that of the control. Also, a slight trend of increasing surface stain with increasing coating concentration was noticeable (Fig. 5).

C. Evaluation of ATH modified PU catheters ex vivo

Three PU catheters modified with 1 mg/ml ATH were inserted into three rabbits. The catheters were removed after 32–106 days and then evaluated with the described EIA either with or without SDS-sodium phosphate (SDS-SP) washing (Table I). A bare unmodified PU catheter was used as a negative control.Table icon

TABLE I. Analysis of antithrombin surface density for covalent antithrombin-heparin (ATH) modified catheter segments before and after implantation in rabbits.

ATH surface density (ng/cm2)ConditionsWithout SDS-SP washingWith SDS-SP washingRabbit No. 1Before implant0.20 ± 0.060.28 ± 0.06Day 321.13 ± 0.040.94 ± 0.07Rabbit No. 2Before implant0.45 ± 0.040.39 ± 0.02Day 1061.23 ± 0.101.02 ± 0.09Rabbit No. 3Before implant0.44 ± 0.010.43 ± 0.16Day 1060.59 ± 0.060.46 ± 0.04Unmodified PU catheter0.0 ± 0.00.0 ± 0.0

D. Discussion

This is the first study using an adapted EIA method to measure the density of an ATH covalent protein complex on CVC surfaces. The ATH density on the catheter segment was extrapolated from the standards containing a known density of ATH detected by sheep antihuman AT-III antibodies on microtiter well surfaces. The utilization of an HRP-conjugated antibody as the detecting antibody provides both quantitative data through an adapted EIA and qualitative evaluation using immunohistochemical staining. Standard curves using an ATH dilution series were repeated for numerous assays and the consistently low coefficients of variation, of less than 10% each time, attested to the robustness of this assay.

The ATH surface density among the PU catheters was more variable than that on microtiter well surfaces and it was independent of the ATH concentration used during coating. Coverage of ATH on catheters determined through qualitative immunohistochemical assays was not entirely uniform (Fig. 5) and may indicate that some nonspecific plasma protein adsorption occurs. The suboptimal modification chemistry, together with variation in synthetic ATH products and PU-C70 catheters, may attribute to the interbatch heterogeneity. Additional studies in our labs are ongoing to optimize ATH modification procedures on various polymers. The quantitative and qualitative immunoassay methods developed here will be applied to assess the effectiveness of these optimized modification procedures.The results of ATH modification on catheters ex vivo were consistent with the observation in a previous study.1010. Y. J. Du, J. L. Brash, G. McClung, L. R. Berry, P. Klement, and A. K. C. Chan, J. Biomed. Mater. Res. A 80A, 216 (2007). https://doi.org/10.1002/jbm.a.30977 After implantation in vivo, there was an increase in the binding of the antithrombin antibody on ATH modified PU catheters left in situ for 32 and 106 days. The amount of binding and observed surface density was similar from 32 to 106 days demonstrating that these changes seemed to be independent of the implantation duration. Following SDS-sodium phosphate washes, there was a decrease in ATH only on the implanted catheters and not prior to implantation for both 32 and 106 days. The PU-C70 catheters may nonspecifically adsorb rabbit plasma protein, which may be detectable by the sheep antihuman AT antibody, because the optical signals were marginally increased, even with BSA-coated PU catheters (Fig. 3). However, the vigorous SDS-SP washing should remove loosely bound proteins, resulting in a measured ATH surface density that is lower than that before the washing procedure but still higher than the corresponding control that was never exposed to rabbit plasma proteins. It is known that the ATH complex interacts with extrinsic AT via its heparin moiety.1313. L. Berry, A. Stafford, J. Fredenburgh, H. O’Brodovich, L. Mitchell, J. Weitz, M. Andrew, and A. K. C. Chan, J. Biol. Chem. 273, 34730 (1998). https://doi.org/10.1074/jbc.273.52.34730 Thus, the ATH coating on the PU catheter may form a further complex with rabbit AT. Indeed, using 125I-radiolabeled AT, our group has demonstrated that ATH-coated and heparin-coated catheters bound extrinsic radiolabeled AT.1010. Y. J. Du, J. L. Brash, G. McClung, L. R. Berry, P. Klement, and A. K. C. Chan, J. Biomed. Mater. Res. A 80A, 216 (2007). https://doi.org/10.1002/jbm.a.30977 Binding of rabbit plasma proteins to the surfaces may also have induced mild conformation changes in ATH epitopes, thereby somewhat enhancing the measured OD signals. The addition of high concentrations of plasma protein (2.7% human albumin) to soluble ATH also exhibited some enhancing effects for signal in this assay.

The current study provides promising results to support the development of a simple, direct, high throughput, yet analyte-specific adapted EIA as a routine assay. This system may also be scaled-up for industrial quality controls. Using analyte-specific, HRP-conjugated detecting antibodies, this methodology simultaneously evaluates the quantity and quality of the biomaterial surface modifications on the same catheter samples. There are limited methods to directly measure the quantity or quality of bioactive surface modifications on biomaterial surfaces, particularly on catheters and other device geometries. In terms of methods used to gather valuable insights about proteins on solid surfaces, some techniques need sample properties that limit the analysis, some provide only semiquantitative results, and many cannot differentiate between various protein types.

The application of classical biochemical methods for the quantification of proteins, such as Lowry, Bradford, and the bicinchoninic acid (BCA) assays, along with immunoblotting methods are restricted to elutable proteins.33–3533. K. Salchert, T. Pompe, C. Sperling, and C. Werner, J. Chromatogr. A 1005, 113 (2003). https://doi.org/10.1016/S0021-9673(03)00932-434. R. M. Cornelius, J. Sanchez, P. Olsson, and J. L. Brash, J. Biomed. Mater. Res. 67A, 475 (2003). https://doi.org/10.1002/jbm.a.1011835. J.-H. Seo, K. Sakai, and N. Yui, Acta Biomater. 9, 5493 (2013). https://doi.org/10.1016/j.actbio.2012.10.015 The reason is that nonelutable immobilized proteins are either covalently bound or actively adsorbed to surfaces and may not be easily removed. Iodination radiolabeling, the most widely used radiolabeling technique, is a well-established method with various commercial labeling products available which serves as a gold standard for direct quantification of protein adsorption due to its extremely high sensitivity (ng/cm2).15,23,36–3815. K. N. Sask, L. R. Berry, A. K. C. Chan, and J. L. Brash, J. Biomed. Mater. Res. A 100A, 2821 (2012). https://doi.org/10.1002/jbm.a.3421823. Y. Luan, D. Li, Y. Wang, X. Liu, J. L. Brash, and H. Chen, Langmuir 30, 1029 (2014). https://doi.org/10.1021/la403498w36. S. R. Sousa, M. M. Brás, P. Moradas-Ferreira, and M. A. Barbosa, Langmuir 23, 7046 (2007). https://doi.org/10.1021/la062956e37. T. A. Horbett, Colloids Surf. B Biointerfaces 191, 110986 (2020). https://doi.org/10.1016/j.colsurfb.2020.11098638. H. Zhang, Advances in Polyurethane Biomaterials (Elsevier, New York, 2016), pp. 23–73. Radiolabeling, however, requires a special experimental environment, and reference bioactive reagents should be radioactively labeled before coating the synthetic surface. It is, therefore, impractical to use the radiolabeling technique for monitoring the quality of surface modifications in many settings and for a large number of surface modified samples. High-performance liquid chromatography (HPLC), as a technique to determine immobilized amounts of proteins at interfaces, has shown valid and reliable results.33,3933. K. Salchert, T. Pompe, C. Sperling, and C. Werner, J. Chromatogr. A 1005, 113 (2003). https://doi.org/10.1016/S0021-9673(03)00932-439. T. Huang, K. Anselme, S. Sarrailh, and A. Ponche, Int. J. Pharm. 497, 54 (2016). https://doi.org/10.1016/j.ijpharm.2015.11.013 HPLC can be used for samples with irregular shapes, can detect absolute amounts of proteins, and allows for the simultaneous quantification of proteins out of mixed samples without the use of labels or secondary binding reactions,3333. K. Salchert, T. Pompe, C. Sperling, and C. Werner, J. Chromatogr. A 1005, 113 (2003). https://doi.org/10.1016/S0021-9673(03)00932-4 but it can be costly, complex, and does not work for all samples.Other methods that can provide information on protein interactions with surfaces include surface plasmon resonance (SPR),22,2322. R. J. Green, R. A. Frazier, K. M. Shakesheff, M. C. Davies, C. J. Roberts, and S. J. B. Tendler, Biomaterials 21, 1823 (2000). https://doi.org/10.1016/S0142-9612(00)00077-623. Y. Luan, D. Li, Y. Wang, X. Liu, J. L. Brash, and H. Chen, Langmuir 30, 1029 (2014). https://doi.org/10.1021/la403498w quartz crystal microbalance with dissipation (QCM-D),23–2523. Y. Luan, D. Li, Y. Wang, X. Liu, J. L. Brash, and H. Chen, Langmuir 30, 1029 (2014). https://doi.org/10.1021/la403498w24. X. Li, R. Wang, F. Wicaksana, Y. Zhao, C. Tang, J. Torres, and A. G. Fane, Colloids Surf. B Biointerfaces 111, 446 (2013). https://doi.org/10.1016/j.colsurfb.2013.06.00825. A. Dolatshahi-Pirouz, S. Skeldal, M. B. Hovgaard, T. Jensen, M. Foss, J. Chevallier, and F. Besenbacher, J. Phys. Chem. C 113, 4406 (2009). https://doi.org/10.1021/jp808488f ellipsometry,36,4036. S. R. Sousa, M. M. Brás, P. Moradas-Ferreira, and M. A. Barbosa, Langmuir 23, 7046 (2007). https://doi.org/10.1021/la062956e40. E. Finot, L. Markey, F. Hane, M. Amrein, and Z. Leonenko, Colloids Surf. B Biointerfaces 104, 289 (2013). https://doi.org/10.1016/j.colsurfb.2012.12.013 x-ray photoelectron spectroscopy (XPS),36,41,4236. S. R. Sousa, M. M. Brás, P. Moradas-Ferreira, and M. A. Barbosa, Langmuir 23, 7046 (2007). https://doi.org/10.1021/la062956e41. M. S. Wagner, S. L. McArthur, M. Shen, T. A. Horbett, and D. G. Castner, J. Biomater. Sci. Polym. Ed. 13, 407 (2002). https://doi.org/10.1163/15685620232025393842. S. Ray and A. G. Shard, Anal. Chem. 83, 8659 (2011). https://doi.org/10.1021/ac202110x mass spectrometry,4141. M. S. Wagner, S. L. McArthur, M. Shen, T. A. Horbett, and D. G. Castner, J. Biomater. Sci. Polym. Ed. 13, 407 (2002). https://doi.org/10.1163/156856202320253938 radioimmunoassay (RIA),4343. M. J. Danilich, K. Kottke-Marchant, J. M. Anderson, and R. E. Marchant, J. Biomater. Sci. Polym. Ed. 3, 195 (1992). https://doi.org/10.1163/156856292X00123 fluorescent labeling,4444. E. Velzenberger, I. Pezron, G. Legeay, M.-D. Nagel, and K. E. Kirat, Langmuir 24, 11734 (2008). https://doi.org/10.1021/la801727p time-of-flight secondary ion mass spectrometry (ToF-SIMS),4545. M. S. Wagner, T. A. Horbett, and D. G. Castner, Langmuir 19, 1708 (2003). https://doi.org/10.1021/la0260382 force spectroscopy,4444. E. Velzenberger, I. Pezron, G. Legeay, M.-D. Nagel, and K. E. Kirat, Langmuir 24, 11734 (2008). https://doi.org/10.1021/la801727p and optical waveguide light mode spectroscopy (OWLS).4646. N. Kovacs, D. Patko, N. Orgovan, S. Kurunczi, J. J. Ramsden, F. Vonderviszt, and R. Horvath, Anal. Chem. 85, 5382 (2013). https://doi.org/10.1021/ac3034322 A variation of an enzyme-linked immunosorbent assay (ELISA)28,4728. K. Merritt, C. R. Edwards, and S. A. Brown, J. Biomed. Mater. Res. 22, 99 (1988). https://doi.org/10.1002/jbm.82022020347. L. Guicai, S. Xiaoli, Y. Ping, Z. Ansha, and H. Nan, Solid State Ionics 179, 932 (2008). https://doi.org/10.1016/j.ssi.2008.02.053 has been used to quantify proteins on surfaces, but this has been for proteins physically adsorbed to the surface, unlike in this work where proteins covalently immobilized on materials are quantified. The popularity of two widely used methods, i.e., SPR and QCM-D (as a mass sensor) has been growing among investigators because of their ability to monitor protein adsorption in real-time rendering information on the kinetic adsorption behavior of proteins.23,4823. Y. Luan, D. Li, Y. Wang, X. Liu, J. L. Brash, and H. Chen, Langmuir 30, 1029 (2014). https://doi.org/10.1021/la403498w48. R. E. Speight and M. A. Cooper, J. Mol. Recognit. 25, 451 (2012). https://doi.org/10.1002/jmr.2209 In addition, QCM-D data provide insight into the conformational changes in the adsorbed proteins through viscoelasticity measurements.16,23,4916. E. A. Vogler, Biomaterials 33, 1201 (2012). https://doi.org/10.1016/j.biomaterials.2011.10.05923. Y. Luan, D. Li, Y. Wang, X. Liu, J. L. Brash, and H. Chen, Langmuir 30, 1029 (2014). https://doi.org/10.1021/la403498w49. X. Wang, G. Liu, and G. Zhang, Langmuir 28, 14642 (2012). https://doi.org/10.1021/la303001j However, in both SPR and QCM-D, a particular sensor surface is needed limiting measurements to these sensor materials or thin-film polymer coatings, and it is also not possible to extend this to materials with complex shapes.The developed EIA described here has been applied to determine the quantity and quality of an antithrombin-heparin (ATH) anticoagulant complex on catheter surfaces and this has the potential to be extended to various blood contacting devices. In other work, we have performed fundamental studies of ATH surface immobilization on polymers including polyurethane and polydimethylsiloxane and have also applied modifications to microfluidic lung assist devices for oxygenation of blood.31,50,5131. K. N. Sask, L. R. Berry, A. K. C. Chan, and J. L. Brash, Langmuir 28, 2099 (2012). https://doi.org/10.1021/la203821g50. J. M. Leung, L. R. Berry, A. K. C. Chan, and J. L. Brash, J. Biomater. Sci. Polym. Ed. 25, 786 (2014). https://doi.org/10.1080/09205063.2014.90766951. J. M. Leung et al., J. Mater. Chem. B 3, 6032 (2015). https://doi.org/10.1039/C5TB00808E These studies have quantified ATH using protein radiolabelling, however, as discussed here there are challenges to the use of radioactive proteins and this EIA will be advantageous for future work to translate these technologies.In addition to applications conjugating ATH and other anticoagulants to materials, this EIA can be applied for detecting many other bioactive molecules on their own and for multifunctional materials. For example, surface modifications that immobilize proteins to provide antiplatelet, fibrinolytic, immunomodulatory, and other active functions to materials can be assessed by the EIA method with appropriate antibodies. There are numerous chemical and biological strategies to conjugate bioactive agents to surfaces, and a technique that has been gaining significant interest over the past decade is the use of mussel-inspired polydopamine (PDA) coatings for versatile immobilization of proteins and other molecules.51,5251. J. M. Leung et al., J. Mater. Chem. B 3, 6032 (2015). https://doi.org/10.1039/C5TB00808E52. J. H. Ryu, P. B. Messersmith, and H. Lee, ACS Appl. Mater. Interfaces 10, 7523 (2018). https://doi.org/10.1021/acsami.7b19865 Due to the limitations described previously for common protein assays such as the BCA assay, it is difficult to obtain quantitative data with coating techniques including PDA since proteins are not easily eluted from these materials. This EIA method has advantages in its ability to quantify various plasma proteins directly on biomaterials, catheters, and other device surfaces in complex fluids and after implantation, simply by selecting the appropriate antibody.

Finally, it is not only the quantity of protein immobilized on materials that are important to device functionality but also their structural details including conformation and orientation. Further development of this EIA through the use of antibodies specific to particular functional regions on proteins has significant potential to provide additional insight into interactions at the biointerface that may influence cell activation, thrombosis, and other adverse events.

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