Preparation Of Monoclonal Antibody Against Human NT-proBNP And Preliminary Development Of An Immunosensor For Determining NT-proBNP

B-Type natriuretic peptide (BNP) as well as the N-terminal fragment of the prohormone (NT-proBNP) is reported to be powerful markers for prognosis and risk stratification of heart failure, reflecting ventricular volume expansion and pressure overload. In 2002, the level of NT-proBNP in serum has been considered as reliable indicator for heart failure. And then, on Nov. 19th of this year, the Food and Drug administration (FDA) in USA cleared for marketing the NT-proBNP, a new laboratory test, the Elecsys proBNP Immunoassay made by Roche Diagnostics for use as an aid in diagnosing congestive heart failure, which is the first fully automated test for this use. Now, NT-proBNP was clinically approved to be widely used in diagnosing heart failure as a powerful marker for myocardium. So far, this technique is possessed of three international corporations only. This NT-proBNP kit clinically was used to diagnose congestive heart failure in a few famous hospitals in China mainland, due to the high importing expenses from USA. Therefore, it is urgent and necessary to develope a novel self-owned NT-proBNP immunoassay in China.Primary research and results as follows:1. Construction of high efficiency prokaryotic expression vector of human NT-proBNP and preparation of immunology standard1.1 Construction of high efficiency expression of human NT-proBNP geneThe human high efficiency expression vector of NT-proBNP gene in Escherichia coli (E.coli BL21 DE3) was constructed by combining artificial synthesis and bridging splicing polymerase chain reaction. According to the BNP gene order offered by GENEBANK(CR54976), we obtained sequence containing 228 base coding amino acids from 27 to 103 of NT-proBNP. We used Graphical codon usage analyzer (http://guca.schoedl.del) to analyze this coding sequence and found that a part of codon the in gene sequence encoding amino acids occur seldom in E.coli BL21. If conventional reverse transcription PCR was applied, it is possible that the constructed recombinant plasmid will not expressed in Escherichia coli perfectly. Thus the GeneSOEing PCR technique was used in our experiment. Modified gene sequence was divided into several parts of small sequence about 40 bases to synthesize. The each small sequence has partial overlapping with each other, so every fragment is the primer and template, at the same time the total DNA modified is synthesized to the hobby of E.coli.1.2 High efficiency prokaryotic expression and purification of human NT-proBNP geneWe constructed NT-proBNP prokaryotic expression plasmid by making NT-proBNP gene order reconstructed to hobby of E.coli into prokaryotic expression plasmid pET32a and pET42a. After induced by using 1.0 mmol/L isopropy-β-D-thiogalactoside (IPTG) 5h at 37℃, pET32a-NT-proBNP and pET42a-NT-proBNP expressed fusion protein weighting about 30KD and 40KD, respectively. The amounts of expressing protein occupy more than 30 percent of the total protein in E.coli. After breaking bacterium, expressing protein was soluble and presenting in supernatant. After isolated by cation exchange, his-tag affinity column, and GST affinity chromatograph, the purified NT-proBNP protein (90%-95%) were obtained.1.3 Preparation of immunology detection standard of human NT-proBNPThis recombinant fusion NT-proBNP protein made by many purity methods and digested by enterokinase from our laboratory is more immunocompetence and stable than that from Roche Corporation. Especially, the purity of NT-proBNP-83 reaches 95% by HPLC detection, which proves to be suitable for immunology detection standard.2. Preparation and identification of monoclonal antibody against human NT-proBNPApplying purified NT-proBNP protein as immunogen, we obtained 7 strains of monoclonal antibody against NT-ProBNP by using modified immune programme and hybridoma technique. Among 5 strains are suitable for diagnostic reagent by subclass identification. After analysis by using immunohistochemistry, ELISA and Western blotting methods, the monoclonal antibody obtained in our experiment prove to be specific to immunogen, NT-proBNP protein.At the same time, we designed and synthesized NT-proBNP polypeptide containing three antigen epitope and crossing link with KHL by epitope on-line analysis and referring NT-proBNP epitope adopted by immunodiagnostic reagent. In addition to immuning animals to produce antibody antagonist specific epitope, this synthesized NT-proBNP polypeptide can also be used to identify the antibody obtained conversely. Now, we have obtained 2 strains of IgG antibody against the same antigen epitope, amino acids 24-35 of NT-proBNP proteins. The other 3 strains still deserve to be invested further. Thus, we can produce at least 3 strains of IgG antibody against the specific epitope of NT-proBNP, which will be the important foundation of developing the new NT-proBNP immunodiagnostic kit. 3. Preliminary development of an immunosensor for the determination of the human NT-proBNPIn the fabrication of antibody nano-immunosensor, electrodeposition technology has been used to deposit a gold nanoparticles film on the surface of the glassy carbon electrode in order to adsorb monoclonal antibody against human NT-proBNP on the surface gold nanoparticles. The change of electrical signal caused by the capture of E.coli on the electrode surface was detected by Cyclic Voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We detected the level of human NT-proBNP in serum from a few patients'samples by CV and the results were better consistent with the clinical diagnosis, which proves that this method based on the immunosensor for the determination of NT-proBNP is feasible and perspective in clinical diagnosis.