RESUMEN
This paper reports the development of a novel surface plasmon resonance (SPR) immunosensor for ultra-sensitive quantitative determination of human articular cartilage oligomeric matrix protein (COMP), a major component of the extracellular matrix and an exploratory biomarker. Capture antibodies against human COMP (anti-COMP16F12) were covalently immobilized on an 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayer (SAM)-coated SPR sensor disk and a dual sandwich-type signal amplification strategy using biotinylated detection antibodies against COMP (anti-COMP17C10-biot) and streptavidin-conjugated quantum dots (SAvâQDs) were used for the development of an immunosensor. The binding of high-mass SAvâQDs via biotin-streptavidin interaction to the surface of the immunosensor resulted in a drastic increase in the sensitivity. The developed immunosensor was able to detect concentrations of COMP in a range from 2.80 to 680.54 fM with a limit of detection (LOD) and a limit of quantification (LOQ) of 0.15 and 0.50 fM, respectively. The immunosensor exhibited good repeatability (relative standard deviation (RSD) 8.05%) and reproducibility (RSD 9.88%) as well as excellent operational stability (2.14 % decrease in SPR signal after 13 days). In addition, the analysis of secretomes of human knee articular cartilage explants from patients with osteoarthritis revealed that the immunosensor has good accuracy (analytical error less than 5 %). These results indicate that the immunosensor developed may be suitable for quantitative determination of COMP derived from articular cartilage and other synovial joint tissues in clinical studies.
Asunto(s)
Técnicas Biosensibles , Resonancia por Plasmón de Superficie , Humanos , Resonancia por Plasmón de Superficie/métodos , Proteína de la Matriz Oligomérica del Cartílago , Técnicas Biosensibles/métodos , Estreptavidina , Reproducibilidad de los Resultados , Inmunoensayo/métodos , BiomarcadoresRESUMEN
Advancements that occurred during the last years in the diagnosis of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis infection, have prompted increased survival rates of patients. However, limitations related to the inefficiency of an early detection still remain; some techniques and laboratory methods do not have enough specificity and most instruments are expensive and require handling by trained staff. In order to contribute to a prompt and effective diagnosis of tuberculosis, we report the development of a portable, user-friendly, and low-cost biosensor device for its early detection. By using a label-free surface plasmon resonance (SPR) biosensor, we have established a direct immunoassay for the direct detection and quantification of the heat shock protein X (HspX) of Mtb, a well-established biomarker of this pathogen, directly in pretreated sputum samples. The method relies on highly specific monoclonal antibodies that are previously immobilized on the plasmonic sensor surface. This technology allows for the direct detection of the biomarker without amplification steps, showing a limit of detection (LOD) of 0.63 ng mL-1 and a limit of quantification (LOQ) of 2.12 ng mL-1. The direct analysis in pretreated sputum shows significant differences in the HspX concentration in patients with tuberculosis (with concentration levels in the order of 116-175 ng mL-1) compared with non-tuberculosis infected patients (values below the LOQ of the assay).
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas Bacterianas/análisis , Esputo/microbiología , Tuberculosis , Humanos , Límite de Detección , Mycobacterium tuberculosis , Sistemas de Atención de Punto , Tuberculosis/diagnósticoRESUMEN
In this study, heterologous indirect competitive enzyme-linked immunosorbent assay (icELISA) was introduced into the screening of hybridomas for the development of broad-specific monoclonal antibodies (mAbs) against organophosphorus (OP) pesticides. After immunization, two formats of icELISA based on the homologous hapten antigen and four heterologous hapten antigens were conducted for hybridoma screening. Two mAbs 2G6 and 7B2 with good recognition toward three OP pesticides (parathion, methyl-parathion, and fenitrothion) were produced. Results of the icELISA showed that the two mAbs exhibited high sensitivity against three OP pesticides, with IC50 ranging from 2.93 to 19.71 ng mL-1. Moreover, a non-competitive surface plasmon resonance (SPR) immunosensor was used for characterizing the binding properties of the mAbs to OP pesticides. After kinetic analysis, equilibrium dissociation constant (KD) values of mAbs 2G6 and 7B2 were calculated as 1.45 × 10-9 M and 4.26 × 10-9 M for parathion, 6.75 × 10-9 M and 4.17 × 10-9 M for methyl-parathion, and 2.44 × 10-8 M and 1.19 × 10-8 M for fenitrothion, respectively. Whereas, both icELISA and SPR-based immunoassay indicated that the two mAbs could not recognize other five OP analogs. Since SPR-based immunoassay provides comprehensive information of two molecules directly interacting with each other, it is a valuable tool during the development and characterization of broad-specific mAbs. Graphical abstract á .
Asunto(s)
Anticuerpos Monoclonales/química , Afinidad de Anticuerpos , Técnicas Biosensibles/métodos , Compuestos Organofosforados/química , Plaguicidas/química , Resonancia por Plasmón de Superficie/métodos , Animales , Líquido Ascítico , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Haptenos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Agua/químicaRESUMEN
In this research, we developed a direct-flow surface plasmon resonance (SPR) immunosensor for ampicillin to perform direct, simple, and fast measurements of this important antibiotic. In order to better evaluate the performance, it was compared with a conventional amperometric immunosensor, working with a competitive format with the aim of finding out experimental real advantages and disadvantages of two respective methods. Results showed that certain analytical features of the new SPR immunodevice, such as the lower limit of detection (LOD) value and the width of the linear range, are poorer than those of a conventional amperometric immunosensor, which adversely affects the application to samples such as natural waters. On the other hand, the SPR immunosensor was more selective to ampicillin, and measurements were more easily and quickly attained compared to those performed with the conventional competitive immunosensor.
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Resonancia por Plasmón de Superficie , Ampicilina , Antibacterianos , Técnicas Biosensibles , InmunoensayoRESUMEN
Ractopamine (RCT) is banned for use in animals in many countries, and it is urgent to develop efficient methods for specific and sensitive RCT detection. A label-free indirect competitive surface plasmon resonance (SPR) immunosensor was first developed with a primary antibody herein and then improved by a secondary antibody for the detection of RCT residue in swine urine. Meanwhile, a pre-incubation process of RCT and the primary antibody was performed to further improve the sensitivity. With all the key parameters optimized, the improved immunosenor can attain a linear range of 0.3-32 ng/mL and a limit of detection (LOD) of 0.09 ng/mL for RCT detection with high specificity. Furthermore, the improved label-free SPR immunosenor was compared thoroughly with a conventional enzyme-linked immunosorbent assay (ELISA). The SPR immunosensor showed advantages over the ELISA in terms of LOD, reagent consumption, analysis time, experiment automation, and so on. The SPR immunosensor can be used as potential method for real-time monitoring and screening of RCT residue in swine urine or other samples. In addition, the design using antibody pairs for biosensor development can be further referred to for other small molecule detection.
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Ensayo de Inmunoadsorción Enzimática , Animales , Técnicas Biosensibles , Fenetilaminas , Resonancia por Plasmón de Superficie , PorcinosRESUMEN
Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy-immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content.