Anti-human C-reactive Protein Research Articles

An integrated immunochromatographic device for C-reactive protein detection using hierarchical dendritic gold nanostructure films

Immunochromatographic test strips typically consist of sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. Even minute variations in the assembly of these components can lead to inconsistent sample-reagent interactions, thereby reducing reproducibility. In addition, the nitrocellulose membrane is susceptible to damage during assembly and handling. To address this issue, we propose to replace the sample pad, conjugate pad, and nitrocellulose membrane with hierarchical dendritic gold nanostructure (HD-nanoAu) films to develop a compact integrated immunochromatographic strip. The strip uses quantum dots as a background fluorescence signal and employs fluorescence quenching to detect C-reactive protein (CRP) in human serum. A 5.9 μm thick HD-nanoAu film was electrodeposited on an ITO conductive glass by the constant potential method. The wicking kinetics of the HD-nanoAu film was thoroughly investigated, and the results indicated that the film exhibited favorable wicking properties, with a wicking coefficient of 0.72 μm ms−0.5. The immunochromatographic device was fabricated by etching three interconnected rings on HD-nanoAu/ITO to designate sample/conjugate (S/C), test (T), and control (C) regions. The S/C region was immobilized with mouse anti-human CRP antibody (Ab1) labeled with gold nanoparticles (AuNPs), while the T region was preloaded with polystyrene microspheres decorated with CdSe@ZnS quantum dots (QDs) as background fluorescent material, followed by mouse anti-human CRP antibody (Ab2). The C region was immobilized with goat anti-mouse IgG antibody. After the samples were added to the S/C region, the excellent wicking properties of the HD-nanoAu film facilitated the lateral flow of the CRP-containing sample toward the T and C regions after binding to AuNPs labeled with CRP Ab1. In the T region, CRP-AuNPs-Ab1 formed sandwich immunocomplexes with Ab2, and the fluorescence of QDs was quenched by AuNPs. The ratio of fluorescence intensity in the T region to that in the C region was used to quantify CRP. The T/C fluorescence intensity ratio was negatively correlated with the CRP concentration in the range of 26.67–853.33 ng mL−1 (corresponding to 300-fold diluted human serum), with a correlation coefficient (R2) of 0.98. The limit of detection was 15.0 ng mL−1 (corresponding to 300-fold diluted human serum), and the range of relative standard deviation: 4.48–5.31%, with a recovery rate of 98.22–108.33%. Common interfering substances did not cause significant interference, and the range of relative standard deviation: 1.96–5.51%. This device integrates multiple components of conventional immunochromatographic strips onto a single HD-nanoAu film, resulting in a more compact structure that improves the reproducibility and robustness of detection, making it promising for point-of-care testing applications.

Oriented Antibody Covalent Immobilization for Label-Free Impedimetric Detection of C-Reactive Protein via Direct and Sandwich Immunoassays.

The detection and monitoring of biological markers as disease indicators in a simple manner is a subject of international interest. In this work, we report two simple and sensitive label-free impedimetric immunoassays for the detection of C-reactive protein (CRP). The gold electrode modified with boronic acid–terminated self-assembled monolayers afforded oriented immobilization of capture glycosylated antibody (antihuman CRP monoclonal antibody, mAb). This antibody-modified surface was able to capture human CRP protein, and the impedance signal showed linear dependence with CRP concentration. We confirmed the immobilization of anti-CRP mAb using surface sensitive X-ray photoelectron spectroscopy (XPS) and electrochemical impedance. The oriented covalent immobilization of mAb was achieved using glycosylated Fc (fragment, crystallizable) region specific to boronic acid. The direct immunoassay exhibited a linear curve for concentration range up to 100 ng ml−1. The limit of detection (LoD) of 2.9 ng ml−1, limit of quantification (LoQ) of 9.66 ng ml−1, and sensitivity of 0.585 kΩ ng−1 ml cm−2 were obtained. The sandwich immunoassay was carried out by capturing polyclonal anti-CRP antibody (pAb) onto the CRP antigen immunoreaction. The impedance signal after pAb capture also showed linear dependence with CRP antigen concentration and acted as a CRP antigen detection signal amplifier. The detection of the CRP antigen using sandwich pAb immunoassay improved LoD to 1.2 ng ml−1, LoQ to 3.97 ng ml−1, and enhanced the sensitivity to 0.885 kΩ ng−1 ml cm−2. The real sample analysis, using newborn calf serum, showed excellent selectivity and % recovery for the human CRP ranging from 91.2 to 96.5%. The method was reproducible to 4.5% for direct immunoassay and 2.3% for sandwich immunoassay.

Electroimmunodetection of cardiac C-reactive protein for determining myocardial Injury.

C-reactive protein (CRP) is an acute phase reactant to be a marker of inflammation and has been correlated with the cardiac injury. An immunoassay was performed using anti-human CRP antibody on an InterDigitated electrode (IDE) sensor to determine and specify CRP concentration for diagnosing the condition of myocardial inflammation. To promote the detection, gold nanoparticle (GNP) was seeded on the aminated-IDE surface. Anti-CRP was hitched on the GNP-seeded surface and identified the abundance of CRP. The limit of quantification was found as 100 fM, and the higher current response was noticed by increasing CRP concentrations with the sensitivity at 1 pM. Furthermore, CRP-spiked human serum did not interfere the determination of CRP and increased the current response, indicating suitability for a real-life sample. Similarly, the control experiments with nonimmune antibody Troponin I are not showing the definite current responses, proving the selective identification of CRP. This method of diagnosing is needful to determine the cardiovascular injury at the right time.

Homogeneous functional self-assembled monolayers: Faradaic impedance baseline signal drift suppression for high-sensitivity immunosensing of C-reactive protein

The limit of detection (LOD) of affinity sensors based on alkanethiol self-assembled monolayers (SAMs) system can be improved by either signal amplification and/or noise reduction. The latter includes baseline signal drift arising, in part, from, monolayer imperfections (and variations in this through repeats) as well as electrical noise of both the sensor and transducer. In reagentless “label free” assays signal drift carries with it the possibility of assay false-positive results (if signal drift is positive) or false-negatives (if specific signal is swamped by downward drift). Faradaic electrochemical impedance spectroscopy (FEIS) sensors based on SAM interfaces have been shown to exhibit considerable baseline signal drift, which consequently affects their assaying capabilities. This study reports on the development of a simple two-step pre-treatment method for generating functional SAMs of 11-mercaptoundecanoic acid (MUA) on Au with a highly suppressed baseline signal drift. For electrochemical characterisation of the SAMs, the charge-transfer resistance (Rct), defect presence (pinhole radius and separation), capacitance, and dielectric constant were evaluated. The interface pre-treatment generates films of substantially improved homogeneity that are subsequently functionalised with anti-human C-reactive protein. CRP, an acute-phase protein, is detectable down to femtomolar levels without any amplification; this is a 2–3 order of magnitude lower detection limit than that typically accessible for analyses of this type. The developed protocols thus present a convenient and general route to suppress baseline signal drifts and improve the limits of detection of affinity sensors based on Faradaic impedance.

Optimization of the secretory expression of recombinant human C-reactive protein in Pichia pastoris.

Human C-reactive protein (CRP), a classical human acute-phase plasma protein, is not only a sensitive systemic inflammatory marker but also an independent risk predictor of cardiovascular diseases. However, existing heterologous expression systems for expressing CRP is not efficient and cost-effective for large-scale industrial production of CRP to meet the growing market demand for CRP. This study aims to improve the secretion of recombinant CRP by Pichia pastoris via optimizing signal peptides, promoters and carbon sources. The CRP genes with encoding four different signal peptides were designed and synthesized. The genes were cloned into pPICZαA or pPICZ B, respectively via splicing by overlap extension polymerase chain reaction (SOE-PCR) technology and expressed in P. pastoris X-33, regulated by the alcohol oxidase I promoter (pAOX1). The CRP led by the α-mating factor secretion signal peptide (α-MF) was secreted at the highest level in these signal peptides. Then, a constitutive construct and expression of the CRP genes were achieved by switching to the glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). Subsequently, different carbon sources and at different concentrations were used to further improve the secretion of CRP. The expression of CRP with the α-MF driven by the pGAP gave the highest yield of secreted CRP, about 3mg/l of culture on the optimized culture conditions. The purified recombinant CRP exhibited good immunoreactivity determined by ELISA with anti-human CRP monoclonal antibody. The efficient engineering strategy established in this work might provide potential uses in large-scale industrial production of human CRP in the future.

Graphene-based rapid and highly-sensitive immunoassay for C-reactive protein using a smartphone-based colorimetric reader

A novel immunoassay (IA) has been developed for human C-reactive protein (CRP), an important biomarker and tissue preserving factor for infection and inflammation. Graphene nanoplatelets (GNP) and 3-aminopropyltriethoxysilane (APTES) were admixed and covalently attached to a polystyrene based-microtiter plate (MTP), pretreated with KOH. The resulting surface served as a stable layer for the covalent attachment of the anti-human CRP antibody. The IA procedure was based on the one-step kinetics-based sandwich IA employing a minimum number of process steps, whereas the enzymatic reaction solution was monitored by a smartphone-based colorimetric reader. With a limit of detection and a limit of quantification of 0.07ngmL−1 and 0.9ngmL−1, it precisely detected CRP spiked in diluted human whole blood and plasma as well as the CRP levels in clinical plasma samples. The results obtained for “real-world” patient samples agreed well with those of the conventional immunosorbent assay and the clinically-accredited analyzer-based IA. The antibody-bound GNP-functionalized MTPs retained its original activity after 6 weeks of storage in 0.1M PBS, pH 7.4 at 4°C.

Detection of the inflammation biomarker C-reactive protein in serum samples: towards an optimal biosensor formula.

The development of an electrochemical immunosensor for the biomarker, C-reactive protein (CRP), is reported in this work. CRP has been used to assess inflammation and is also used in a multi-biomarker system as a predictive biomarker for cardiovascular disease risk. A gold-based working electrode sensor was developed, and the types of electrode printing inks and ink curing techniques were then optimized. The electrodes with the best performance parameters were then employed for the construction of an immunosensor for CRP by immobilizing anti-human CRP antibody on the working electrode surface. A sandwich enzyme-linked immunosorbent assay (ELISA) was then constructed after sample addition by using anti-human CRP antibody labelled with horseradish peroxidase (HRP). The signal was generated by the addition of a mediator/substrate system comprised of 3,3,5',5'-Tetramethylbenzidine dihydrochloride (TMB) and hydrogen peroxide (H2O2). Measurements were conducted using chronoamperometry at −200 mV against an integrated Ag/AgCl reference electrode. A CRP limit of detection (LOD) of 2.2 ng·mL−1 was achieved in spiked serum samples, and performance agreement was obtained with reference to a commercial ELISA kit. The developed CRP immunosensor was able to detect a diagnostically relevant range of the biomarker in serum without the need for signal amplification using nanoparticles, paving the way for future development on a cardiac panel electrochemical point-of-care diagnostic device.

One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min

This article reveals a rapid sandwich enzyme-linked immunosorbent assay (ELISA) for the highly sensitive detection of human C-reactive protein (CRP) in less than 30min. It employs a one-step kinetics-based highly simplified and cost-effective sandwich ELISA procedure with minimal process steps. The procedure involves the formation of a sandwich immune complex on capture anti-human CRP antibody-bound Dynabeads in 15min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81ngml−1 with a limit of detection of 0.4ngml−1 and an analytical sensitivity of 0.7ngml−1. It detects CRP spiked in diluted human whole blood and serum with high analytical precision, as confirmed by conventional sandwich ELISA. Moreover, the results of the developed ELISA for the determination of CRP in the ethylenediaminetetraacetic acid plasma samples of patients are in good agreement with those obtained by the conventional ELISA. The developed immunoassay has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

Relaxation of biofunctionalized magnetic nanoparticles in ultra-low magnetic fields

In this work, the spin-spin relaxation rate, 1/T2, and spin-lattice relaxation rate, 1/T1, of protons’ spins induced by biofunctionalized magnetic nanoparticles and ferrofluids are investigated using a high-Tc superconducting quantum interference device-detected magnetometer in ultra-low fields. The biofunctionalized magnetic nanoparticles are the anti-human C-reactive protein (antiCRP) coated onto dextran-coated superparamagnetic iron oxides Fe3O4, which is labeled as Fe3O4-antiCRP. The ferrofluids are dextran-coated iron oxides. It was found that both 1/T2 and 1/T1 of protons in Fe3O4-antiCRP are enhanced by the presence of magnetic nanoparticles. Additionally, both the 1/T1 and 1/T2 of Fe3O4-antiCRP are close to that of ferrofluids, which are dextran-coated Fe3O4 dispersed in phosphate buffer saline. Characterizing the relaxation of Fe3O4-antiCRP can be useful for biomedical applications.

Factor H-related Protein 4 Activates Complement by Serving as a Platform for the Assembly of Alternative Pathway C3 Convertase via Its Interaction with C3b Protein

Human complement factor H-related protein (CFHR) 4 belongs to the factor H family of plasma glycoproteins that are composed of short consensus repeat (SCR) domains. Although factor H is a well known inhibitor of the alternative complement pathway, the functions of the CFHR proteins are poorly understood. CFHR4 lacks SCRs homologous to the complement inhibitory domains of factor H and, accordingly, has no significant complement regulatory activities. We have previously shown that CFHR4 binds C-reactive protein via its most N-terminal SCR, which leads to classical complement pathway activation. CFHR4 binds C3b via its C terminus, but the significance of this interaction is unclear. Therefore, we set out to clarify the functional relevance of C3b binding by CFHR4. Here, we report a novel role for CFHR4 in the complement system. CFHR4 serves as a platform for the assembly of an alternative pathway C3 convertase by binding C3b. This is based on the sustained ability of CFHR4-bound C3b to bind factor B and properdin, leading to an active convertase that generates C3a and C3b from C3. The CFHR4-C3bBb convertase is less sensitive to the factor H-mediated decay compared with the C3bBb convertase. CFHR4 mutants containing exchanges of conserved residues within the C-terminal C3b-binding site showed significantly reduced C3b binding and alternative pathway complement activation. In conclusion, our results suggest that, in contrast to the complement inhibitor factor H, CFHR4 acts as an enhancer of opsonization by promoting complement activation.

Immune Responses to HSP65/60 in Periodontal Disease

Chronic periodontitis (CP) is a chronic inflammatory condition which destroys the supporting tissues of teeth and increases in prevalence with age. Immune responses against heat shock proteins (HSP) can be cross-reactive among bacterial and human antigens. There is evidence that microbial HSP65 and human HSP60 are involved in periodontal disease. The aim of this study is to investigate immune responses to the human HSP60 and microbial HSP65 in patients with CP and relate these to the level of inflammation and smoking status. We collected serum samples from 30 patients with chronic gingivitis (CG) and 30 patients with CP. In each group, eight subjects were current smokers. ELISA was used to determine the levels of serum anti-HSP and C-reactive protein (CRP) in each group. Peripheral blood mononuclear cells were also isolated and stimulated with HSPs. Significant lymphoproliferation was seen in CP when stimulated with human HSP60. CRP and serum anti-human HSP60 IgG were elevated in CP compared to the CG, but not serum anti-microbial HSP 65 IgG. In view of the potential confounding effects of smoking in CP, a group of current smokers (n = 16) was also recruited to investigate whether smoking affects HSP immune responses. There was no significant difference in HSP-induced lymphoproliferation between smokers and non-smokers in either the CG or CP. There was a significant correlation between CRP and lymphoproliferative responses to Human HSP60 irrespective of smoking status. This study shows that serum anti-human HSP60 IgG and serum CRP are raised in untreated CP. In CP, serum CRP levels correlated significantly with human HSP60-induced lymphoproliferation, but not with anti-HSP antibody levels.

Study of CRP immobilization on nanostructured silicon

Abstract C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein, which is widely used as an indicator of inflammatory states due to rapid increase of its plasma concentration up to 1000 times compared to normal values. Detection of CRP levels in a rapid, simultaneous and multiplex format is therefore of great interest for diagnostics. Microarray technology could provide such a multiplex format of CRP levels detection. Different nanostructured porous silicon (PS) surfaces were obtained and used for the immobilization of CRP and anti-human CRP antibodies in order to achieve an optimum microarray assay. Comparative analysis of the attachment degree and preservation of the biomolecules activity on the silicon surfaces and functionalized glass slides is also described.

Anti C-Reactive Protein Immobilization Onto Poly(glycidyl Methacrylate) Microspheres and its Use for Diagnosis

A method for a polymeric carrier based test was developed for the diagnosis of C-reactive protein (CRP) as an alternative to commercial kits. Poly(glycidyl methacrylate) (PGMA) particles were synthesized by dispersion polymerization method from the glycidyl methacrylate (GMA) monomer. The monomer transformation value was 88.67% by weight. In the first step the epoxide rings were opened to form amine groups on the particle surfaces. The optimized conditions were found for the antigen to be immobilized onto the surface-modified microspheres. Agglutination tests were carried out under in vitro conditions with the anti-human CRP immobilized microspheres with samples from patient serum. Relatively high sensitivity was obtained with a short reaction time between doing the test after receiving patient serum compared with the selected commercial kit now in use. By referring to the agglutination intensity scores, using only 10 mg/L samples of plasma, the CRP concentration was detectable with these new kits.

Fiber optic biosensor for the detection of C-reactive protein and the study of protein binding kinetics

Application of a fiber optic biosensor (FOB) to the real-time investigation of the interaction kinetics between FITC-conjugated monoclonal sheep anti-human C-reactive protein (CRP) antibody and CRP isoforms on the surface of optical fiber is described. Recently, both the native pentameric CRP (pCRP), an acute phase protein belonging to pentraxin family, and an isoform of pCRP, modified CRP (mCRP), have been suggested to have proinflammation effects on vascular cells in acute myocardial infarction (AMI). In current studies, we generate mCRP from pCRP, and use several methods including fluorescence spectral properties, circular dichroism, analytical ultracentrifuge, and Western blotting to demonstrate their differences in physical and chemical properties as well as the purity of pCRP and mCRP. In addition, we design and implement an FOB to study the real-time qualitative and quantitative biomolecular recognition of CRP isoforms. Specifically, the association and dissociation rate constants of the reaction between FITC-conjugated monoclonal sheep anti-human CRP antibody and the pCRP and mCRP are determined. The feasibility of our current approach to measure the association and dissociation rate constants of the reaction between tested CRP isoforms was successfully demonstrated.

Microvascular Function, Metabolic Syndrome, and Novel Risk Factor Status in Women With Cardiac Syndrome X

To characterize microvascular function, candidate risk pathways, and metabolic syndrome prevalence in women with cardiac syndrome X, 52 nondiabetic women with angiographically normal epicardial arteries but >1 mm of planar ST depression during exercise testing (patients) and 24 healthy controls of similar age were recruited. In addition to fasting blood samples and anthropometric measurements, forearm cutaneous microvascular function after iontophoresis of acetylcholine and sodium nitroprusside was assessed by laser Doppler imaging. Despite body mass index correction and a larger proportion on statin therapy, patients had high levels of insulin (p=0.016), triglycerides (p=0.018), intercellular adhesion molecule-1 (p=0.021), von Willebrand factor (p=0.005), and leptin (p=0.005) and lower levels of high-density lipoprotein cholesterol (p=0.042) compared with controls. Consistent with these data, 30% of patients but only 8% of controls fulfilled criteria for the metabolic syndrome as defined by the National Cholesterol Education Program (p=0.015). Endothelium-dependent and -independent microvascular functions were markedly impaired in patients (p<0.001), and the odds ratio for cardiac syndrome X was 7.38 (95% confidence interval 2.2 to 24.7) if the acetylcholine response was <8,710 flux units. In conclusion, women with cardiac syndrome X more commonly have metabolic syndrome and related adiposity, metabolic, and inflammatory derangements. They also have significantly impaired skin microvascular function as assessed by laser Doppler imaging, consistent with generalized vascular dysfunction, a finding with potential diagnostic implications.

Plasma concentration of C-reactive protein and risk of ischemic stroke and transient ischemic attack: the Framingham study.

The role of C-reactive protein (CRP) as a novel plasma marker of atherothrombotic disease is currently under investigation. Previous studies have mostly related CRP to coronary heart disease, were often restricted to a case-control design, and failed to include pertinent risk factors to evaluate the joint and net effect of CRP on the outcome. We related plasma CRP levels to incidence of first ischemic stroke or transient ischemic attack (TIA) in the Framingham Study original cohort. There were 591 men and 871 women free of stroke/TIA during their 1980 to 1982 clinic examinations, when their mean age was 69.7 years. CRP levels were measured by using an enzyme immunoassay on previously frozen serum samples. Analyses were based on sex-specific CRP quartiles. Risk ratios (RRs) were derived, and series of trend analyses were performed. During 12 to 14 years of follow-up, 196 ischemic strokes and TIAs occurred. Independent of age, men in the highest CRP quartile had 2 times the risk of ischemic stroke/TIA (RR=2.0, P=0.027), and women had almost 3 times the risk (RR=2.7, P=0.0003) compared with those in the lowest quartile. Assessment of the trend in risk across quartiles showed unadjusted risk increase for men (RR=1.347, P=0.0025) and women (RR=1.441, P=0.0001). After adjustment for smoking, total/HDL cholesterol, systolic blood pressure, and diabetes, the increase in risk across CRP quartiles remained statistically significant for both men (P=0.0365) and women (P=0.0084). Independent of other cardiovascular risk factors, elevated plasma CRP levels significantly predict the risk of future ischemic stroke and TIA in the elderly.

Trypanosoma cruzi: Detection of a Surface Antigen Cross-Reactive to Human C-Reactive Protein

Coutinho, C. M. L. M., Cavalcanti, G. H., Bonaldo, M. C., Mortensen, R. F., and Araújo-Jorge, T. C. 1998.Trypanosoma cruzi: Detection of a surface antigen cross-reactive to human C-reactive protein.Experimental Parasitology90, 143–153. C-reactive protein (CRP) is an acute phase protein secreted by liver hepatocytes, and is also found on the surface of lymphocytes and as a membrane-associated protein expressed on rat liver macrophages and human monocytes. C-reactive protein levels increase in the sera of children infected withTrypanosoma cruzi, during the acute phase of Chagas' disease, but its role in the course of this infection is unknown. Experiments designed to detect the binding of CRP to circulating forms ofT. cruzifailed to observe it because anti-human CRP antibodies bind to the parasite. The present work intended to further clarify this novel question related to the anti-CRP cross-reactivity with the parasite. Indirect immunofluorescence, immunoenzymatic, flow cytometry, and Western blot assays showed that three different polyclonal anti-human CRP antibody preparations bind toT. cruzisurface. This binding is dose-dependent, saturable, and is inhibited when anti-CRP antibodies from different species were allowed to compete, indicating the specificity of the reactivity. The antibodies recognized a protein band below 23 kDa in Western blot analysis of parasite extracts. The divalent cation chelators EDTA and EGTA impaired the antigen recognition by the antibodies. The binding to parasite surface was also observed with some available monoclonal antibodies raised against human CRP. A polyclonal anti-human CRP presented an inhibitory effect on invasion of heart muscle cells byT. cruzi. Our results indicate that a molecule antigenically related to CRP, a possible CRP-like molecule, is expressed on the surface ofT. cruzi.

Development of monkey C-reactive protein (CRP) assay methods

Monkey-specific C-reactive protein (CRP) assay methods (enzyme-linked immunosorbent assay (ELISA) and turbidimetric immunoassay (TIA)) were developed. The anti-monkey CRP serum was prepared by immunization of rabbits with the immune complex formed between the acute-phase serum from turpentine oil-inoculated monkeys and goat anti-human CRP serum. The specificity of the rabbit anti-monkey CRP serum was confirmed by immunoelectrophoresis and Western blotting. The purity of monkey CRP prepared by chromatography procedures was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The serum CRP levels in nine normal monkeys, as measured by sandwich ELISA were ranged from 0.26 to 1.42 μg/ml (mean 0.71±0.37). The CRP levels in five acute-phase sera of turpentine oil-inoculated monkeys were 248–451 μg/ml (mean 371.2±73.8). This monkey-specific CRP assay method was found more sensitive than the human-specific CRP assay method in detecting monkey CRP by TIA.

Immunohistochemical localization of C‐reactive protein‐binding sites in human atherosclerotic aortic lesions by a modified streptavidin‐biotin‐staining method

One-step fluorescein-conjugated polyclonal antibody technique has shown that C-reactive protein (CRP) was located only extracellularly in human atherosclerotic lesions. In this report a more sensitive streptavidin-biotin technique was applied to detect the localization of CRP in human atherosclerotic lesions. Immunohistochemical staining with polyclonal and monoclonal anti-human CRP antibodies both produced a brown color extracellularly in the necrotic lesions, and intracellularly in CD68+ foam cells. The latter suggests an uptake of CRP-lipid complexes by macrophages. The staining is human CRP-specific because it was eliminated by preabsorption of the monoclonal antibody with pure human CRP, or by substitution of the primary antibody with non-immune rabbit serum. By overlaid CRP-binding study, a positive stain was observed on intimal smooth muscle cells and foam cells, suggesting that they have CRP-binding sites unless the CRP-binding activity was generated de novo through the fixation procedure. Accordingly, it is hypothesized that CRP may facilitate the uptake of lipids by macrophages accumulating in atherosclerotic lesions. Further, CRP might participate in cytolysis, which enlarges the necrotic area, and/or in phagocytosis that scavenges the necrotic tissue.

C-Reactive Protein Induces Human Peripheral Blood Monocytes to Synthesize Tissue Factor

AbstractThe acute inflammatory response is frequently accompanied by serious thrombotic events. We show that C-reactive protein (CRP), an acute-phase reactant that markedly increases its serum concentration in response to inflammatory stimuli, induced monocytes to express tissue factor (TF), a potent procoagulant. Purified human CRP in concentrations commonly achieved in vivo during inflammation (10 to 100 µg/mL) induced a 75-fold increase in TF procoagulant activity (PCA) of human peripheral blood mononuclear cells (PBM), with a parallel increase in TF antigen levels. CRP-induced PCA was completely blocked by a monoclonal antibody against human TF but not by irrelevant murine IgG. Dot blot analysis showed a significant increase of TF mRNA after 4 hours of incubation with CRP, followed by a peak of PCA within 6 and 8 hours. Actinomycin D and cycloheximide blocked CRP-stimulated PCA, suggesting that de novo TF protein synthesis was required. Endotoxin (LPS) contamination of CRP was excluded as the mediator of TF synthesis because: (1) CRP was Limulus assay negative; (2) induction of TF PCA by CRP was not blocked by Polymyxin B, in contrast to LPS-induced PCA; (3) antihuman CRP IgG inhibited CRP-induced PCA, but not LPS-induced PCA; (4) CRP was able to stimulate TF production in LPS-pretreated PBM refractory to additional LPS stimulation; and, (5) unlike LPS, CRP was incapable of inducing TF in human umbilical vein endothelial cells. We suggest that CRP-mediated TF production in monocytes may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states.