Tannic acid coating gauze immobilized with thrombin with ultra-high coagulation activity and antimicrobial property for uncontrollable hemorrhage.

Rapid and safe hemostasis is crucial for the survival of bleeding patients in prehospital care. It is urgent to develop high performance hemostatic material to control the massive hemorrhage in the military field and accidental trauma. In this work, an efficient protein hemostat of thrombin was immobilized onto commercial gauze, which was mediated by self-polymerization and anchoring of tannic acid (TA). Through TA treatment, the efficient immobilization of thrombin was achieved, preserving both the biological activity of thrombin and the physical properties of the dressing, including absorbency, breathability, and mechanical performance. Moreover, in the presence of TA coating and thrombin, Gau@TA/Thr could obviously shortened clotting time and enriched blood components such as plasma proteins, platelets, and red blood cells, thereby exhibiting an enhanced in vitro coagulation effect. In SD rat liver volume defect and artery transection hemorrhage models, Gau@TA/Thr still had outstanding hemostatic performance. Besides, the Gau@TA/Thr gauze had inherent antibacterial property and demonstrated excellent biocompatibility. All results suggested that Gau@TA/Thr would be a potential candidate for treating uncontrollable hemorrhage in prehospital care.

A graphene oxide-assisted protein immobilization paper-tip immunosensor with smartphone and naked eye readout for the detection of okadaic acid.

BACKGROUND: Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated shellfish. At present, much effort has been made to graft immunoassay onto a paper substrate to make paper-based sensors for rapid and simple detection of shellfish toxin. However, the complicated washing steps and low protein fixation efficiency on the paper substrate need to be further addressed. RESULTS: A novel paper-tip immunosensor for detecting OA was developed combined with smartphone and naked eye readout. The trapezoid paper tip was consisted of quantitative and qualitative detection zones. To improve the OA antigen immobilization efficiency on the paper substrate, graphene oxide (GO)-assisted protein immobilization method was introduced. Meanwhile, Au nanoparticles composite probe combined with the lateral flow washing was developed to simplify the washing step. The OA antigen-immobilized zone, as the detection zone Ⅰ, was used for quantitative assay by smartphone imaging. The paper-tip front, as the detection zone Ⅱ, which could qualitatively differentiate OA pollution level within 45 min using the naked eye. The competitive immunoassay on the paper tip exhibited a wide linear range for detecting OA (0.02-50 ng∙mL-1) with low detection limit of 0.02 ng∙mL-1. The recovery of OA in spiked shellfish samples was in the range of 90.3 %-113.%. SIGNIFICANCE: These results demonstrated that the proposed paper-tip immunosensor could provide a simple, low-cost and high-sensitivity test for OA detection without the need for additional large-scale equipment or expertise. We anticipate that this paper-tip immunosensor will be a flexible and versatile tool for on-site detecting the pollution of marine products.

Construction and application of H1R ligand screening materials based on SMA stabilization and his-tag covalent immobilization of membrane proteins.

The histamine H1 receptor (H1R) plays a pivotal role in allergy initiation and undergoes the necessity of devising a high-throughput screening approach centered on H1R to screen novel ligands effectively. This study suggests a method employing styrene maleic acid (SMA) extraction and His-tag covalent bonding to immobilize H1R membrane proteins, minimizing the interference of nonspecific proteins interference while preserving native protein structure and maximizing target exposure. This approach was utilized to develop a novel material for high-throughput ligand screening and implemented in cell membrane chromatography (CMC). An H1R-His-SMALPs/CMC model was established and its chromatographic performance (selectivity, specificity and lifespan) validated, demonstrating a significant enhancement in lifespan compared to previous CMC models. Subsequently, this model facilitated high-throughput screening of H1R ligands in the compound library and preliminary activity verification of potential H1R antagonists. Identification of a novel H1R antagonist laid the foundation for further development in this area.

Site-specific immobilization of Cysteinyl leukotriene receptor 1 through enzymatic DNA-protein conjugation strategy for lead screening.

Immobilization of functional protein, especially G protein-coupled receptors (GPCRs), is particularly significant in various fields such as the development of assays for diagnosis, lead compound screening, as well as drug-protein interaction analysis. However, there are still some challenges with the immobilized proteins such as undefined loads, orientations, and the loss of activity. Herein, we introduced a DNA conjugation strategy into the immobilization of Cysteinyl leukotriene receptor 1(CysLTR1) which enables exquisite molecular control and higher activity of the receptor. We used the bacterial relaxases VirD2 as an immobilized tag fused at the C terminus of CysLTR1. Tyrosine residue(Y29) at the core binding site of the VirD2 tag can react with the single-strand piece of DNA(T-DNA) in the form of a covalent bond. Inspired by this strategy, we developed a new immobilization method by mixing the T-DNA-modified silica gel with the cell lysate containing the expressed VirD2-tagged CysLTR1 for 1 hour. We found that the successful formation of DNA-protein conjugate enables the immobilization of CysLTR1 fast, site-specific, and with minimal loss of activity. The feasibility of the immobilized CysLTR1 was evaluated in drug-protein binding interaction by frontal analysis and adsorption energy distribution analysis. The binding of pranlukast, zafirlukast, and MK571 to the immobilized CysLTR1 was realized, and the association constants presented good agreement between the two methods. Rosmarinic acid was retained in the immobilized CysLTR1 column, and the in-vitro test revealed that the compound binds to the receptor in one type of binding site mode. Despite these results, we concluded that the DNA-protein conjugate strategy will probably open up the possibilities for capturing other functional proteins in covalent and site-specific modes from the complex matrices and the immobilized receptor preserves the potential in fishing out lead compounds from natural products.

Affinity purification/immobilization of poly histidine-tagged proteins by nickel-functionalized porous chitosan membranes.

Although immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for purifying his-tagged proteins, it has limitations such as expensive commercial resins and non-specific binding of unwanted proteins to the nickel immobilized on the resin. In this study, biocompatible chitosan and porous chitosan membranes as alternative resins were synthesized for protein immobilization and purification, but finally porous chitosan membrane was selected due to its higher porosity and consequently higher nickel adsorption. Once the membrane was functionalized with nickel ions and its metal adsorption confirmed by EDS and ICP methods, it was used to immobilize and purify recombinant ß-NGF as a protein model with his-tag tail in batch-fashion. Protein binding and purification were also approved by FTIR and UV-Vis spectroscopy and SDS-PAGE technique. Our results indicated that the protein of interest could bind to the nickel-functionalized porous chitosan membrane with high efficiency at pH=7. Furthermore, for protein purification, the pH value of 6 and an imidazole concentration of 750 mM were suggested for the final elution buffer. In conclusion, nickel-functionalized porous chitosan membrane could be a suitable alternative to IMAC for low cost and specific protein immobilization and purification.

High-Throughput DNA-Encoded Libraries Affinity Selection Platform for Binder Identification with Solid Support Protein Immobilization.

DNA-encoded libraries (DELs) have demonstrated to be one of the most powerful technologies within the ligand identification toolbox, widely used either in academia or biotech and pharma companies. DEL methodology utilizes affinity selection (AS) as the approach to interrogate the protein of interest for the identification of binders. Here we present a high-throughput, fully automated AS platform developed to fulfill industrial standards and compatible with different assay formats to improve the reproducibility of the AS process for DEL binders identification. This platform is flexible enough to virtually set aside all kinds of DELs and AS methods and conditions using immobilized proteins. It bears the two main immobilization methods to support of the proteins of interest: magnetic beads or resin tip columns. A combination of a broad variety of protocol options with a wide range of different experimental conditions can be set up with a throughput of 96 samples at the same time. In addition, small modifications of the protocols provide the platform with the versatility to run not only the routine DEL screens, but also test covalent libraries, the successful immobilization of the proteins of interest, and many other experiments that may be required. This versatile AS platform for DEL can be a powerful instrument for direct application of the technology in academic and industry settings.

Protein-polymer bioconjugation, immobilization, and encapsulation: a comparative review towards applicability, functionality, activity, and stability.

Polymer-based biomaterials have received a lot of attention due to their biomedical, agricultural, and industrial potential. Soluble protein-polymer bioconjugates, immobilized proteins, and encapsulated proteins have been shown to tune enzymatic activity, improved pharmacokinetic ability, increased chemical and thermal stability, stimuli responsiveness, and introduced protein recovery. Controlled polymerization techniques, increased protein-polymer attachment techniques, improved polymer surface grafting techniques, controlled polymersome self-assembly, and sophisticated characterization methods have been utilized for the development of well-defined polymer-based biomaterials. In this review we aim to provide a brief account of the field, compare these methods for engineering biomaterials, provide future directions for the field, and highlight impacts of these forms of bioconjugation.

Polydopamine-mediated immobilization of BMP-2 onto electrospun nanofibers enhances bone regeneration.

Dealing with bone defects is a significant challenge to global health. Electrospinning in bone tissue engineering has emerged as a solution to this problem. In this study, we designed a PVDF-b-PTFE block copolymer by incorporating TFE, which induced a phase shift in PVDF fromαtoß, thereby enhancing the piezoelectric effect. Utilizing the electrospinning process, we not only converted the material into a film with a significant surface area and high porosity but also intensified the piezoelectric effect. Then we used polydopamine to immobilize BMP-2 onto PVDF-b-PTFE electrospun nanofibrous membranes, achieving a controlled release of BMP-2. The scaffold's characters were examined using SEM and XRD. To assess its osteogenic effectsin vitro, we monitored the proliferation of MC3T3-E1 cells on the fibers, conducted ARS staining, and measured the expression of osteogenic genes.In vivo, bone regeneration effects were analyzed through micro-CT scanning and HE staining. ELISA assays confirmed that the sustained release of BMP-2 can be maintained for at least 28 d. SEM images and CCK-8 results demonstrated enhanced cell viability and improved adhesion in the experimental group. Furthermore, the experimental group exhibited more calcium nodules and higher expression levels of osteogenic genes, including COL-I, OCN, and RUNX2. HE staining and micro-CT scans revealed enhanced bone tissue regeneration in the defective area of the PDB group. Through extensive experimentation, we evaluated the scaffold's effectiveness in augmenting osteoblast proliferation and differentiation. This study emphasized the potential of piezoelectric PVDF-b-PTFE nanofibrous membranes with controlled BMP-2 release as a promising approach for bone tissue engineering, providing a viable solution for addressing bone defects.

Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

Vascular stent with immobilized anti-inflammatory chemerin 15 peptides mitigates neointimal hyperplasia and accelerates vascular healing.

Endovascular stenting is a safer alternative to open surgery for use in treating cerebral arterial stenosis and significantly reduces the recurrence of ischemic stroke, but the widely used bare-metal stents (BMSs) often result in in-stent restenosis (ISR). Although evidence suggests that drug-eluting stents are superior to BMSs in the short term, their long-term performances remain unknown. Herein, we propose a potential vascular stent modified by immobilizing clickable chemerin 15 (C15) peptides on the stent surface to suppress coagulation and restenosis. Various characterization techniques and an animal model were used to evaluate the surface properties of the modified stents and their effects on endothelial injury, platelet adhesion, and inflammation. The C15-immobilized stent could prevent restenosis by minimizing endothelial injury, promoting physiological healing, restraining the platelet-leukocyte-related inflammatory response, and inhibiting vascular smooth muscle cell proliferation and migration. Furthermore, in vivo studies demonstrated that the C15-immobilized stent mitigated inflammation, suppressed neointimal hyperplasia, and accelerated endothelial restoration. The use of surface-modified, anti-inflammatory, endothelium-friendly stents may be of benefit to patients with arterial stenosis. STATEMENT OF SIGNIFICANCE: Endovascular stenting is increasingly used for cerebral arterial stenosis treatment, aiming to prevent and treat ischemic stroke. But an important accompanying complication is in-stent restenosis (ISR). Persistent inflammation has been established as a hallmark of ISR and anti-inflammation strategies in stent modification proved effective. Chemerin 15, an inflammatory resolution mediator with 15-aa peptide, was active at picomolar through cell surface receptor, no need to permeate cell membrane and involved in resolution of inflammation by inhibiting inflammatory cells adhesion, modulating macrophage polarization into protective phenotype, and reducing inflammatory factors release. The implications of this study are that C15 immobilized stent favors inflammation resolution and rapid re-endothelialization, and exhibits an inhibitory role of restenosis. As such, it helps the decreased incidence of ISR.

Highly efficient GPCR immobilization with enhanced fouling resistance, salt tolerance, and chromatographic performance.

The feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR2A) and endothelin A receptor (ETAR), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k'), half peak width (W1/2), tailing factor (Tf), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non-specific interactions and enhanced fouling resistance, salt tolerance, and chromatographic performance were clearly observed. We believe it is the first work to introduce PEGylation in GPCR immobilization and provide comprehensive proof-of-concept studies to illustrate the improved antifouling property, salt tolerance, and chromatographic performance. This method could be generally applicable in other immobilized protein-based technology for reliable biodetection.

Conformational Changes in Surface-Immobilized Proteins Measured Using Combined Atomic Force and Fluorescence Microscopy.

Biological organisms rely on proteins to perform the majority of their functions. Most protein functions are based on their physical motions (conformational changes), which can be described as transitions between different conformational states in a multidimensional free-energy landscape. A comprehensive understanding of this free-energy landscape is therefore of paramount importance for understanding the biological functions of proteins. Protein dynamics includes both equilibrium and nonequilibrium motions, which typically exhibit a wide range of characteristic length and time scales. The relative probabilities of various conformational states in the energy landscape, the energy barriers between them, their dependence on external parameters such as force and temperature, and their connection to the protein function remain largely unknown for most proteins. In this paper, we present a multimolecule approach in which the proteins are immobilized at well-defined locations on Au substrates using an atomic force microscope (AFM)-based patterning method called nanografting. This method enables precise control over the protein location and orientation on the substrate, as well as the creation of biologically active protein ensembles that self-assemble into well-defined nanoscale regions (protein patches) on the gold substrate. We performed AFM-force compression and fluorescence experiments on these protein patches and measured the fundamental dynamical parameters such as protein stiffness, elastic modulus, and transition energies between distinct conformational states. Our results provide new insights into the processes that govern protein dynamics and its connection to protein function.

Actinomycin-X2-Immobilized Silk Fibroin Film with Enhanced Antimicrobial and Wound Healing Activities.

Actinomycin is a family of chromogenic lactone peptides that differ in their peptide portions of the molecule. An antimicrobial peptide, actinomycin X2 (Ac.X2), was produced through the fermentation of a Streptomyces cyaneofuscatus strain. Immobilization of Ac.X2 onto a prepared silk fibroin (SF) film was done through a carbodiimide reaction. The physical properties of immobilized Ac.X2 (antimicrobial films, AMFs) were analyzed by ATR-FTIR, SEM, AFM, and WCA. The findings from an in vitro study showed that AMFs had a more broad-spectrum antibacterial activity against both S. aureus and E. coli compared with free Ac.X2, which showed no apparent strong effect against E. coli. These AMFs showed a suitable degradation rate, good hemocompatibility, and reduced cytotoxicity in the biocompatibility assay. The results of in vivo bacterially infected wound healing experiments indicated that wound inflammation was prevented by AMFs, which promoted wound repair and improved the wound microenvironment. This study revealed that Ac.X2 transformation is a potential candidate for skin wound healing.

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein.

Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pKa value(s), and the amount of immobilized protein.Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

Controlled Temporal Release of Serum Albumin Immobilized on Gold Nanoparticles.

Proteins adsorbed to gold nanoparticles (AuNPs) form bioconjugates and are critical to many emerging technologies for drug delivery, diagnostics, therapies, and other biomedical applications. A thorough understanding of the interaction between the immobilized protein and AuNP is essential for the bioconjugate to perform as designed. Here, we explore a correlation between the number of solvent-accessible thiol groups on a protein and the protein desorption rate from the AuNP surface in the presence of a competing protein. The chemical modification of human serum albumin (HSA) was carried out to install additional free thiols using Traut's reagent and create a library of HSA analogues by tailoring the molar excess of the Traut's reagent. We pre-adsorbed HSA variants onto the AuNP surface, and the resulting bioconjugates were then exposed to IgG antibody, and protein exchange was monitored as a function of time. We found that the rate of HSA displacement from the AuNP correlated with the experimentally measured number of accessible free thiol groups. Additionally, bioconjugates were synthesized using thiolated analogues of bovine serum albumin (BSA) and suspended in serum as a model for a complex sample matrix. Similarly, desorption rates with serum proteins were modulated with solvent-accessible thiols on the immobilized protein. These results further highlight the key role of Au-S bonds in the formation of protein-AuNP conjugates and provide a pathway to systematically control the number of free thiols on a protein, enabling the controlled release of protein from the surface of AuNP.

Effect of microchannel geometry and linker molecules on surface immobilization efficiency of proteins in microfluidic devices.

One of the key metrics in the design of biosensors is the presence of an effective capture layer. Surface-immobilized proteins (especially as a part of antibody-antigen combinations) are the most commonly used capture ligands in biosensors. The surface coverage of these proteins in flow-based biosensors are affected by both the linker chemistry used to attach them as well as the microchannel geometry. We used streptavidin as a model protein to compare glutaraldehyde, EDC-NHS, sulfo-SMCC and sulfo-NHS-biotin as linkers inside straight, serpentine and square-wave microchannel geometries. We found that straight microchannels achieve the highest degree of protein immobilization compared to serpentine and square-wave microchannels, irrespective of the linker chemistry used. We also showed that for a given microchannel geometry, sulfo-NHS-biotin leads to the highest immobilization of streptavidin among all the linkers.

Strategy based on multiplexed brush architectures for regulating the spatiotemporal immobilization of biomolecules.

Functional surfaces that enable both spatial and temporal control of biomolecules immobilization have attracted enormous attention for various fields including smart biointerface materials, high-throughput bioarrays, and fundamental research in the biosciences. Here, a flexible and promising method was presented for regulating the spatiotemporal arrangement of multiple biomolecules by constructing the topographically and chemically diverse polymer brushes patterned surfaces. A series of polymer brushes patterned surfaces, including antifouling brushes patterned surface, epoxy-presenting brushes patterned surface without and with antifouling background layer, were fabricated to control the spatial distribution of protein and cell adhesion through specific and nonspecific means. The fluorescence measurements demonstrated the effectiveness of spatially regulating the density of surface-immobilized protein through controlling the areal thickness of the poly (glycidyl methacrylate) (PGMA) brush patterns, leading to various complex patterns featuring well-defined biomolecule concentration gradients. Furthermore, a multiplexed surface bearing epoxy groups and azido groups with various areal densities was fabricated for regulating the spatiotemporal arrangement of different proteins, enabling binary biomolecules patterns with higher degrees of functionality and complexity. The presented strategy for the spatiotemporal control of biomolecules immobilization would boost the development of dynamic and multifunctional biosystems.

Controlled co-immobilisation of proteins via 4'-phosphopantetheine-mediated site-selective covalent linkage.

In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4'-phosphopantetheine (4'-PP) to residue serine-36. We found that the long flexible 4'-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.

Structurally Altered, Not Wild-Type, Pentameric C-Reactive Protein Inhibits Formation of Amyloid-ß Fibrils.

The structure of wild-type pentameric C-reactive protein (CRP) is stabilized by two calcium ions that are required for the binding of CRP to its ligand phosphocholine. CRP in its structurally altered pentameric conformations also binds to proteins that are denatured and aggregated by immobilization on microtiter plates; however, the identity of the ligand on immobilized proteins remains unknown. We tested the hypotheses that immobilization of proteins generated an amyloid-like structure and that amyloid-like structure was the ligand for structurally altered pentameric CRP. We found that the Abs to amyloid-ß peptide 1-42 (Aß) reacted with immobilized proteins, indicating that some immobilized proteins express an Aß epitope. Accordingly, four different CRP mutants capable of binding to immobilized proteins were constructed, and their binding to fluid-phase Aß was determined. All CRP mutants bound to fluid-phase Aß, suggesting that Aß is a ligand for structurally altered pentameric CRP. In addition, the interaction between CRP mutants and Aß prevented the formation of Aß fibrils. The growth of Aß fibrils was also halted when CRP mutants were added to growing fibrils. Biochemical analyses of CRP mutants revealed altered topology of the Ca2+-binding site, suggesting a role of this region of CRP in binding to Aß. Combined with previous reports that structurally altered pentameric CRP is generated in vivo, we conclude that CRP is a dual pattern recognition molecule and an antiamyloidogenic protein. These findings have implications for Alzheimer's and other neurodegenerative diseases caused by amyloidosis and for the diseases caused by the deposition of otherwise fluid-phase proteins.

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

Biomaterials used for blood contacting devices are inherently thrombogenic. Antithrombotic agents can be used as surface modifiers on biomaterials to reduce thrombus formation on the surface and to maintain device efficacy. For quality control and to assess the effectiveness of immobilization strategies, it is necessary to quantify the surface-immobilized antithrombotic agent directly. There are limited methods that allow direct quantification on device surfaces such as catheters. In this study, an enzyme immunoassay (EIA) has been developed to measure the density of a synthetic antithrombin-heparin (ATH) covalent complex immobilized on a catheter surface. The distribution of the immobilized ATH was further characterized by an immunohistochemical assay. This analyte-specific EIA is relatively simple and has high throughput, thus providing a tool for quantitative analysis of biomaterial surface modifications. These methods may be further modified to evaluate plasma proteins adsorbed and immobilized on various biomaterial surfaces of complex shapes, with a range of bioactive functionalities, as well as to assess conformational changes of proteins using specific antibodies.