anti-CRP Antibody Research Articles

Soluble lectin-like oxidized low density lipoprotein receptor-1 in type 2 diabetes mellitus

The lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) can be proteolytically cleaved and released as soluble forms (sLOX-1). We have determined serums LOX-1 in type 2 diabetes and evaluated the effect of glucose and advanced glycation end products (AGEs) on sLOX-1 in vitro and in vivo. Endothelial cells were incubated with glucose or AGEs, and sLOX-1 in cell medium was measured. Serum sLOX-1 was measured in 219 diabetic patients and 187 controls by ELISA. The effect of lowering glucose and AGEs on sLOX-1 was determined in 38 poorly controlled diabetic patients after improvement in glycemic control. Incubation of endothelial cells with AGE-BSA led to a dose-dependent increase in sLOX-1, whereas the effect of glucose on sLOX-1 was less marked. Serum sLOX-1 was 9% higher in diabetic patients compared with controls (P<0.01). In the poorly controlled patients, serum sLOX-1 decreased by 12.5% after improvement in glycemic control (P<0.05). The magnitude of reduction in sLOX-1 correlated with the improvement in hemoglobin A1c and AGEs but not with the reduction in oxidized LDL. sLOX-1 level is increased in type 2 diabetes. Both glucose and AGEs are important determinants of LOX-1 expression, and lowering glucose and AGEs leads to a reduction in sLOX-1.

Binding behavior of CRP and anti-CRP antibody analyzed with SPR and AFM measurement

Atomic force microscope (AFM) was exploited to take picture of the molecular topology of C-reactive protein (CRP) in phosphate-buffered saline (PBS) solution. An explicit molecular image of CRP demonstrated a pentagonal structure composed of five subunits. Dimensions of the doughnut-shaped CRP molecule measured by AFM were about 25 nm in outside diameter and 10 nm in central pore diameter, and the height of CRP molecule was about 4 nm which was comparable to the value determined by X-ray crystallography. Bis( N-succinimido)-11,11′-dithiobis (undecyl succinate) (DSNHS) was synthesized for use as a linker for immobilizing anti-CRP antibody (anti-CRP) onto the gold surface of a surface plasmon resonance (SPR) sensor chip. DSNHS formed self-assembled monolayer (SAM) on the gold surface. By use of an AFM tip, a pattern of ditch was engraved within the SAM of DSNHS, and anti-CRP was immobilized on the engraved SAM through replacement of N-hydroxysuccinimide group on the outside surface of DSNHS by the amine group of anti-CRP. Formation of CRP/anti-CRP complex on the gold surface of SPR sensor chip was clearly demonstrated by measuring SPR angle shift. A consecutive series of SAM, SAM/anti-CRP, and SAM/anti-CRP/CRP complexes was generated on a SPR sensor chip, and the changes in depth of the ditch were monitored by taking AFM images of the complexes. Comparative analysis of the depth differences indicates that binding of CRP to anti-CRP occurs in a planar mode.

Identification of a novel centrosomal protein Crp F46 involved in cell cycle progression and mitosis

A novel centrosome-related protein Crp F46 was detected using a serum F46 from a patient suffering from progressive systemic sclerosis. We identified the protein by immunoprecipitation and Western blotting followed by tandem mass spectrometry sequencing. The protein Crp F46 has an apparent molecular mass of ~ 60 kDa, is highly homologous to a 527 amino acid sequence of the C-terminal portion of the protein Golgin-245, and appears to be a splice variant of Golgin-245. Immunofluorescence microscopy of synchronized HeLa cells labeled with an anti-Crp F46 monoclonal antibody revealed that Crp F46 localized exclusively to the centrosome during interphase, although it dispersed throughout the cytoplasm at the onset of mitosis. Domain analysis using Crp F46 fragments in GFP-expression vectors transformed into HeLa cells revealed that centrosomal targeting is conferred by a C-terminal coiled-coil domain. Antisense Crp F46 knockdown inhibited cell growth and proliferation and the cell cycle typically stalled at S phase. The knockdown also resulted in the formation of poly-centrosomal and multinucleate cells, which finally became apoptotic. These results suggest that Crp F46 is a novel centrosome-related protein that associates with the centrosome in a cell cycle-dependent manner and is involved in the progression of the cell cycle and M phase mechanism.

C-reactive protein, anti-C-reactive protein antibodies and clinical atherosclerosis

There has been considerable interest in the relationship between C-reactive protein (CRP) and atherosclerosis. We have previously demonstrated that individuals, especially those with rheumatoid arthritis and systemic lupus erythematosus, may produce antibodies to CRP. This study was therefore undertaken to determine the possible association between anti-CRP antibodies and atherosclerosis. A total of 103 individuals were identified with or without atherosclerosis, and without clinical rheumatic diseases. They were evaluated with respect to cholesterol, HDL, LDL, high-sensitivity (hs)CRP, and anti-CRP antibody levels, as well as use of statin medications. Individuals with atherosclerosis were much more likely to be taking a statin, and thus have lower lipid levels. However, there was no association between hsCRP or anti-CRP antibody levels with atherosclerosis, statin use, or each other. These observations suggest that anti-CRP antibody is not involved in atherosclerosis, and may represent an epiphenomenon.

Elevated uric acid and cardiovascular disease. How strong is the evidence of a pathogenetic link?

In a recent issue of this Journal, Montalcini and coworkers demonstrated that an increase in serum uric acid levels may be an independent risk factor for atherosclerosis in a series of healthy postmenopausal women [1]. The possible causative effect of modifications in uric acid metabolism in the pathogenesis of cardiovascular disease (CVD) was also reviewed by Manzato in the same issue of the journal [2], bringing to the attention of the scientific community the old concept of the possible role of uric acid as an additional player in the development of atherosclerosis and arterial thrombosis. Epidemiological studies showed that elevated serum uric acid may be a risk factor for CVD independent of other abnormalities commonly observed in patients with the metabolic syndrome, such as obesity, dyslipidaemia, hypertension, insulin resistance and glucose intolerance [3–5]. The study by Montalcini et al. [1] tested the pro-atherosclerotic effects of hyperuricaemia by using high-resolution ultrasound, a widely accepted technique able to noninvasively detect early atherosclerotic modifications of arterial wall in vivo, and specifically an increase in carotid intima-media thickness. It has been previously shown that this structural change correlates with future clinical events, therefore providing reliable information on the prognostic role of cardiovascular risk factors [6]. The mechanisms potentially involved in the pro-atherosclerotic effect of uric acid are still under investigation and, as mentioned by Manzato [2], several hypotheses have been made to explain this issue. One possible pathogenetic mechanism relates to the effect exerted by uric acid on vascular and inflammatory cells. It has been demonstrated that uric acid, in a range of concentration of 120–240 μmol/l (6–12 mg/dl), is able to upregulate Creactive protein (CRP) mRNA expression in human umbilical vein endothelial cells (HUVEC) in vitro, thus inducing CRP release into culture media [7]. In the same experiments uric acid was shown to stimulate human vascular smooth muscle cell (VSMC) proliferation and migration, and to inhibit endothelial cell proliferation and nitric oxide (NO) release. All these effects of uric acid could be blocked by incubation of vascular cells with anti-CRP antibodies [7]. Taken together these data suggest that uric acid may potentially contribute to vasoconstriction and platelet activation and to atherosclerotic plaque growth, and that these effects are mediated by locally produced CRP. Work by the same group showed that hyperuricaemia, obtained in rats by blocking uricase by oxonic acid, was also able to induce a decrease in serum NO levels together with an increase in systolic blood pressure, and that this effect was reversed after allopurinol treatment for 7 days. In additional experiments, uric acid was shown to dose dependently inhibit both basal and vascular endothelial growth factor (VEGF)-induced NO production by bovine endothelial cells in vitro, thus suggesting that exposure to uric acid may induce endothelial dysfunction and a pro-atherosclerotic phenotype in these cells [8]. Kanellis and coworkers also demonstrated that uric acid induced an increase in rat aortic VSMC monocyte chemoattractant protein-1 (MCP-1) expression in a timeand dose-dependent manner, with a peak at 24 h. Overexpression of MCP-1 mRNA and protein occurred as early as 3 h after the incubation of VSMC with uric acid and was associated with activation of the transcription factors NF-κB and activator protein-1 (AP-1), as well as with the activation of the mitogen-activated protein kinase (MAPK) extracellular signalling molecules ERK p44/42 and p38, and with an increase in cyclooxygenase-2 (COX2) mRNA expression [9]. Inhibition of ERK p44/42, p38 and COX-2 each suppressed uric acid-induced MCP-1 production [9]. These data clearly show that uric acid, in its soluble form, can elicit an inflammatory response in VSMC, thus providing further evidence of its potential role in monocyte recruitment, VSMC activation and finally in atherosclerotic plaque growth and rupture. A large body of experimental data demonstrated that inflammatory processes play a crucial role in all steps of atherosclerotic plaque formation and disruption and in its thrombotic complications, and that these processes are sustained by a variety of cells such as endothelial cells, VSMC and macrophages [10]. CRP is one of the most important inflammatory mediators. It is mainly produced by the liver in response to the proinflammatory cytokine IL-6 which, in turn, is syntheIntern Emerg Med (2007) 2:320–321 DOI 10.1007/s11739-007-0087-x

Anti-C-reactive protein antibodies in chronic hepatitis C infection: Correlation with severity and autoimmunity

The aim of this work was to detect circulating anti-C-reactive protein (CRP) antibodies in serum samples of patients with chronic hepatitis C virus (HCV) and to investigate a possible association with other autoimmune manifestations. A total of 94 patients with chronic HCV infections and 108 healthy controls were enrolled. All patients underwent a baseline evaluation: immunological assessment of cryoglobulin,antinuclear antibodies (ANA), rheumatoid factor (RF), anticardiolipin (aCLA), and anti-CRP antibodies. Patients with HCV underwent a liver biopsy scored according to the modified Knodell score. Anti-CRP antibodies were detected in 17% of HCV patients compared with 6.4% of the healthy controls (p < 0.025). When HCV patients positive for anti-CRP antibodies were compared with patients who were negative for anti-CRP antibodies, the prevalence of positive RF was significantly higher, 50% versus 17.9% (p < or = 0.05). Cryoglobulinemia was also significantly more frequent in patients who were positive for anti-CRP antibodies, 75% versus 32%, p < or = 0.01. ANA and aCLA did not differ significantly between the two groups. The presence of anti-CRP antibodies was associated with greater liver disease severity (histology activity index, 9 +/- 3.3 versus 6 +/- 2.9, p = 0.01). An increased prevalence of anti-CRP antibodies was manifested in HCV-infected patients. The presence of anti-CRP antibodies correlated with the presence RF, cryoglobulinemia, and severity of liver disease.

Single step diagnosis system using the FRET phenomenon induced by antibody-immobilized phosphorylcholine group-covered polymer nanoparticles

We report here a single step molecular diagnosis system using phosphorylcholine group-covered nanoparticles (PC-NPs). The most favorable characteristics were (i) the suppression of non-specific protein adsorption by the PC group-covered surface and (ii) molecular diagnosis by the capture of the target antigen. The fundamental strategy was based on antigen–antibody reactions on the PC-NPs. In particular, we installed a fluorescence resonance energy transfer (FRET) system on the immobilized antibodies. The immobilized antibody was labeled by donor or acceptor molecules separately, which were fluorescent molecules. These molecules induce the FRET phenomenon if they are close to each other upon capture of the target antigen by the PC-NPs. Therefore, the resulting fluorescence was readable using the FRET phenomenon. In this paper, C-reactive protein (CRP) was used as the target antigen, and the anti-CRP antibody was labeled by fluorescent molecules. The labeled antibody was then immobilized onto the PC-NPs. The resulting fluorescence intensity was well correlated with the change in the concentration of CRP. The protocol for molecular diagnosis is quite simple, and only involves mixing the FRET-installed NPs and target molecules such as CRP. Single step molecular diagnosis via FRET may be of great importance for designing sensor devices.

Autoantibodies against Protective Molecules—C1q, C‐Reactive Protein, Serum Amyloid P, Mannose‐Binding Lectin, and Apolipoprotein A1

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of several autoantibodies. Among the multiple factors involved in SLE development, apoptotic defects and impaired clearance of cellular debris have gained considerable interest, as they contribute to autoantigen overload. Several molecules of the innate immunity, also participate in the removal of damaged and apoptotic cells. Among them are C1q, C-reactive protein (CRP), serum amyloid P protein (SAP), mannose-binding lectin (MBL), and apolipoprotein A1 (APO A1). To evaluate the prevalence of autoantibodies against CRP, SAP, MBL, APO A1, and C1q among SLE patients, and their relationship with disease activity, a total of 150 SLE patients were screened for the presence of elevated antibody titers against C1q, CRP, SAP, MBL, and APO A1, utilizing the enzyme-linked immunosorbent assay (ELISA) method. Disease activity was assessed using the ECLAM or SLEDAI scores. The study population comprised two groups of patients: 100 patients with quiescent disease (median ECLAM score 2) comprised the first group, and 50 patients with active disease (median SLEDAI score 16) comprised group 2. Elevated titers of anti-CRP antibodies were significantly elevated only in group 1 (10% versus 4% of controls). Antibodies against SAP were evaluated only among patients in group 1, and were found at a significant high prevalence (20%). Elevated titers of anti-MBL antibodies were significantly elevated only in group 1 (15% versus 3.6%); and antibodies directed against APO A1 were significantly elevated in 21% of group 1, and 50% of group 2 patients. Elevated titers of anti-C1q were evaluated only in group 2, and were found at a significant prevalence of 66%. Significant correlation with disease activity was found only for anti-APO A1 antibodies, and only in group 1. Several patients harbored more than one of the autoantibodies tested. In patients with SLE, autoantibodies directed against protective molecules, that is, acute-phase proteins involved in the disposal of cellular and nuclear debris, can be detected. These autoantibodies may play a pathogenic role in the development or perpetuation of autoimmunity in SLE.

Antibodies against C-Reactive Protein Cross-React with 60-Kilodalton Heat Shock Proteins

C-reactive protein (CRP) is an acute-phase reactant frequently used in histochemistry as a marker of ongoing inflammation. Furthermore, CRP is a powerful biomarker for the prediction of coronary artery disease risk. Heat-shock protein 60 (Hsp60) and CRP are complement-activating molecules, and the effect of their interactions on the regulation of complement activation was studied. However, during the first experiments, we learned that polyclonal anti-CRP antibodies cross-react with Hsp60. Therefore, the aim of our present study was to analyze the cross-reactivity of anti-CRP antibodies (Ab) with Hsp60 in solid-phase enzyme immune assays, in epitope studies using a series of overlapping synthetic peptides, and in Ouchterlony analyses. We found that three different commercial rabbit polyclonal antibodies and two monoclonal (9C9 and CRP-8) anti-CRP antibodies specifically recognize recombinant human Hsp60 and recombinant Mycobacterium tuberculosis Hsp65, respectively. Hsp60 was found to inhibit the binding of anti-CRP polyclonal Ab to Hsp60. Six epitope regions of Hsp60 were recognized by the anti-CRP antibodies, and one region (amino acids [AA] 218 to 232) was recognized by monoclonal antibodies CRP-8 and 9C9. This epitope region of Hsp60 displays 26.6% amino acid identity to CRP AA region 77 to 90. These data suggest that the B-cell epitopes shared between CRP and Hsp60 give rise to a true mimicry-based cross-reaction and the induction of cross-reactive antibodies. Our study underlines the importance of thorough study design and careful interpretation of results while using polyclonal anti-CRP antibodies for histochemistry, especially at low dilutions. Furthermore, analytical interference with Hsp60 in CRP assays should also be tested.

High prevalence of anti-CRP antibodies in patients with SLE

High prevalence of anti-CRP antibodies in patients with SLE

Apolipoprotein E polymorphism has no cross sectional association with C‐reactive protein levels in women

Introduction: The purpose of this study was to compare the level of high sensitive C-Reactive protein (CRP) in those with the E3/E4 genotype to those with the E2/E3 or E3/E3 genotypes. Methods: Sixty-sixty females were assessed for ApoE genotype and CRP levels (mean age 38.2 ± 11.6 years). CRP was assessed using a two-site chemiluminescent enzyme immunometric assay with one monoclonal and one polyclonal anti-CRP antibody. Results: Women with the E3/E4 (n=27) genotype had significantly lower CRP than those without the E4 allele (n=39) (2.02 ± 2.13 vs 3.37 ± 2.33, p<0.01). Discussion: Contrary to the hypothesis that E4 carriers would have higher levels of systemic inflammation, these data show lower CRP levels in this group compared to those without the E4 allele. How this relates to the increased cardiovascular mortality rate in this group is unclear, but highlights a complex interaction between ApoE genotype, inflammation, other cardiovascular risk factors and cardiovascular mortality.

Antibody Immobilization on Functional Monolayers Using a Quartz Crystal Microbalance

This paper evaluated immobilization of anti-C-reactive protein (CRP) monoclonal antibody on a quartz crystal microbalance (QCM) when 2-aminoethanethiol (AET), 4,4'-dithiodibutyric acid (DDA), and 11-mercaptoundecanoic acid (MUA) were deposited on the gold surface of QCM. In all monolayers, anti-CRP antibodies were immobilized such as Langmuir types because it had been introduced with a corresponding active group. According to the Langmuir isotherm equation amax, the maximum immobilized amounts of anti-CRP antibody were 4.27, 2.72, and 3.74 pmol/cm2, respectively. Although the immobilized amount of anti-CRP antibody was highest on the AET monolayer, the amount of antigen-antibody binding between the anti-CRP antibody and the CRP was highest on the MUA monolayer. CRP was detected from negative to positive levels when the calibration curve was achieved using MUA monolayer

C-Reactive Protein Alters Antioxidant Defenses and Promotes Apoptosis in Endothelial Progenitor Cells

C-reactive protein (CRP) has been suggested to participate in the development of atherosclerosis, in part, by promoting endothelial dysfunction and impairing endothelial progenitor cell (EPC) survival and differentiation. In the present study, we evaluated the effects of CRP on antioxidative enzymes, reactive oxygen species production, telomerase activity, and apoptosis in human circulating EPCs. EPCs, isolated from peripheral venous blood, were cultured in the absence or presence of native pentameric azide and lipopolysaccharide (LPS)-free CRP (0, 5, 15, and 20 microg/mL), N-acetylcysteine (NAC), hydrogen peroxide (H2O2) or monoclonal anti-CRP antibodies. Fluorescence-activated cell sorter (FACS) analysis was used for the measurement of intracellular H2O2 and superoxide (O2(-)) by loading cells with 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). Apoptosis was evaluated with Annexin V immunostaining and cytosolic cytochrome c expression. Western blot analysis was used for the determination of manganese superoxide dismutase (MnSOD) and glutathione peroxidase expression, and polymerase chain reaction enzyme-linked immunosorbent assay was used to assess telomerase activity. Incubation of EPCs with CRP caused a concentration dependent increase in reactive oxygen species (ROS) production and apoptosis, with an effect quantitatively similar to H2O2. This effect was attenuated during coincubation with NAC or anti-CRP antibodies. Furthermore, CRP altered EPC antioxidative enzyme levels, demonstrating a reduced expression of glutathione peroxidase and a significant increase in MnSOD expression. Transfection of EPCs with MnSOD-RNAi resulted in a reduction in CRP-induced ROS production, apoptosis, and telomerase inactivation. CRP, at concentrations known to predict cardiovascular events, may serve to impair EPC antioxidant defenses, and promote EPC sensitivity toward oxidant-mediated apoptosis and telomerase inactivation. These data further support a direct role of CRP in the development and/or progression of atherothrombosis.

CRP determination based on a novel magnetic biosensor

The c-reactive protein (CRP) is a very significant human blood marker for inflammatory processes and is routinely determined for many clinical purposes. The widespread and well established detection method for this approximately 115 kDa hepatic protein is the high-sensitivity ELISA assay (hsCRP-ELISA) in blood serum. New approaches in medical CRP diagnosis (e.g. for CVD, inflammatory bowel disease) require rapid quantification in native matrices. A novel CRP determination method based on magnetic detection is described and tested for human blood serum, saliva and urine. The detection principle is based on two different anti-CRP antibodies (monoclonal, IgG) for CRP trapment and labelling. The linear detection range of this immunosensor ranged from 25 ng/ml to 2.5 microg/ml and is therefore much more sensitive than typical hsCRP-ELISA-assays.

Determination of C-reactive protein with an ultra-sensitivity immunochemiluminometric assay

C-reactive protein (CRP), the classic acute phase reactant, is strongly associated with increased risk of cardiovascular events. The demand for measuring serum CRP levels has been predicted to increase. We developed an ultra-sensitivity in-house immunometric assay on polystyrene beads for measuring CRP and studied its analytical and clinical performance. The assay used a pair of monoclonal anti-CRP antibodies and detected CRP in a 1-step immunometric assay with a chemiluminescence signal. The calibration was traceable to the World Health Organization reference material. The assay covered a linear range of 0.01 to 50.00 mg/L. The analytical detection limit calculated from the mean level plus 3 SD of the zero calibrator was 0.004 mg/L. The within-run imprecision was 7.0%, 5.2%, and 4.1% for mean CRP levels of 0.02 mg/L, 1.44 mg/L, and 11.04 mg/L, respectively. The between-run imprecision was 9.2%, 7.0%, and 6.0% for mean CRP levels of 0.02 mg/L, 1.49 mg/L, and 10.90 mg/L, respectively. The average recovery was 102.0% ( n = 6). The assay correlated well with a high-sensitivity latex-enhanced nephelometric assay (regression line y = 0.865 x + 1.333, r = 0.974, S y/ x = 3.415, n = 47 for 0–50.00 mg/L and y = 1.076 x − 0.080, r = 0.985, S y/ x = 0.989, n = 29 for 0–20.00 mg/L). The central 95 percentile reference interval for Han Chinese residing in Taiwan was 0.02–4.33 mg/L ( n = 469). There was no significant difference in serum CRP levels between healthy male and female subjects (median, 0.34 and 0.31 mg/L, respectively); however, CRP levels increased moderately with age ( r = 0.276, P < .05). The reference values for the Chinese population were about 5-fold lower than those for the United States population. This ultra-sensitivity immunochemiluminometric assay for CRP is rapid and accurate and can be used to assess cardiovascular risk.

Uric Acid–Induced C-Reactive Protein Expression

Recent experimental and human studies have shown that hyperuricemia is associated with hypertension, systemic inflammation, and cardiovascular disease mediated by endothelial dysfunction and pathologic vascular remodeling. Elevated levels of C-reactive protein (CRP) have emerged as one of the most powerful independent predictors of cardiovascular disease. In addition to being a marker of inflammation, recent evidence suggests that CRP may participate directly in the development of atherosclerotic vascular disease. For investigating whether uric acid (UA)-induced inflammatory reaction and vascular remodeling is related to CRP, the UA-induced expression of CRP in human vascular smooth muscle cells (HVSMC) and human umbilical vein endothelial cells (HUVEC) was examined, as well as the pathogenetic role of CRP in vascular remodeling. It is interesting that HVSMC and HUVEC expressed CRP mRNA and protein constitutively, revealing that vascular cells are another source of CRP production. UA (6 to 12 mg/dl) upregulated CRP mRNA expression in HVSMC and HUVEC with a concomitant increase in CRP release into cell culture media. Inhibition of p38 or extracellular signal-regulated kinase 44/42 significantly suppressed UA-induced CRP expression, implicating these pathways in the response to UA. UA stimulated HVSMC proliferation whereas UA inhibited serum-induced proliferation of HUVEC assessed by 3H-thymidine uptake and cell counting, which was attenuated by co-incubation with probenecid, the organic anion transport inhibitor, suggesting that entry of UA into cells is responsible for CRP expression. UA also increased HVSMC migration and inhibited HUVEC migration. In HUVEC, UA reduced nitric oxide (NO) release. Treatment of vascular cells with anti-CRP antibody revealed a reversal of the effect of UA on cell proliferation and migration in HVSMC and NO release in HUVEC, which suggests that CRP expression may be responsible for UA-induced vascular remodeling. This is the first study to show that soluble UA, at physiologic concentrations, has profound effects on human vascular cells. The observation that UA alters the proliferation/migration and NO release of human vascular cells, mediated by the expression of CRP, calls for careful reconsideration of the role of UA in hypertension and vascular disease.

SPR-based immunosensor for the CRP detection—A new method to detect a well known protein

The c-reactive protein (CRP) is one of the significant human blood serum markers for inflammatory processes. The serum presence of this hepatic approximately 115 kDa protein of five identical subunits accompanies several diseases (e.g. CVD, inflammatory bowel diseases) and is nowadays detected by high-sensitivity ELISA assays in blood serum. To enable CRP detection in other matrices, an SPR-based (surface plasmon resonance) immunosensor for the CRP detection has been established. A linear detection range of 2-5 microg CRP per ml was found, using two different antiCRP antibodies (monoclonal, IgG) for CRP trapment and detection. Furthermore, the kinetic antibody association and dissociation constants of one antibody (antiCRP, clone C2) could be determined.

C-reactive protein binds to the 3β-OH group of cholesterol in LDL particles

C-reactive protein (CRP) has been suggested to contribute to the development of atherosclerosis. We previously found binding of CRP to cholesterol in modified low density lipoprotein (LDL) particles. Here, we characterize the interaction between CRP and cholesterol in more detail. When lipids of native LDL were separated by thin-layer chromatography, CRP bound only to cholesterol. When various cholesterol analogues were compared for their ability to bind CRP, we found that any modification of the 3β-OH group blocked binding of CRP to cholesterol. Similarly, enrichment of LDL with cholesterol but not with its analogues triggered the binding of CRP to LDL. Finally, with the aid of anti-CRP monoclonal antibodies and by molecular modeling, we obtained evidence for involvement of the phosphorylcholine-binding site of CRP in cholesterol binding. Thus, CRP can bind to cholesterol, and the interaction is mediated by the phosphorylcholine-binding site of CRP and the 3β-hydroxyl group of cholesterol.

Ultrasensitive confocal fluorescence microscopy of C-reactive protein interacting with FcgammaRIIa.

C-Reactive protein (CRP) is an acute phase protein with a suggested pathogenic role in cardiovascular disease. Previous reports proposed that the low-affinity IgG receptor FcgammaRIIa is the major receptor for CRP. However, these reports were met with criticism because the use of anti-CRP antibodies in the detection of CRP binding to FcgammaRIIa may have caused false-positive results. To resolve this controversy, we used ultrasensitive fluorescence microscopy to study the association, dissociation, and equilibrium of CRP binding to FcgammaRIIa. CRP indeed binds to FcgammaRIIa, with low association rates and dissociation rates. Anti-CRP antibodies markedly enhance binding, as is evident from the decrease of the equilibrium dissociation coefficient by 2 orders of magnitude. Our study demonstrates the virtues of single fluorophore labeling and highlights the pitfalls of immunolabeling in investigating CRP/Fc receptor interactions. Importantly, this article provides the first quantitative characterization of CRP binding to FcgammaRIIa and explains and reconciles the diverse and conflicting data presented in the literature.

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab′) 2-IgG antibody (70 μg/mL) was immobilized on the QCM. In case of anti-CRP F(ab′) 2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co- n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab′) 2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 μg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab′) 2-IgG antibody and MPC polymer.