| N-terminal pro-brain natriuretic peptide (NT-proBNP) with 76 amino acids, cleaved from its amino acid precursor protein BNP (pro-BNP), is a better marker for heart failure and can reflect the ventricular volume expansion, ventricular overload and the degree of cardiac injury. As early as 2000, an editorial article“BNP: soon to become a routine measure in the care of patients with heart failure”, published in Heart, made it clear that the level of brain natriuretic peptide or B-type natriuretic peptide (brain natriuretic peptide, B-type natriuretic peptide; BNP) can be used as a prognostic marker in heart failure patients and a new method monitoring the heart failure. In 2002, brain natriuretic peptide, namely NT-proBNP, has been recognized as a landmark heart failure with specific markers in the international community. At present, only a few large medical productions prepare NT-proBNP diagnostic reagents abroad, which are expensive and difficult to use popular. Meanwhile, no sale of NT-proBNP testing for antibodies and standards was found in the market. Therefore, the development of NT-proBNP testing antibodies and calibrations will make up for domestic application and improve the advantage of price compared to the imported reagents. This work itself is a huge social economic value, and will be benefit for establishment of a fully independent intellectual property right of NT-proBNP electrochemical immunosensor technology.In recent years, the technology of immunsensor based on immune and electrochemical detection has achieved greatly development. This method can not only be used in quantitative analysis, but also develop applications of rapid and cheap detection for community hospitals or beside bed, which has received much attention. The concentration of NT-proBNP is 0.1ng/ml in normal human serum and up to 3-3ng/ml in patients with heart failure. The technology of immunsensor is usually available to detect the proBNP, which can detect the level of NT-proBNP at concentration of 0.5ng/ml. It needs to establish a reliable determination of NT-proBNP method sensitive to the levels below the normal human serum NT-proBNP. Also these are urgent problems, such as how to extend the life of the sensor and solve the testing renewable, how to improve the anti-interference ability of electrode to enhance the specificity of detection for the clinical application. In order to improve the sensitivity of the biological sensors, many researchers have used different markers to mark antigens or antibodies and made good progress in this work. In addition to enzyme, metal or semiconductor nano particles and other new types of biological material with electricity have been used as markers. In our research, we successfully prepared antibodies for immunoassay of NT-proBNP and standard for clinical testing. Compared with Roche’s NT-proBNP, the antibodies prepared in our method is highly consistent with this detection reagent (95%). On the basis, the regeneration of immunosensor and rapid testing can be realized by the antibody-coupled magnetic particles in conjunction with electrodes of controllable magnetic field. High activity of NT-proBNP Fab antibody prepared through antibody fragment can be used for immunosensor detection to eliminate the false positive, which was brought by non-specific binding in non-labeled test. Multi-layer carbon nanotubes (CNTs) were used as solid phase for NT-proBNP antibody to improve surface area of adsorption. While gold chains and complex of horseradish peroxidase enzyme pairing with NT-proBNP, in the form of signal amplification greatly, would improve the sensitivity of detection. Through the above study, we solved several key issues for detection of NT-proBNP by immunosensor, which would help the establishment of NT-proBNP detection with immunosensor technology in clinical diagnosis.Results:1. Preparation of heart failure marker NT-proBNP immunoassay of antibodies and calibrators1.1 Preparation of calibratorsPurified human NT-proBNP protein was expressed in recombinant Escherichia coli (E.coli BL21 DE3). And further study was focused on the form of preservation and the best form of stability preservation. These results demonstrated that after freeze-dried preservation in PBS containg 0.3% Proclin300 for 6 months, reduction of protein detected was <5%. And the reduction of protein was <10% after diluted with calf serum solution for one month, with good preservation and perfect testing results. 1.2 Purification and identification of polyclonal antibodiesThe technology of purifying polyclonal antibody was established by specific affinity chromatography based on peptides-coupled NHS activating filler combined with Protein A affinity chromatography. The content of Specific IgG in polyclonal antibody was only1%, which was purified by the above technology. And 20mg of purified antibody could be acquired from per 100ml of immune serum. The purified antibody with high affinity was a similar epitope-specific monoclonal antibody.1.3 Purification and identification of monoclonal antibodiesPre-purified monoclonal antibody from ascites was treated with by SiO2 adsorption, which was then purified by the improved Protein A packing Mabselect. The purity of purified antibody was >95% identified by SDS-PAGE. This method can effectively remove the lipoprotein in antibody to improve the purity and specificity of monoclonal antibodies, which are more suitable for antibody labeling and fragmentation of transformation.1.4 HRP labeling of NT-proBNP antibodyHRP-labeled monoclonal antibody was prepared by optimized HRP labeling method, which could reach the good marking and improve the sensitivity of detection.1.5 The establishment and evaluation of ELISA for detection of human NT-proBNPELISA kit for NT-proBNP was prepared by NT-proBNP monoclonal antibody, polyclonal antibody and calibrator of detection. The higher limit of detection for this kit was 30000pg/ml and lower limit to 100pg/ml. After the detecting the 234 clinical samples, the results made from this kit was highly consistent with Roche’s ELC NT-proBNP kit (99%, p<0.01), verifying the ability of prepared NT-proBNP testing natural antibody in serum.2. The specificity and free regeneration of immunsensor for detecting NT-proBNP2.1 Preparation of Fab antibody from monoclonal antibodyTo acquire Fab antibody, papain was used to digesting the monoclonal antibody. After digestion, purified Fab antibody was obtained by antigen-coupled NHS activating filler combined with Protein A purification. This Fab antibody has high activity with completely removing the Fc fragment.2.2 Application of Fab antibody to improve specificity of NT-proBNP immunosensorDouble-antibody sandwich method established by double monoclonal antibody was used to detect the level of NT-proBNP in serums from 40 patients with rheumatic diseases and 216 neative serums. Among these serums, 6 and 2 samples were detected as negative by the Roche false-positive specimens. Fab of the monoclonal antibody combined with immunosensor was used, and all of these 8 Roche false-positive specimens were detected normally, which eliminate false positive results caused by non-marking system immunosensor for detection of rheumatoid factor, complement system, as well as anti-mouse antibodies.2.3 Biotin of NT-proBNP antibodyMonoclonal antibody was biotinylated by Pierrce biotin kit, which was in conjunction with magnetic particles coupled with avidin. Then, a controlled electromagnetic electrode was used to construct immune electrode for detection o NT-proBNP.2.4 Construction of NT-proBNP regeneration-free immunosensorTo test NT-proBNP by immunosensor, the formation of the reaction system to capture biotinylated antibody - antigen - the second antibody was used to form immune complexes, in which avidin-coated magnetic particles was used as solid phase. Controlled by the magnetic field electrode designed to detect, elution, and then test the form of continuous testing, this detection was designed to regeneration. 2.5 Evaluation of NT-proBNP immunosensor based on renewable-free detection by Fab antibodyThe design and construction of a new type of controllable magnetic field electrode was based on the technology of electrochemical immunosensor, which was also combined with biotinylated NT-proBNP antibody, avidin-coated magnetic particles. With this electrode, the detection of a standard NT-proBNP and NT-proBNP in clinical samples was fast, easy and free regeneration. High activity of NT-proBNP Fab antibody prepared through antibody fragment can be used for immunosensor detection to eliminate the false positive, which was brought by non-specific binding in non-labeled test. The results showed that the sensitivity of detection was 0.03ng/ml (0.04~2.5ng/ml, R=0.9827) after using of immunosensor constructed in this method, which had a good detecting results. Compared with full fragment antibody, Fab antibody eliminated 4% false positive. Moreover, this detection method promote the technology of electrochemical immunosensor tuning from equipment to the development of reagents with more convenient and practically. So the application of electrochemical immunosensor technology in clinical is more forward. 3. Ultra-sensitive detection of immunosensor for NT-proBNP3.1 Preparation and application of multi-layer carbon nanotubes (CNTs) solid phaseIn order to fixed multi-layer carbon nanotube electrode to the surface of testing effectively, we first used the non-covalent way to treat CNTs with acid, and then with BSA. CNTs combined with BSA protein can be adsorbed on the gold electrode surface, while avoid the use of covalent binding of CNTs way to affecte the consequences of electron transfer. And the gold electrode with BSA can be further combined with acolloidal gold, coupling the purpose of antibody molecules. By cyclic voltammetry (CV) study, the prepared electrode has good current response.3.2 Preparation and application of Gold chains, horseradish peroxidase second antibody complexHAuCl4 solution and NT-proBNP second antibody horseradish peroxidase (HRP) was formed into chain complexes, in which ascorbic acid was used as a reaction to the media. The results reached the expected effects by transmission electron microscopy (TEM) detection for their preparation. Compared with ordinary colloidal gold second antibody, this method greatly improved the detection sensitivity.3.3 Evaluation of NT-proBNP immunosensor based on carbon nanotubes and gold chain and multi-horseradish peroxidase complexMulti-layer carbon nanotubes (CNTs) were used as solid phase for NT-proBNP antibody to improve surface area of adsorption, which elevate the fixed amount of antibody in the electrode surface. At the same, complex of gold chain and HRP in conjunction with second antibody, amplifying signal, would improve the detection sensitivity greatly. After examining the electrochemical properties of the electrode surface with CV, the performance of the immunosensor were investigated in detail. The results demonstrated that this immunosensor sensitivity was 6pg/ml (0.02~100ng/ml, R=0.997), which was higher than the sensitivity of clinical detection requirements (0.1ng/ml). And the immunosensor was also proved to be good selectivity and free regenerative performance, which was expected to be further used in clinical research applications. |
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