Binding Of C-reactive Protein Research Articles

Biomimetic affinity membrane roll column for rapid purification of C-reactive protein

To in-depth explore the action mechanism of C-reactive protein (CRP) and precisely study its signaling pathways, it is essential to acquire high-purity CRP while preserving its intact structure and functionality. In this study, we propose and fabricate a high-density 2-methacryloyloxyethyl phosphorylcholine (MPC)-modified membrane roll column (MPC-MRC) using a surface-initiated atom transfer radical polymerization (SI-ATRP) approach, which can overcome these limitations (long incubation time and low adsorption capacity) of conventional enrichment materials. The MPC-MRC incorporates a high-density 2-hydroxyethyl methacrylate polymer brush to prevent non-specific protein adsorption and multiple MPC polymer brush layers for high-performance enrichment of CRP in the company of calcium ions. Furthermore, the MPC-MRC exhibits high permeability, hydrophilicity, and mechanical strength. Compared to previous technologies, this novel material demonstrates significantly higher CRP binding capacity (310.3 mg/g), shorter processing time (only 15 min), and lower cost (only 12 USD/column). Notably, the MPC-MRC enables fast and effective purification of CRP from both human and rat serum, exhibiting good selectivity, recovery (> 91.3 %), and purity (> 95.2 %). Thus, this proposed purification approach based on MPC-MRC holds great potential for target protein enrichment from complex samples, as well as facilitating in-depth studies of its biological functions.

A study to determine systemic anti-inflammatory mediators in chronic obstructive pulmonary disorder

Background: Chronic obstructive pulmonary disorder (COPD) is a prevalent and debilitating respiratory condition characterized by chronic inflammation within the airways and lung tissues. By elucidating the specific mediators involved and their potential therapeutic implications, this research seeks to contribute to a better understanding of the inflammatory mechanisms underlying COPD and explore novel avenues for managing the disease. Aims and Objectives: The objective of study is to determine systemic anti-inflammatory mediators in COPD and its correlation with disease severity. Materials and Methods: The study analyzed systemic levels of several anti-inflammatory mediators, namely, soluble interleukin 1 receptor II (sIL-1RII), soluble tumor necrosis factor receptor p55 (sTNF-R55), and sTNF-R75, as well as C-reactive protein (CRP) and lipopolysaccharide binding protein (LBP), in 55 patients with stable COPD (median forced expiratory volume in 1 s [FEV1] of 34% predicted, ranging from 15% to 78%) and compared them with 23 control subjects. In addition, changes in these mediators were studied in 13 COPD patients (median FEV1 of 34% predicted, ranging from 19% to 51%) during the first 7 days of hospitalization for an exacerbation of the disease. Results: The results revealed that patients with stable COPD showed evidence of a systemic inflammatory process, characterized by an elevated leukocyte count, increased levels of CRP and LBP, and moderate increases in both sTNF-Rs. However, the sIL-1RII levels did not differ significantly between patients and controls. During the treatment of disease exacerbations, systemic levels of CRP (at day 3) and LBP (at day 7) significantly decreased compared to day 1, while sIL-1RII levels increased. Conclusion: Based on these findings, it is suggested that there is an imbalance in systemic levels of pro- and anti-inflammatory mediators in patients with stable COPD. The observed increase in the anti-inflammatory mediator sIL-1RII during treatment of exacerbations may play a role in the clinical improvement observed in these patients.

Investigating the Association Between Diet-Induced "Leaky Gut" and the Development of Prediabetes.

Chronic consumption of a high-calorie diet compromises the gut microbiota and the integrity of the intestinal wall, which causes translocation of bacterial lipopolysaccharides (LPS) into the blood. This elicits the secretion of pro-inflammatory cytokines, resulting in inflammation. However, how a high-fat high carbohydrate diet affects intestinal permeability and its possible role in the development of prediabetes have not been investigated. This study investigated the effects of HFHC diet-induced prediabetes on gut microbiota and intestinal permeability in male Sprague Dawley rats. The animals were randomly assigned into the non-prediabetic (NPD) and diet-induced prediabetic (PD) groups (n=6) for 20 weeks. Then, the fecal samples were analyzed to measure the gut microbiota level of Firmicutes, Bacteroidetes, and Proteobacteria in both animal groups. Blood glucose, plasma insulin, serum zonulin, plasma LPS, soluble CD14, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), and intestinal fatty-acid binding protein (IFABP) concentrations were measured. The PD group had a reduction in the Firmicutes and an increase in Bacteroidetes and Proteobacteria levels compared to those in the NPD group. Blood glucose, insulin concentration, serum zonulin, and plasma sCD14 concentrations in the PD group increased significantly, while plasma LPS concentrations were similar to the NPD group. Concentrations of plasma TNF-α, IL-6, CRP, and IFABP, an intracellular protein expressed in the intestine, increased in PD compared to the NPD group. the study results cumulatively suggest that chronic consumption of the HFHC diet may be associated with the dysregulation of gut microbiota, leading to increased intestinal permeability.

C-reactive protein binds to short phosphoglycan repeats of Leishmania secreted proteophosphoglycans and activates complement.

Human C-reactive protein (CRP) binds to lipophosphoglycan (LPG), a virulence factor of Leishmania spp., through the repeating phosphodisaccharide region. We report here that both major components of promastigote secretory gel (PSG), the filamentous proteophosphoglycan (fPPG) and the secreted acid phosphatase (ScAP), are also ligands. CRP binding was mainly associated with the flagellar pocket when LPG deficient Leishmania mexicana parasites were examined by fluorescent microscopy, consistent with binding to secreted material. ScAP is a major ligand in purified fPPG from parasite culture as demonstrated by much reduced binding to a ScAP deficient mutant fPPG in plate binding assays and ligand blotting. Nevertheless, in sandfly derived PSG fPPG is a major component and the major CRP binding component. Previously we showed high avidity of CRP for LPG ligand required multiple disaccharide repeats. ScAP and fPPG only have short repeats but they retain high avidity for CRP revealed by surface plasmon resonance because they are found in multiple copies on the phosphoglycan. The fPPG from many species such as L. donovani and L. mexicana bound CRP strongly but L. tropica and L. amazonensis had low amounts of binding. The extent of side chain substitution of [-PO4-6Galβ1-4Manα1-] disaccharides correlates inversely with binding of CRP. The ligand for the CRP on different species all had similar binding avidity as the half maximal binding concentration was similar. Since the PSG is injected with the parasites into host blood pools and phosphoglycans (PG) are known to deplete complement, we showed that CRP makes a significant contribution to the activation of complement by PSG using serum from naive donors.

C-reactive protein - My perspective on its first half century, 1930-1982.

C-reactive protein (CRP) was discovered in 1930 in the sera of patients during the acute phase of pneumococcal pneumonia and was so named because it bound to the C-polysaccharide of the pneumococcal cell wall. During the next half century many questions raised by this discovery were answered. Phosphorylcholine was found to be the moiety of the C-polysaccharide to which CRP bound. The molecular structure of CRP was elucidated: five identical subunits arranged in cyclic symmetry, giving rise to the term pentraxin. Initially felt to be not normally present in the blood, CRP was found to be a component of normal serum in trace amounts. Its site of origin was determined to be the hepatocyte. It became clear that the presumed humoral mediator responsible for CRP induction was of leukocytic origin. Binding of CRP to its ligand activated the complement system, one of the important effector mechanisms of innate immunity. CRP was found to stimulate phagocytosis of some bacterial species via binding to Fc receptors and was found to be protective in vivo against the pneumococcus in mice. It appeared likely that a related function of CRP was clearance of necrotic tissue. CRP was recognized as being a highly evolutionary conserved molecule. Its discovery during the acute phase of pneumococcal pneumonia led to its being dubbed an acute phase protein. What we today call "the acute phase response", refers to the large number of behavioral, physiologic, biochemical, and nutritional changes that occur during inflammatory states.

Serum biomarkers for nutritional status as predictors in COVID-19 patients before and after vaccination.

The aim of this study was to characterize serum protein biomarkers for nutritional status that may be used as predictors for disease symptomatology in COVID-19 patients before and after vaccination. In pre-vaccine cohorts, proteomics analysis revealed significant differences between groups, with serum proteins alpha-1-acid glycoproteins (AGPs) 1 and 2, C-reactive protein (CRP) and retinol binding protein (RBP) increasing with COVID-19 severity, in contrast with serum albumin, transthyretin (TTR) and serotransferrin (TF) reduction as the symptomatology increased. Immunoassay reproduced and validated proteomics results of serum proteins albumin and RBP. In post-vaccine cohorts, the results showed the same pattern as in pre-vaccine cohorts for serum proteins AGPs, CRP, albumin and TTR. However, TF levels were similar between groups and RBP presented a slight reduction as COVID-19 symptomatology increased. In these cohorts, immunoassay validated proteomics results of serum proteins albumin, TTR and TF. Additionally, immune response to α-Gal in pre-vaccine cohorts varied in predominant immunoglobulin type profile, while post-vaccine groups presented mainly anti-α-Gal protective IgG antibodies. The study identified serum nutritional biomarkers that could potentially predict an accurate prognostic of COVID-19 disease to provide an appropriate nutritional care and guidance in non-vaccinated and vaccinated individuals against SARS-CoV-2. These results highlight the importance of designing personalized nutrition protocols to improve diet along with the application of prebiotics or probiotics for the control of COVID-19 and other infectious diseases.

Conformational state of C-reactive protein is critical for reducing immune complex-triggered type I interferon response: Implications for pathogenic mechanisms in autoimmune diseases imprinted by type I interferon gene dysregulation

Presence of autoantibodies targeting nuclear constituents, i.e., double-stranded DNA and small nuclear ribonucleoproteins (snRNPs), remain a cornerstone in systemic lupus erythematosus (SLE). Fcγ receptor IIa (FcγRIIa) dependent uptake of nucleic acid containing immune complexes (ICs) by plasmacytoid dendritic cells (PDCs) can activate toll-like receptors (TLRs) such as TLR7 and TLR9 resulting in type I interferon (IFN) production. Previously, the classical liver-derived acute-phase reactant C-reactive protein (CRP) has been suggested to reduce IC-induced type I IFN production, whereas monomeric (mCRP) vs. pentameric (pCRP) mediated effects have not yet been unraveled. Herein, peripheral blood mononuclear cells (PBMCs) or enriched blood DCs from healthy volunteers were stimulated with SLE sera, snRNP-IgG (ICs), or TLR ligands with or without pCRP, mCRP, or anti-FcγRIIa antibody. Type I IFNs and cytokine responses were investigated using quantitative PCR, ELISA, and flow cytometry.pCRP inhibited IFN gene expression in PBMCs and enriched DCs after incubation with ICs, compared to ICs alone, whereas mCRP had significantly less inhibitory effect. The effect was independent on the order in which IC or CRP was added to the cells. In addition, pCRP inhibited IFN induced by other TLR stimulators, implicating broader inhibitory effects induced by pCRP. We demonstrate pronounced immunoregulatory functions of CRP whereas the inhibitory properties were evidently dependent on CRP's intact conformational state. The inhibition of type I IFNs was not due to competition of FcγRs, or binding of CRP to the ICs. Our findings have implications for autoimmune IC-mediated conditions imprinted by type I IFN gene dysregulation.

Association of pro-inflammatory cytokines, inflammatory proteins with atherosclerosis index in obese male subjects.

Investigation the association of pro-inflammatory markers interleukin (IL)-1β and IL- 10 expression, serum levels of C-reactive protein (CRP), cyclooxygenase-2 (COX2), High-density lipoprotein (HDL), Apolipoprotein A1 (ApoA1), and ATP Binding Cassette Subfamily A Member 1 (ABCA1) inflammatory proteins with atherosclerosis index (homocysteine) in normal-weight and obese male subjects. 59 males including 30 obese (Body mass index (BMI) of≥30kg/m2) and 29 normal-weight (BMI of 18.5-24.9kg/m2) were joined to this study. Plasma levels of IL-1β and IL-10 (pg/mL), CRP (pg/mL), COX-2 (ng/mL), APOA1 (mg/dL), ABCA1 (ng/mL), HDL, Cholesterol, and Triglyceride (TG) (mg/dL), and homocysteine (µmol/L) was measured. Association of these biomarkers with homocysteine was determined. Obese subjects had higher serum levels of IL10, IL1β, CRP, COX-2, TG, and cholesterol concentrations (allp<0.05 except IL-10 and cholesterol) and low levels of HDL, APOA1, and ABCA1 (non-significant differences) in comparison to normal-weight group. Homocysteine levels were high in obese men with no significant differences between the two groups. In obese subjects, homocysteine had a significant inverse correlation with APOA1, ABCA1, and HDL, and a strong and moderate positive correlation was found with CRP and TG levels, respectively. High level of homocysteine and its correlation with inflammation proteins and markers in obese subjects appear to be contributed with atherosclerosis development.

Functional iron status of chronic kidney disease patients at the University of Ilorin Teaching Hospital, Ilorin, Nigeria.

Functional iron deficiency has been found to be a common cause of poor response to erythropoiesis stimulating agents in anaemic patients with chronic kidney disease (CKD). Assess the functional iron status of patients with chronic kidney disease. This was a hospital based cross sectional study. The study subjects were chronic kidney disease patients with age and sex matched healthy controls. Full blood count, serum ferritin, soluble transferring receptor, C-reactive protein, serum iron and total iron binding capacity were measured in the patients and healthy controls.Data was analyzed with statistical package for the social sciences software version 22.0. And the level of statistical significance was set at p. value < 0.05. The mean ± SD of the age of patient with CKD was 55.0 + 15.4 years, while that of controls was 52.7 + 13.6 years. The mean serum ferritin, serum iron, TIBC and CRP were significantly higher in patients compared with controls (p<0.001, 0.023, <0.001 and 0.001) respectively. Functional iron deficiency was seen in 19.5% of patients with CKD. The predominant form of iron deficiency in our study was functional iron deficiency.

Ultrasensitive aptamer-functionalized Cu-MOF fluorescent nanozyme as an optical biosensor for detection of C-reactive protein

In the present work, an aptasensing method based on integration of RNA on Cu-MOF was developed for detection of C-Reactive Protein (CRP). Cu-MOF showed stimulated fluorescence and mimetic peroxidase enzymatic activity at the time and can be used as dual-signal transduction. CRP binding RNA was used as a highly selective recognition element and immobilized on the Cu-MOF. The immobilized RNA can block the peroxidase activity and fluorescence of the signal traducer probe. Adding CRP to the RNA/Cu-MOF will release RNA from the surface of Cu-MOF and recover both the stimulated fluorescence and peroxidase activity. A biosensor was built for detection of CRP using the two modes of transduction, either colorimetry or fluorometry. A dynamic linear range was obtained from 0.1 to 50 ng mL −1with a limit of detection (LOD) as small as 40 pg mL −1was calculated in fluorescence mode and 240 pg mL −1 as LOD in colorimetry mode. The LODs are lower than the LOD of nephelometric techniques used in clinical practice and is comparable to the normal clinical cutoff value in high-sensitivity CRP assays (1 μg/mL). The aptasensor was successfully applied for detection of CRP in Covid-19 patients with spike recoveries between 84 and 102% and RSD from 0.94% to 2.05%.

Structurally Altered, Not Wild-Type, Pentameric C-Reactive Protein Inhibits Formation of Amyloid-β 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-β peptide 1-42 (Aβ) reacted with immobilized proteins, indicating that some immobilized proteins express an Aβ epitope. Accordingly, four different CRP mutants capable of binding to immobilized proteins were constructed, and their binding to fluid-phase Aβ was determined. All CRP mutants bound to fluid-phase Aβ, suggesting that Aβ is a ligand for structurally altered pentameric CRP. In addition, the interaction between CRP mutants and Aβ prevented the formation of Aβ fibrils. The growth of Aβ 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 Aβ. 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.

Low Testosterone Level and Risk of Adverse Clinical Events among Male Patients with Chronic Kidney Disease: A Systematic Review and Meta-Analysis of Cohort Studies.

The phenomenon of low testosterone level is extremely common in male patients with chronic kidney diseases (CKDs). This meta-analysis aimed to evaluate whether the low circulating testosterone could independently predict adverse outcomes among male patients with chronic kidney diseases (CKDs). The data till May 2022 were systematically searched from Pubmed, Web of Science, and Embase from inception. Studies meeting the PICOS (population, intervention/exposure, control/comparison, outcomes, and study design) principles were included in this meta-analysis. Study-specific effect estimates were pooled using fixed-effects (I2 > 50%) or random-effects models (I2 < 50%). Ultimately, 9 cohort studies covering 5331 patients with CKDs were involved in this meta-analysis. The results suggested that per 1-standard deviation (SD) decrease in total testosterone independently increased the risk of all-cause mortality by 27% [hazard risk (HR) 1.27, 95% confidence interval (CI) 1.16–1.38], cardiovascular mortality by 100% (HR 2.00, 95% CI 1.39–2.86), cardiovascular events by 20% (HR 1.20, 95% CI 1.04–1.39), and infectious events by 41% (HR 1.41, 95% CI 1.08–1.84). Besides, with per 1-SD decrease in free testosterone, the risk of overall adverse events increased by 66% (HR 1.66, 95% CI 1.34–2.05). Stratified analyses indicated that the negative relationship of the total testosterone with all-cause death risk was independent of factors involving age, race, body mass index, diabetes, hypertension, C-reactive protein, creatinine, and sex hormone binding globulin. In conclusion, it was identified that low endogenous testosterone could serve as an independent predictor of adverse clinical events among male patients with CKDs.

Insights into Otitis Media: Dissecting the interaction of C-Reactive Protein with Non-Typeable Haemophilus influenzae

Otitis Media (OM) is the inflammation of the middle ear (ME). Non-typeable Haemophilus influenzae (NTHi) is one of the leading otopathogens in causing OM. Phosphocholine (PCho) on the NTHi lipopolysaccharide influences host-pathogen interaction. C-Reactive Protein (CRP), an acute phase protein recognizes PCho, and can mediate bacterial killing. However, some strains of NTHi survive even in the presence of CRP. We aim to study the interaction of CRP with NTHi to understand its role in bacterial survival and OM. NTHi can efficiently infect the Junbo mouse, a characterised model of chronic and acute OM. CRP levels were highest 1 day post-intranasal inoculation in the ME fluid (MEF) and nasal passage (NP) washes. We show CRP is a localized response to NTHi as serum CRP levels were unaffected in NTHi inoculated and non-inoculated mice at 1, 3 and 7-day post intranasal inoculation. Further, we confirm the presence of NTHi influences CRP levels in the MEF and NP washes. We show CRP binding is influenced by the position and expression of PCho on the NTHi surface. Serum bactericidal assays indicate that the expression and position of PCho affects NTHi survival. The removal of CRP from the serum restores NTHi survival. The expression of PCho also influences opsonophagocytosis activity in macrophages, thereby confirming the importance of PCho in NTHi survival. The CRP-NTHi interaction is currently under investigation to advance our understanding of its role in the complex biological processes that influence bacterial killing and the onset, progression and resolution of OM caused by NTHi.

Nonencapsulated Streptococcus pneumoniae cause Bacteremia through Inhibition of C-Reactive Protein Binding

Abstract Naturally occurring nonecapsulated Streptococcus pneumoniae (NESp) are found worldwide and can cause invasive infections. In contrast, encapsulated pneumococci that fail to express capsule are rapidly cleared from the blood of infected mice. We previously demonstrated that NESp expressing the Ami-like proteins AliC and AliD are able to evade complement-mediated killing. Data indicated that factor H (FH) failed to bind to NESp, and thus did not reduce complement activation as seen in FH binding pneumococci. Proteomic data have shown that deletion of aliC and aliD altered pneumococcal protein expression. Additionally, NESp expressing AliC and AliD caused invasive pneumococcal disease in C1q-deficient mice. The purpose of this study was to examine the regulated pneumococcal genes in complement evasion by NESp. Flow cytometry was used to examine binding of C3b to NESp. A mouse model of infection was used to investigate complement interaction with NESp in vivo. NESp expressing AliC and AliD were able to persist for at least four days following intravenous infection of mice. Deletion of aliC and aliD resulted in the clearance of NESp within 24 hours post-infection. There was a four-fold increase in C3b deposition on the mutant NESp compared to the wildtype. Choline binding protein AC (CbpAC), a variant of pneumococcal protein A (PspA), was found to have reduced expression in the aliC and aliD deleted mutant. C-reactive protein (CRP) showed increased binding to NESp in which cbpAC was deleted. Regulated expression of CbpAC in NESp is able to overcome the innate immune response and lead to persistence of NESp within the host by reducing C1q deposition and arresting classical complement activation. Supported by Departmental research funds.

Amelioration of C-Reactive Protein and Lectin Like Oxidized Low Density Lipoprotein Receptor Complex Induced Endothelial Dysfunction by Oligomeric Proanthocyanidins.

C-reactive protein (CRP) is a well-established biochemical marker for atherosclerosis. Modification of LDL inside the artery wall favors the elevation of this acute phase protein. Hence, this mechanism is considered an important factor to trigger the monocyte to macrophages differentiation which results in the formation of foam cells. Therefore, this key event should be targeted and focused on how this complex (OxLDL + CRP) proceeds to endothelial dysfunction. Oligomeric proanthocyanidins (OPC) is a well-known cardioprotective flavon-3-ols. The present study is challenged between the cardioprotective roles of OPC against the deleterious effect of OxLDL + CRP complex upon endothelial cells. Protein-protein docking was carried out between CRP and LOX-1. This docked protein complex was again docked with OPC to show the inhibitory mechanism of CRP binding with LOX-1. OPC showed a promising inhibitory mechanism against OxLDL + CRP complex. Docking studies showed that in the absence of ligands (OPC), binding of CRP and LOX-1 was greater and vice versa in the presence of ligands. Based on these molecular docking results, in vitro studies have been carried out. The monolayer of endothelial cells was incubated with THP-1 monocytes for 48h, induced with OxLDL (10μg/ml) + CRP (15μg/ml) and cotreated with OPC (100μg/ml). Morphological changes, cell migration assay, and capillary tube forming assay were carried out. Myeloperoxidase levels were estimated to determine the adhesion of monocytes onto EC monolayer. RT-PCR analysis of L-Selectin was also done. The quantification of NO levels and analysis of mRNA expressions of eNOS was to determine the nitric oxide demand caused due to OxLDL + CRP complex. LOX-1, scavenger receptor levels were analyzed by mRNA expression. Proinflammatory markers such as IL-6, MCP-1, and IL-1β were studied. Accumulation of ROS levels was measured fluorimetrically using DCF-DA staining. Mitochondrial membrane potential was determined by JC-1 dye and cell cycle analysis was done by FACS analysis. To emphasis the results, the OPC-treated group showed decreased levels of proinflammatory markers, LOX-1 and L-selectin levels. Endothelial nitric oxide levels were increased upon OPC treatment and reduction in the ROS levels was also observed. Endothelial cells apoptosis was prevented by OPC. To conclude, OxLDL + CRP complex inhibitory effects of OPC could maintain the normal homeostasis.

Equivalent circuit model of a non-faradaic impedimetric ZnO nano-crystal biosensor

• Sensing of C-reactive protein using a novel co-planar electrode PET-ZnO biosensor. • Development of an EEC model of the biosensor platform. • EEC modelling demonstrates predicted properties of a non-faradaic biosensor. • Quantified EDL charge modulations arising due to CRP binding at the ZnO surface. • Dominance of each sensor component identified to support control of parasitics. This paper describes the development of an equivalent electrical circuit (EEC) model of a unique co-planar electrode PET-ZnO biosensor platform. This non-faradaic ZnO biosensor had a PET insulating layer between the electrodes and the active ZnO sensor surface and impedance spectroscopy was used to measure varying concentrations of C-reactive protein (CRP). Analysis of the impedance magnitude bode plot reveals a slope indicative of a capacitive behaviour, where R 2 is 0.99. Furthermore, extraction of the complex impedance parameters shows capacitive reactance (Xc) being the dominant parameter as a result of capacitive transduction mechanism. The EEC was based on the generalized Randles model and adapted to represent the non-faradaic ZnO biosensor. Experimental data was fitted to the model at 100 kHz to establish values for the component parts of the biosensor system. With these values the model showed good correlation between experimental and modelled data across the frequency range studied, within the limitations of the measurement instrument. The model quantified charge modulations arising due to bimolecular binding of CRP within the electrical double layer (EDL) formed at the ZnO-PBS interface. The CRP bio-molecular interaction within the EDL can cause high charge carrier density accumulation in the semiconductor and as a result the capacitance of the EDL is increased. The EEC model quantifies this phenomenon, presented as a parallel capacitance connected to the ZnO-Antibody interfacial capacitance. The model was adapted to two variants of the biosensor design, with differing ZnO nanoparticle concentrations forming the sensor surface. The variations on the components modelled concurred with expected changes established from biosensor surface analysis. Using the EEC model, the dominance of each constituent sensor element on measurement stability is quantified identifying the significant contributors to experimental and sensor-to-sensor repeatability to support control of parasitic effects.

Biochemically prepared C-reactive protein conformational states differentially affect C1q binding

C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.

Digital plasmonic immunosorbent assay for dynamic imaging detection of protein binding

A highly sensitive imaging sensor and digital plasma immunosorbent assay, based on a gold–titanium plasmonic nanopore array, has been reported. The sensor can quantitatively determine a low concentration of proteins and exhibits superior performance by combining a classical Poisson statistical algorithm and the digital surface plasmon resonance (SPR) imaging technique. In this study, we used a device to detect C-reactive protein (CRP) and anti-CRP antibody binding information from SPR images. Our technique avoids complex spectroscopic detection and was applied to detect the inflammatory marker CRP protein with an LOD of 2.36 ng/ml using white light. Moreover, we analyzed the protein-binding kinetics and demonstrated that our device could also be used to determine the equilibrium dissociation constant in protein interactions through ultrasensitive dynamic imaging of the CRP protein during interaction with anti-CRP antibodies. Owing to the superior performance of the plasmonic nanopore array, the proposed device is promising for future portable optical sensing with visible light for the early detection of diseases.

Development of a Point-of-Care System Based on White Light Reflectance Spectroscopy: Application in CRP Determination.

The development of methods and miniaturized systems for fast and reliable quantitative determinations at the Point-of-Care is a top challenge and priority in diagnostics. In this work, a compact bench-top system, based on White Light Reflectance Spectroscopy, is introduced and evaluated in an application with high clinical interest, namely the determination of C-Reactive protein (CRP) in human blood samples. The system encompassed all the necessary electronic and optical components for the performance of the assay, while the dedicated software provided the sequence and duration of assay steps, the reagents flow rate, the real-time monitoring of sensor response, and data processing to deliver in short time and accurately the CPR concentration in the sample. The CRP assay included two steps, the first comprising the binding of sample CRP onto the chip immobilized capture antibody and the second the reaction of the surface immunosorbed CRP molecules with the detection antibody. The assay duration was 12 min and the dynamic range was from 0.05 to 200 μg/mL, covering both normal values and acute inflammation incidents. There was an excellent agreement between CRP values determined in human plasma samples using the developed device with those received for the same samples by a standard diagnostic laboratory method.

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors in vitro.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.