Development and Evaluation of Europium-Based Quantitative Lateral Flow Immunoassay for the Chronic Kidney Disease Marker Cystatin-C.

This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.

Modulation of Cystatin C in Human Macrophages Improves Anti-Mycobacterial Immune Responses to Mycobacterium tuberculosis Infection and Coinfection With HIV.

Tuberculosis owes its resurgence as a major global health threat mostly to the emergence of drug resistance and coinfection with HIV. The synergy between HIV and Mycobacterium tuberculosis (Mtb) modifies the host immune environment to enhance both viral and bacterial replication and spread. In the lung immune context, both pathogens infect macrophages, establishing favorable intracellular niches. Both manipulate the endocytic pathway in order to avoid destruction. Relevant players of the endocytic pathway to control pathogens include endolysosomal proteases, cathepsins, and their natural inhibitors, cystatins. Here, a mapping of the human macrophage transcriptome for type I and II cystatins during Mtb, HIV, or Mtb-HIV infection displayed different profiles of gene expression, revealing cystatin C as a potential target to control mycobacterial infection as well as HIV coinfection. We found that cystatin C silencing in macrophages significantly improves the intracellular killing of Mtb, which was concomitant with an increased general proteolytic activity of cathepsins. In addition, downmodulation of cystatin C led to an improved expression of the human leukocyte antigen (HLA) class II in macrophages and an increased CD4+ T-lymphocyte proliferation along with enhanced IFN-γ secretion. Overall, our results suggest that the targeting of cystatin C in human macrophages represents a promising approach to improve the control of mycobacterial infections including multidrug-resistant (MDR) TB.

Identification and molecular profiling of a novel homolog of cystatin C from rock bream (Oplegnathus fasciatus) evidencing its transcriptional sensitivity to pathogen infections.

Cystatins are reversible inhibitors of cysteine proteases which show an omnipresent distribution in the life on earth. Although, cystatins with mammalian origin were well characterized and their roles in physiology were reported in details, those from teleostean origin are still underrepresented in literature. However, role of cystatins in fish physiology and immune defense is highlighted in few recent reports. In this study, a cystatin C holmologue from rock bream (Oplegnathus fasciatus); termed RbCytC was identified and molecularly characterized. The complete coding sequence of RbCytC was 387 bp in length, which codes for a polypeptide with 129 amino acids, including a signal peptide of 19 amino acids. The consensus cystatin family signatures including a G residue, turning up towards the N-terminus region, QVVAG motif, locating at the middle of the sequence and the PW motif at the c terminal region was found to be well conserved in RbCytC. Phylogenetic analysis using different cystatin counterparts affirmed the close evolutionary relationship of RbCytC with its teleostan homologs which belong to family 2 cystatins. The predicted molecular model of RbCytC resembled most of the structural features of empirically elucidated tertiary structures for chicken egg white cystatin. According to the qPCR assays, RbCytC showed detectable expression in all fish tissues used in the experiment, with markedly pronounced expression level in liver. Moreover, its basal mRNA expression was up-regulated in liver and spleen tissues by experimental rock bream iridovirus infection, whereas down regulated in the same tissues, post live Edwardsiella tarda injection. Collectively, outcomes of our study validate the structural homology of RbCytC with known cystatin C similitudes, especially those of teleosts and suggest its potential roles in proteolytic processes of rock bream physiology as well as in host immune defense mechanisms.

Disposable electrochemical immunosensor for analysis of cystatin C, a CKD biomarker.

Specific monitoring of cystatin C (CysC) levels in biological fluids is critical for diagnosis, treatment and mechanistic understanding of a spectrum of diseases, particularly chronic kidney disease (CKD). Despite evidences that CysC correlates with the high risk and/or progression of CKD, its use in clinical practice is still scarce. In this context, we report the development of a simple and sensitive immunosensor for the detection of CysC. The biosensor combines the technology of cost-effective screen-printed electrodes with the high specificity of a sandwich immunoassay. Optimized conditions showed that the sensor operates in a linear range between 10 and 100 ng mL-1, with a detection limit and a sensitivity of 6.0 ng mL-1 and 6.4 ±â€¯0.3 µA ng mL-1 cm-2, respectively. Moreover, the sensor provided precise results (RSD ≤ 6.2%) and the quantification of CysC in CKD serum samples revealed to be in agreement with the values obtained by a particle-enhanced nephelometric immunoassay. In this light, the proposed immunosensor qualifies for clinical application, constituting a step forward in the development of fast, sensitive and cost-effective diagnostic tools that can improve the current medical care settings of CKD patients.

Functional characterization of Cystatin C in orange-spotted grouper, Epinephelus coioides.

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1ß and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.

The identification of discontinuous epitope in the human cystatin C - Monoclonal antibody HCC3 complex.

Human cystatin C (hCC) is a cysteine proteinase inhibitor involved in pathophysiological processes of dimerization and amyloid formation. These processes are directly associated with a number of neurodegenerative disorders such as Alzheimer disease or hereditary cystatin C amyloid angiopathy (HCCAA). One of the ideas on how to prevent amyloid formation is to use immunotherapy. HCC3 is one of a group of antibodies binding to hCC and reducing the in vitro formation of cystatin C dimers. Therefore, identification of the binding sites in the hCC-HCC3 complex may facilitate a search of effective drugs against HCCAA as well as understanding the mechanisms of neurodegenerative disorders. In this work we present epitope identification of the hCC-HCC3 complex using methods such as affinity chromatography, epitope excision and extraction MS approach, enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry (HDX MS). Comprehensive analysis of the obtained results allowed us to identify the epitope sequence with the key fragment covering hCC L1 loop and two potential epitopic fragments - α-helical part, hCC (17-28) and ß4 strand in C-terminal part of hCC. The presence of the L1 loop in the epitope sequence accounts for the significant reduction of hCC dimer formation in the presence of HCC3 antibody. SIGNIFICANCE OF THE STUDY: Deciphering the mechanism of the cystatin C aggregation process and detailed analysis of the interactions between hCC, or its pathogenic variant, and monoclonal antibodies, potentially constituting aggregation inhibitors, might be of great value as there still is a complete lack of any kind of efficient therapy for young people with the pathogenic mutation of hCC.

Molecular characterization, expression and functional analysis of cystatin C in Japanese flounder (Paralichthys olivaceus).

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases found in a wide arrange of organisms. Studies have shown that cystatins play important roles under both physiological and pathological conditions in mammals. However, much less is known about fish cystatins. In this study, we described the identification and analysis of the gene encoding cystatin C in Japanese flounder (Paralichthys olivaceus). This gene had a high homology with the sequence of cystatin C in many fish species and had a signal peptide and three conserved functional sites. The results of qRT-PCR showed that the gene was highly expressed in the liver. Lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid all increased its expression after stimulation. Functional analysis showed that the recombinant P. olivaceus cystatin C purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Meanwhile, rPocystatin C could up-regulate the expression of cytokines tumor necrosis factor α and interleukin 10. These results indicated that cystatin C of P. olivaceus might be considered to have the similar immunomodulatory function to mammalian cystatin.

Involvement of cystatin C in immunity and apoptosis.

As an abundantly expressed cysteine protease inhibitor widely distributed in the organisms, cystatin C is involved in various physiological processes. Due to its relatively small molecular weight and easy detection, cystatin C is commonly used as a measure for glomerular filtration rate. In pathological conditions, however, growing evidences suggest that cystatin C is associated with various immune responses against either exogenous or endogenous antigens, which ultimately result in inflammatory autoimmune diseases or tumor development if not properly controlled. Thus the fluctuation of cystatin C levels might have more clinical implications than a reflection of kidney functions. Here, we summarize the latest development of studies on the pathophysiological functions of cystatin C, with focus on its immune regulatory roles at both cellular and molecular levels including antigen presentation, secretion of cytokines, synthesis of nitric oxide, as well as apoptosis. Finally, we discuss the clinical implications and therapeutic potentials of what this predominantly expressed protease inhibitor can bring to us.

Identification and characterization of antibodies elicited by human cystatin C fragment.

Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. One of rather atypical, occurring at a young age amyloidosis is hereditary cystatin C amyloid angiopathy (HCCAA) related to aggregation of L68Q variant of human cystatin C (hCC). Human cystatin C plays a very important role in many aspects of human health; however, its amyloidogenic properties manifested in HCCAA present a real, lethal threat to some populations and any work on factors that can affect possible influencing hCC aggregation is not to overestimate. It was proved that interaction of hCC with monoclonal antibodies suppresses significantly hCC dimerization process. Therefore, immunotherapy seems to be the right approach toward possible HCCAA treatment. In this work, the hCC fragment encompassing residue 60-70 (in 2 variants: linear peptide and multiple antigenic peptide) was used as an immunogen in rabbit immunization. As a result, specific anti-hCC antibodies were found in both rabbit sera. Surprisingly, rabbit antibodies were obtained after immunization with only a short peptide. The obtained antibodies were characterized, and their influence on the aggregation propensity of the hCC molecules was evaluated. The antibodies turned out not to have any significant influence on the cystatin C dimerization process. Nevertheless, we hope that antibodies elicited in rabbits by other hCC fragments could lead to elaboration of effective treatment against HCCAA.

[Preparation of monoclonal antibody against cystatin C and establishment of its immunoassay system].

OBJECTIVE: To prepare human cystatin C( CysC) recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum. METHODS: The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established, and 52 cases of human serum samples were detected by the detection system. RESULTS: Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 µg/mL. The detection limit was 4. 598 ng/mL and IC_(50) was 0. 145 µg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples. CONCLUSION: The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.

Human cystatin C monomer, dimer, oligomer, and amyloid structures are related to health and disease.

Human cystatin C (hCC) is a small protein belonging to the cystatin family of papain-like cysteine proteinase inhibitors. We review the recent literature concerning structural aspects of hCC related to disease. We focus on the mechanisms of hCC dimerization, oligomerization, and amyloid formation. Amyloid formation is associated with a number of neurodegenerative diseases that affect the independence and quality of life of aging populations. hCC is one of the second-wave proteins that have been found to undergo amyloidosis associated with disease. For hCC, this includes cerebral amyloid angiopathy, as well as a disorder resulting in reduced male fertility.

Application of amide hydrogen/deuterium exchange mass spectrometry for epitope mapping in human cystatin C.

Human cystatin C (hCC) is a small cysteine protease inhibitor whose oligomerization by propagated domain swapping is linked to certain neurological disorders. One of the ways to prevent hCC dimerization and fibrillogenesis is to enable its interaction with a proper antibody. Herein, the sites of interaction of hCC with dimer-preventing mouse monoclonal anti-hCC antibodies Cyst28 are studied and compared with the binding sites found for mAb Cyst10 that has almost no effect on hCC dimerization. In addition, hCC epitopes in complexes with native polyclonal antibodies extracted from human serum were studied. The results obtained with hydrogen-deuterium exchange mass spectrometry (HDX MS) were compared with the previous findings made using the excision/extraction MS approach. The main results from the two complementary MS-based approaches are found to be in agreement with each other, with some differences being attributed to the specificity of each method. The findings of the current studies may be important for future design of hCC dimerization inhibitors.

Isolation and characterization of autoantibodies against human cystatin C.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a severe neurodegenerative disorder related to the point mutation in cystatin C gene resulting in human cystatin C (hCC) L68Q variant. One of the potential immunotherapeutic approaches to HCCAA treatment is based on naturally occurring antibodies against cystatin C. A recent growing interest in autoantibodies, especially in the context of neurodegenerative diseases, emerges from their potential use as valuable diagnostic markers and for controlling protein aggregation. In this work, we present characteristics of natural anti-hCC antibodies isolated from the IgG fraction of human serum by affinity chromatography. The electrophoresis (1-D and 2-D) results demonstrated that the isolated NAbs are a polyclonal mixture, but their electrophoretic properties did not allow to classify the new autoantibodies to any particular type of IgG. The Fc-glycan status of the studied autoantibodies was assessed using mass spectrometry analysis. For the isolated NAbs, the epitopic fragments in hCC sequence were identified by MS-assisted proteolytic excision of the immune complex and compared with the ones predicted theoretically. The knowledge of hCC fragments binding to NAbs and other ligands may contribute to the search for new diagnostic methods for amyloidosis of different types and the search for their treatment.

Epitope location for two monoclonal antibodies against human cystatin C, representing opposite aggregation inhibitory properties.

Human cystatin C (hCC), like many other amyloidogenic proteins, dimerizes and possibly makes aggregates by subdomain swapping. Inhibition of the process should suppress the fibrillogenesis leading to a specific amyloidosis (hereditary cystatin C amyloid angiopathy, HCCAA). It has been reported that exogenous agents like monoclonal antibodies against cystatin C are able to suppress formation of cystatin C dimers and presumably control the neurodegenerative disease. We have studied in detail two monoclonal antibodies (mAbs) representing very different aggregation inhibitory potency, Cyst10 and Cyst28, to find binding sites in hCC sequence responsible for the immunocomplex formation and pave the way for possible immunotherapy of HCCAA. We used the epitope extraction/excision mass spectrometry approach with the use of different enzymes complemented by affinity studies with synthetic hCC fragments as a basic technique for epitope identification. The results were analyzed in the context of hCC structure allowing us to discuss the binding sites for both antibodies. Epitopic sequences for clone Cyst28 which is a highly potent dimerization inhibitor were found in N-terminus, loop 1 and 2 (L1, L2) and fragments of ß2 and ß3 strands. The crucial difference between conformational epitope sequences found for both mAbs seems to be the lack of interactions with hCC via N-terminus and the loop 1 in the case of mAb Cyst10. Presumably the interactions of mAbs with hCC via L1 and ß sheet fragments make the hCC structure rigid and unable to undergo the swapping process.

Highly Effective Protein Converting Strategy for Ultrasensitive Electrochemical Assay of Cystatin C.

In this work, a highly effective protein converting strategy based on immunoreaction-induced DNA strand displacement and T7 Exonuclease (T7 Exo)-assisted protein cyclic enzymatic amplification for ultrasensitive detection of cystatin C was described. Herein, Au@Fe3O4 as magnetic separator was labeled by antibody 1-conjungated DNA (DNA1) and the DNA substrate of T7 Exo (DNA3) which initially hybridized with output DNA (S1) to form a stable S1/DNA1 duplex (S1/DNA3). Antibody 2 was labeled by competing DNA (DNA 2). In the presence of cystatin C, sandwich immunoreaction would induce proximity hybridization between DNA2 and DNA3 and thus displace S1 from the S1/DNA3 duplex with formation of a stable DNA2/DNA3 duplex, realizing the conversion of input target cystatin C into output S1. To enhance the conversion ratio, the DNA2/DNA3 duplex was then digested by T7 Exo with release of DNA2 which could act as competing DNA again to displace S1 from the S1/DNA3 duplex in adjacent locations and initiate another cleavage reaction. Through such a cyclic process, each input cystatin C could induce more than one output S1, enhancing detection sensitivity. A hairpin DNA modified electrode was used to capture the output S1, and then, a hybridization chain reaction is triggered on the biosensor surface. Then, thionine as electron mediator was embedded into the dsDNA polymers to produce a detection signal. The electrochemical biosensor exhibited a much wider linear range of 0.01 pg mL(-1) to 30 ng mL(-1) with low detection limit of 3 fg mL(-1). Moreover, this method introduced protein unrelated to nucleic acids into the realm of potential inputs for translation, which might create a new immunoassay method for sensitive detection of protein.

Preparation and Identification of a Monoclonal Antibody Against Cystatin C.

Cystatin C (Cys C) has been shown to be an excellent marker of renal function, especially when evaluating the early stages of acute kidney injury. It is less affected by age, gender, muscle mass, and ethnicity. The detection of Cys C is important and has broad application prospects. Therefore, we have developed a panel of monoclonal antibodies against Cys C that can be used to establish an enzyme-linked immunosorbent assay (ELISA) kit and paired for further use in other methods of detecting Cys C. This study describes the preparation, application, and characterization of monoclonal antibodies used in ELISA. The antibodies were developed by PEG fusion of the SP2/0 cells with splenic B cells from Cys C immunized BALB/c mice. Antibody-producing cells were identified by ELISA and Western blot analysis. By way of cloning and screening, four hybridoma cell lines were established. Simultaneously large-scale monoclonal antibodies produced in mice ascites were prepared. The results showed that the cell clone 8D12 could be used in immunohistochemical staining. With the ELISA additivity test, we got a preliminarily finding that the monoclonal antibodies were not on the same epitope. The antibody matching test showed that 5D7 and 7A8 successfully paired with 8D12, and the optimal reaction conditions were initially identified.

Stabilization, characterization, and selective removal of cystatin C amyloid oligomers.

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.

Efficient production of recombinant cystatin C using a peptide-tag, 4AaCter, that facilitates formation of insoluble protein inclusion bodies in Escherichia coli.

Cystatin C is a cysteine protease inhibitor produced by a variety of human tissues. The blood concentration of cystatin C depends on the glomerular filtration rate and is an endogenous marker of renal dysfunction. Recombinant cystatin C protein with high immunogenicity is therefore in demand for the diagnostic market. In this study, to establish an efficient production system, a synthetic cystatin C gene was designed and synthesized in accordance with the codon preference of Escherichia coli genes. Recombinant cystatin C was expressed as a fusion with a peptide-tag, 4AaCter, which facilitates formation of protein inclusion bodies in E. coli cells. Fusion with 4AaCter-tag dramatically increased the production level of cystatin C, and highly purified protein was obtained without the need for complicated purification steps. The purity and yield of the final product was estimated as 87 ± 5% and 7.1 ± 1.1 mg/l culture, respectively. The recombinant cystatin C prepared by our method was as reactive against anti-cystatin C antibodies as native human cystatin C. Our results suggest that protein production systems using 4AaCter-tag could be a powerful means of preparing significant amounts of antigen protein.

Endogenous aggregates of amyloidogenic cystatin C variant are removed by THP-1 cells in vitro and induce differentiation and a proinflammatory response.

A mutation in the human cystatin C gene leads to familial cerebral amyloid angiopathy. This disease is known as "hereditary cerebral hemorrhage with amyloidosis-Icelandic type" or "hereditary cystatin C amyloid angiopathy." The mutant cystatin C protein forms aggregates and amyloid, within the central nervous system almost exclusively in connection with the vascular system. It was not known whether immune cells could remove mutant cystatin C protein aggregates. Ex vivo mutant cystatin C protein aggregates, both in solution and dried onto a glass surface, induced adhesion to the substrate, differentiated the THP-1 monocyte cell line and led to a proinflammatory response. Aggregates were also taken up by both THP-1 cells and THP-1 derived macrophages. These are the same responses induced by other amyloidogenic protein species, such as amyloid ß protein and amylin, supporting the model of all amyloidogenic proteins being toxic due to common structural motifs. Proinflammatory response induced by the ex vivo mutant cystatin C protein aggregates suggests that vascular inflammation plays an important role in hereditary cerebral hemorrhage with amyloidosis-Icelandic type. Ex vivo protein aggregates of cystatin C might better model cellular behavior than in vitro-generated aggregates or supplement in vitro material.

Quantification of cystatin C by time-resolved fluorometry-based immunoassays.

Plasma cystatin C is increasingly used as a marker of glomerular filtration rate. Most assays for cystatin C are based on turbidimetric or nephelometric detection and studies of other rapid methods are limited. This study aimed to develop and compare differently configured immunoassays for quantification of plasma cystatin C, using recombinant cystatin C and two cystatin C-specific antibodies. Method 1 was a two-step sandwich assay with polyclonal antibody as capture and europium chelate-labeled monoclonal antibody as tracer. Method 2 was a one-step heterogeneous competitive assay using immobilized polyclonal antibody and europium-labeled cystatin C. Method 3 was a one-step homogeneous competitive assay with europium-labeled polyclonal antibody as donor and cyanine 5-labeled cystatin C as acceptor. All three assays were evaluated with plasma samples and their performance was compared to a conventional particle-enhanced turbidimetric immunoassay (PETIA). Method 3 was the easiest to perform, with incubation at ambient temperature for 10 min and 20 µL of sample, while methods 1 and 2 had washing steps, took 40 min and 15 min at 37°C, respectively, but used only 10 µL of 100- or 10-fold diluted sample, respectively. The working ranges for methods 1, 2 and 3 were 0.0005-0.2, 0.05-1.0 and 0.25-20mg/L, respectively. Kinetics for method 3 was the fastest with >95% binding completion and for method 2 the slowest with 60% binding completion. All three methods showed good correlation to PETIA, but produced higher cystatin C levels than PETIA. Methods 1 and 3 offered the most favorable performance characteristics and especially method 3 enabled rapid and simple measurement of circulating cystatin C.