Recombinant antibody fragments allow repeated measurements of C-reactive protein with a quartz crystal microbalance immunosensor

C-reactive protein (CRP) is a serum marker highly upregulated in inflammation after bacterial infection. Robust, reliable and quick quantification of CRP would be a substitute for erythrocyte sedimentation rate (ESR) with superior diagnostic value. Quartz crystal microbalance (QCM) based sensors coated with specific antibodies and integrated into lab-on-chip systems are in development for rapid point of care quantification. In this study, we isolated three CRP specific single chain (sc)Fv antibody fragments using phage display from an antibody gene library. Their affinities ranged from 2.7 × 10^?8 to 1.0 × 10^?8 M when measured by surface plasmon resonance. ScFv antibody fragment LA13-IIE3 showed best affinity, high long-term stability and remarkable resistance to denaturation. This scFv antibody fragment was coupled to a QCM sensor. CRP quantification in up to 15 samples sequentially measured on the same sensor with intermitting regeneration by buffer was demonstrated.     

C-反应蛋白对脂肪细胞脂联素表达的影响

通过体外培养脂肪细胞,研究C-反应蛋白（CRP）对大鼠脂肪细胞脂联素蛋白分泌及mRNA基因表达的影响。取大鼠附睾脂肪垫培养脂肪细胞。用0、10、50ug／mL的CRP刺激脂肪细胞6h,提取细胞RNA,用实时荧光定量RT-PCR技术检测脂联素mRNA表达的变化;收集细胞培养液,运用Western blot技术检测脂联素蛋白分泌的变化。结果显示0、10、50ug／mL的CRP对大鼠脂肪细胞脂联素mRNA表达的影响无差异（P〉0．05）。50、10ug／mL的CRP均可减少大鼠脂肪细胞培养液中脂联素蛋白的分泌量（P〈0．05）。CRP可呈剂量依赖性的降低脂肪细胞脂联素蛋白分泌的水平。而各组CRP未能影响脂肪细胞脂联素mRNA的表达。CRP对脂肪细胞脂联素基因表达和蛋白分泌的研究可以揭示转录后的控制决定了CRP对脂联素的影响。     

Study of the spontaneous dissociation of rabbit C-reactive protein

C-Reactive protein (CRP) is composed of five identical noncovalently linked monomers and characterized as an important acute-phase protein. The CRP subunit obtained by denaturing treatments, which is termed modified CRP, has also been widely studied. In the current work, we found that there exists some degree of natural dissociation of CRP in stock solution. This dissociation is critically dependent on the absence of Ca²⁺. Low pH could enhance the dissociation of CRP, while ionic strength has little effect. Anilinonaphthalenesulfonate (ANS) fluorescence detections indicate that the exposure of hydrophobic surface increases during the dissociation. Acidic pH conditions also induce an increase in ANS fluorescence. This suggests that hydrophobic interactions between CRP subunits may contribute to the study of its pentameric structure. Surface plasmon resonance experiments indicate that monomeric CRP does not specifically bind to phosphatidylcholine-containing membrane as native CRP does. Electron microscopy shows that monomeric CRP binds to negatively charged lipid through electrostatic forces, and such lipid may induce the dissociation of CRP due to the acidic pH in the diffuse double layer near the membrane.     

Membrane activity of a C-reactive protein

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca²⁺. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein’s role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens.     

一种定量检测人血清高敏C反应蛋白的化学发光免疫方法

旨在建立一种可定量检测人血清高敏CRP的化学发光检测方法 (High-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay,hs-CRP CLIA)。首先利用亲和层析和离子交换层析技术从肝硬化病人腹水中纯化出高纯度的天然CRP作为免疫原制备了22株CRP单克隆抗体 (单抗),其中13株单抗在磷酸胆碱配体捕获ELISA中呈阳性,然后利用方正滴定法筛选出单抗10C5和10C11建立了hs-CRP CLIA。试剂盒评估结果显示：该方法对血清中干扰物质IgG、血红蛋白、甘油三酯等无非特异性反应;该方法检测灵敏度高,在0.04~20.38 mg/L范围内定量检测人血清CRP标准品呈良好线性关系 (R2＞0.993);该方法准确性高、可重复性好,平均回收率为99％,批内差为4.2％~5.8％,批间差为9.0％~11.5％;该方法与进口商品化高敏CRP ELISA试剂盒平行比较检测90份血清标本,结果显示两者有良好的可比性 (r=0.968)。综上,建立的hs-CRP CLIA是一种准确、可靠、可定量的高灵敏C反应蛋白检测方法,该方法的临床应用,有利于改善我国心脏病风险评估及肠炎性疾病预后判断。     

Site-directed immobilisation of antibody fragments for detection of C-reactive protein

C-reactive protein, CRP antibody Fab'-fragments have been attached on pre-cleaned gold slides and protein repellent polymers have been used to block the remaining free space in between the antibody fragments. At optimal conditions the antibody fragments are site-directly immobilised on the surface and non-specific binding is reduced. The amount of Fab'-fragments in the polymer host monolayer has been optimised for various buffers. Binding of CRP to Fab'-fragment/polymer layers produced in phosphate buffered saline decreased with NaCl salt concentration. In a 1M NaCl phosphate buffer, the antibodies seem to be randomly oriented on the surface with a similar response to CRP as that of an antibody F(ab)(2)-fragment layer. In a 150 mM NaCl phosphate buffer, on the other hand, the fragments seem to be site-directly oriented and the response to CRP was fivefold. The highest response to CRP was obtained to a layer with a Fab'-fragment concentration of 60 microg/ml. CRP could be detected in a concentration range of 1 ng/ml to 50 microg/ml from a standard solution in phosphate buffer and in a range of 4 ng/ml to 50 microg/ml from serum/PBS. CRP was, moreover, successfully detected in patient samples with good reproducibility. The layer would thus be sensitive enough to analyse the CRP concentration in human serum for predicting cardiovascular disease.     

Expression of membrane-associated C-reactive protein by human monocytes: indications for a selectin-like activity participating in adhesion

We have shown previously that rat liver macrophages (Kupffer cells) express a membrane-bound form of C-reactive protein (mCRP) on their surface which is identical to a galactose-specific particle receptor activity. We now establish the presence of mCRP on human monocyte-macrophages using immunocytochemistry with an anti-neoCRP specific monocloncal antibody and RNA-RNAin situ hybridization to demonstrate the presence of CRP-specific mRNA. Concomitant with mCRP expression, cells exhibit galactose-dependent uptake of particles coated with lactosylated bovine serum albumin. Adhesion experiments on fibronectin-coated surfaces that mCRP on human blood monocytes may act as a selectin-like adhesion molecule, mediating initial carbohydrate-specific contacts which are followed by peptide-specific recognition via integrin receptors.     

动态监测C-反应蛋白及降钙素原在创伤后脓毒血症中的价值

目的 探讨C-反应蛋白及降钙素原在创伤后脓毒血症中的动态变化及对预后的评估价值。方法 选择诸暨市中心医院创伤后脓毒血症患者78例作为研究对象,比较脓毒血症组与对照组之间血清CRP及PCT水平的差异。根据脓毒血症的不同程度将入选的创伤后脓毒血症病例分为单纯脓毒血症、脓毒血症合并器官功能衰竭及感染性休克组,比较三组患者血清CRP水平、血清PCT水平及APACHE-II评分的差异。根据不同的随访结果将脓毒血症患者分为好转、无效及病死组,比较三组入选当日、入选后第3、5天血清CRP及PCT水平的变化。结果 入选当日,脓毒血症组患者血清CRP及PCT水平均明显高于对照组,差异具有统计学意义（t=68.472、40.243,P0.05）,其余两组间比较差异均有统计学意义（P0.05）;血清PCT水平入选当日、入选后第3天、第5天呈逐渐下降趋势,组间比较差异均有统计学意义（P0.05）,而血清PCT水平呈递增趋势,组间比较差异均有统计学意义（P0.05）。结论 血清CRP及PCT水平均能反应创伤后脓毒血症的感染状态,但在评估病情及预后方面,血清PCT更具有优势。     

C-reactive protein functions as a negative regulator of macrophage activation induced by apoptotic DNA

C-reactive protein (CRP), an acute-phase protein with an ability to bind to nuclear antigen, has been reported to regulate cytokine secretion and modulate immune responses. We previously reported that activated syngeneic lymphocyte-derived apoptotic DNA (apopDNA) could induce macrophage activation and contribute to the initiation and progression of lupus nephritis. It is reasonable to hypothesize that CRP might regulate apopDNA-induced macrophage activation. Herein, CRP was shown to promote macrophage-mediated apopDNA uptake by binding to apopDNA (CRP/apopDNA complex). Notably, CRP/apopDNA treatment inhibited the production of inflammatory cytokines and chemokines by macrophages which could be induced by apopDNA alone. Further coculture and transwell studies revealed that CRP/apopDNA-induced macrophages prohibited apopDNA-induced macrophage activation in an IL-10 dependent manner. These results provide insight into the potential mechanism of CRP regulatory activity in macrophage activation induced by apopDNA in the context of lupus nephritis and other autoimmune diseases.     

Quantitative measurements of C-reactive protein using silicon nanowire arrays

A silicon nanowire-based sensor for biological application showed highly desirable electrical responses to either pH changes or receptor-ligand interactions such as protein disease markers, viruses, and DNA hybridization. Furthermore, because the silicon nanowire can display results in real-time, it may possess superior characteristics for biosensing than those demonstrated in previously studied methods. However, despite its promising potential and advantages, certain process-related limitations of the device, due to its size and material characteristics, need to be addressed. In this article, we suggest possible solutions. We fabricated silicon nanowire using a top-down and low cost micromachining method, and evaluate the sensing of molecules after transfer and surface modifications. Our newly designed method can be used to attach highly ordered nanowires to various substrates, to form a nanowire array device, which needs to follow a series of repetitive steps in conventional fabrication technology based on a vapor-liquid-solid (VLS) method. For evaluation, we demonstrated that our newly fabricated silicon nanowire arrays could detect pH changes as well as streptavidin-biotin binding events. As well as the initial proof-of-principle studies, C-reactive protein binding was measured: electrical signals were changed in a linear fashion with the concentration (1 fM to 1 nM) in PBS containing 1.37 mM of salts. Finally, to address the effects of Debye length, silicon nanowires coupled with antigen proteins underwent electrical signal changes as the salt concentration changed.     

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 30 min. 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 15 min, followed by two magnet-assisted washings and one enzymatic reaction. The developed sandwich ELISA detects CRP in the dynamic range of 0.3 to 81 ng ml⁻¹ with a limit of detection of 0.4 ng ml⁻¹ and an analytical sensitivity of 0.7 ng ml⁻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.     

阿维菌素中毒患者血清C反应蛋白的浓度变化及意义

目的探讨口服阿维菌素中毒患者血清C反应蛋白浓度变化与病情严重程度及预后的关系。方法将39例口服阿维菌素中毒患者分为轻度中毒组和中重度中毒组,42例健康体检者为对照组。轻、中重度中毒组患者分别于入院时及入院第3、5天检测血清C反应蛋白浓度,并进行组间比较。结果中毒组血清CRP水平明显高于对照组,差异有统计学意义（P〈0.05）;轻度中毒组血清CRP水平在入院时及入院第3、5天变化较小,各时点血清CRP水平比较无显著统计学意义（P〉0.05）;中重度中毒组血清CRP水平在入院时即显著升高,入院第3天达最高值,各时点血清CRP水平比较差异有统计学意义（P〈0.05）。结论血清C反应蛋白浓度可作为判断口服阿维菌素中毒患者病情严重程度及预后的敏感指标。     

The role of C-reactive protein in ischemia/reperfusion injury and preconditioning in a rat model of myocardial infarction

For the first time the involvement of C-Reactive protein (CRP) in early (acute) and delayed ischemic (IPC) and pharmacological (chemical) preconditioning (CPC) in an in vivo model of rat myocardial infarction was presented. Acute IPC was produced by three 5 minute occlusion (ischemia) periods interspersed with 5 minute reperfusion, followed by 30 minute occlusion of the left coronary artery and 2 hour reperfusion injury. Acute CPC was produced by a k-opioid receptor agonist U50488H (5 mg/kg) applied i.v. 15 minutes before 30 minute ischemia/ 2 hour reperfusion. Delayed preconditioning was produced by 30 minute ischemia/ 2 hour reperfusion, induced 24 hour after either ischemic or pharmacological preconditioning. The myocardial ischemia/reperfusion injury was evaluated on the basis of total and cardiac creatine kinase isoenzyme activity, functional recovery of the heart (ECG), infarct size (% IS/RA) and mortality at the end of the experiments. The results obtained showed that: k-opioid receptor agonist U50488H mimics both the acute and delayed IPC in the above experimental protocol; Both acute IPC and most probably CPC act by opening of K(ATP) channels (the effects were blocked by nonspecific ATP-sensitive K channel blocker glybenclamide), and via activation of protein kinase C (a selective protein kinase C inhibitor chelerythrine blocked the efects); C-reactive protein (CRP) was significantly elevated by 54% in non-preconditioned acute ischemia/reperfusion injury. The elevation was more pronounced (82% increase) 24 hour after non-preconditioned ischemia/reperfusion injury. It reflected very well the increase in cardiac isoenzymes, infarct size and mortality of the rats, and can be used as a marker of the severity of myocardial injury in this model; The increase of CRP was prevented by both IPC and CPC in early, and especially in late preconditioning. This confirms the involvement of CRP as a marker in cardiac ischemic/reperfusion injury. It was concluded that in addition to the established involvement of adenosine, bradykinin, opioid and other receptors, a suppression of myocardial CRP/complement production might be involved in the biological mechanism of preconditioning. This could be a promising perspective in clinical interventions against ischemia/reperfusion injuries of the heart.     

Therapeutic effects of a synthetic peptide of C-reactive protein in pre-clinical tumor models

Previous studies have shown that multilamellar vesicles (MLV) or other carriers containing purified human C-reactive protein (CRP) have therapeutic activity in preclinical tumor models. Here we evaluated the therapeutic effects of MLV containing novel synthetic peptides, derived from the structure of CRP, on the extent of (a) established lung metastases of fibrosarcoma T241 in C57B1/6 mice, (b) survival of C57B1/6 mice bearing established liver metastases of colon carcinoma MCA-38, and (c) primary tumor growth of Renca renal carcinoma in Balb/c mice. In all cases, a single synthetic CRP peptide, RS-83277, demonstrated significant antitumor effects comparable to that seen with intact CRP. Two other synthetic CRP peptides, RS-83287 and RS-83147, showed no therapeutic activity and were comparable to control MLV containing only buffer. None of the peptides contained sequences homologous with that of the phagocyte stimulant, tuftsin. Activity of MLV-encapsulated RS-83277 was dose-dependent, and a comparable dose of the soluble peptide, given either alone or following injection of buffer-MLV, was ineffective. These results demonstrate immunotherapeutic potential for a novel synthetic peptide derived from CRP, and endogenous acute-phase protein.This work was supported in part by grant CA 49950 from the National Cancer Institute, and grant 43618 from the National American Cancer Society     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Two-photon excitation fluorometric measurement of homogeneous microparticle immunoassay for C-reactive protein

Recent developments in infrared laser technology have enabled the design of a compact instrumentation for two-photon excitation microparticle fluorometry (TPX). The microparticles can be used in immunoassays as the antibody-coated solid phase to capture an antigen and then detect it with a fluorescently labeled tracer antibody. Unlike most other methods, TPX technology allows low-volume, homogeneous immunoassays with real-time measurements of assay particles in the presence of a moderate excess of fluorescent tracer. In this study, the TPX assay system was used for the reagent characterization and the measurement of C-reactive protein (CRP) in diluted plasma samples, targeting the assay range useful in infectious disease diagnosis. The pentameric structure of the CRP permitted the optimization of an assay with the lowest detectable concentration of 1 microg/L (7.5 pM) by using a single monoclonal antibody both for capture and as the tracer. With a 1:200 predilution of samples, the measurement range of the assay was 1-150 mg/L, but an additional 1:10 dilution was required for higher concentrations. The TPX method showed a good correlation with the reference result obtained in a routine hospital laboratory, demonstrating the feasibility of the technology for immunodiagnostic applications.     

In vitro selection of a DNA aptamer targeted against Shigella dysenteriae

To identify DNA aptamers demonstrating binding specificity for Shigella dysenteriae, a whole-bacterium Systemic Evolution of Ligands by Exponential enrichment (SELEX) method was applied to a combinatorial library of single-stranded DNA (ssDNA) molecules. After several rounds of selection using S. dysenteriae as the target, the highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homologies and similarities in the secondary structures. Aptamer S 1, which showed particularly high binding affinity in preliminary studies, was chosen for further characterisation. This aptamer displayed a dissociation constant (K_d value) of 23.47 ± 2.48 nM. Binding assays to assess the specificity of aptamer S 1 showed high binding affinity for S. dysenteriae and low apparent binding affinity for other bacteria. The ssDNA aptamers generated may serve as a new type of molecular probe for microbial pathogens, as it has the potential to overcome the tedious isolation and purification requirements for complex targets.     

Very low C-reactive protein in apparently healthy individuals: physiological status or just a reflection of an improved health profile

Abstract

The objective of our study was to determine whether the very low concentrations of C-reactive protein (CRP) detected by high-sensitivity CRP (hs-CRP) assays that one encounters from time to time in apparently healthy individual represent a physiological status or are just a reflection of an improved general health profile. The concentration of hs-CRP was determined by using the Behring BN II nephelometer. The arbitrary cut-off point of hs-CRP (≤0.16mgl^?1) was determined at the lower detection level of the assay. A total of 6588 apparently healthy individuals were screened following exclusion of recent infection/inflammation by using a detailed questionnaire. One hundred and sixty (2.4%) individuals out of the above-mentioned cohort presented hs-CRP concentrations of ≤0.16mgl^?1. They were found to be significantly younger and lean, had an improved lipid profile and an attenuated acute-phase response in terms of lower erythrocyte sedimentation rate and fibrinogen concentration as well as white blood cell count. In addition, these individuals had less atherothrombotic risk factors, except for smoking habits which were as frequent as in those of individuals with a higher hs-CRP concentration. After calculating the concentration of this biomarker following multiple adjustments, the individuals with very low CRP remained with a very low value despite the multiplicity of the adjustments. We raise the possibility that this particular low concentration might represent a physiological status and is not necessarily a result of the improved general health profile per se.