A New Method For Highly Sensitive Chemiluminescence Immunoassay Cytokines

Part one A paramagnetic microspheres based automation-friendly rapid chemiluminescent immunoassay method forsensitive detection of chicken interferon-yThis work developed a new paradigm of automation-friendly and rapid chemiluminescent (CL) immunoassay for highly sensitive detection of Chicken interferon-γ (ChIFN-γ) at the sub-picogram per millilitre level. Paramagnetic microspheres(PMs) were for the first time used to immobilize the ChIFN-γ antibody and further design automation-friendly immunoassay. Compared with labour-intensive, time-consuming and inadequately sensitive biological assay and enzyme-linked immunoassay(ELISA), this method showed obvious advantages such as high sensitivity, rapid assay speed, wide linear range and simple manipulation. Anti-ChIFN-γ immobilized PMs, ChIFN-γ and horseradish peroxidase (HRP)-labeled anti-ChIFN-γ was introduced into the flow cell to form sandwich complex after on-line incubation. After HRP substrate was injected into the flow cell, chemiluminescence reaction was triggered immediately. The CL signals increased linearly with the ChIFN-γ concentration from0.001to0.1ng/mL. The detection limit of this method (0.87pg/mL), which was56.5-fold lower than that of the current minimum value (50pg/mL) reported for ChIFN-γ. This method also showed high specificity, good accuracy and acceptable reproducibility, stability and can be applied in automated rapid detection of practical samples. Moreover, the proposed method this work can be extend to assay other cytokines in chicken or cytokines in other poultry.Part two Graphene oxide based ultrasensitive flow-through chemiluminescent immunoassay for sub-picogram level detection of chicken interferon-yThe quantitative detection of chicken interferon-y (ChIFN-γ) released by T cells after in vitro stimulation is a good evaluation of cell-mediated immunity in chickens after infection or vaccination. In this study, we report a new paradigm of flow-through CL immunoassay for ultrasensitive, rapid and specific determination of ChIFN-γ at sub-pictogram per milliliter level. The biocompatible graphene oxide nanosheet is for the first time introduced into CL immunoassay fields for highly efficient immobilization of capture antibody. The detection limit of the proposed method at a signal to noise ration of3is0.36pg/mL, which is138-fold lower than the current lowestvalue of50pg/mL for ChIFN-γ. Coupling with flow-though system, the whole immunoassay process complete within25min. The resulting ChIFN-γ immunosensor shows excellent detection and fabrication reproducibility, good specificity and stability. The assay results of nature ChIFN-γ samples with the proposed method are in an acceptable agreement with the reference values. Compared to the present assay methods, this method is more flexible, simple, rapid and sensitive. The aim of this work is to demonstrate that the ultrasensitive, specific and rapid CL immunoassay format can become a very potential application for quantifying ChIFN-y and further studying its role in immune response in poultry.Part three Flow-through chemiluminescent immunosensor for chicken interleukin-4based on nanorod TiO2-chitosan matrixThis work proposed a first sample of chemiluminescence(CL) immunosensor for highly sensitive determination of chicken interleukin-4(IL-4) based on the immobilization of anti-ChIL-4on nanorod-likeTiO2matrix. The CL immunosensor was fabricated by dropping the mixure of nanorod-like TiO2, chitosan(CS) and anti-ChIL-4antibody on surface of epoxy-activated glass substrate. The synthesized nanorod-shaped TiO2has large surface area and good biocompatibility, which can be used for highly efficient immobilization of anti-ChIL-4and retaining its bioactivity. Scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) was used to characterize the constructed ChIL-4immunosensor. The highly efficient CL immunosensor showed a linear range of3orders of magnitude from0.1to100ng/mL and a low detection limit of0.045ng/mL. Compared with traditional bioassays,The proposed CL immunoassay system showed higher sensitivity, simpler fabrication, lower cost, and better practicability. In addition, the immunosensor showed excellent specificity, good reproducibility and stability, which provided a promising clinical evaluation standard for evaluation cell-mediated immunity in chickens after infection or vaccination.Part four High sensitive chemiluminescent immunosensor for Bovine IFN-γ based on Chitosan-Platinum colloidIn this work, a high sensitive chemiluminescent immunosensor for bovine interferon-y(BoIFN-γ) was proposed based on the immobilization of BoIFN-y antibody in platinum nanoparticles-chitosan(CS)composite. Firstly, platinum nanoparticles was despersed into the CS solution and then mixed with BoIFN-γ antibody. Next, the reslutant mixture was dropping on epoxy-activated glass substrate surface to fabricated the immunosensor. Platinum nanoparticles provided high specific surface area, good adsorption and biocompatibility for improving the detection sensitivity for BoIFN-γ. Platinum nanoparticles and the constructed biosensor were characterized using transmission electron microscope (TEM), UV-vis and electrochemical impedance spectroscopy (EIS). The BoIFN-γ immunosensor showed a wide linear range of0.1to120ng/mL and a low detection limit of0.026ng/mL (S/N=3). Compared with the traditonal method for BoIFN-γ, the CL immunosensor showed higher sensitivity, wider linear range and fast assy speed. Moreover, the immunosensor showed excellent selectivity, good reproducibility and stability. The research work provides a promising assy method for detecting BoIFN-γ level in bovine after infection or vaccination.