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N-Terminal pro-brain natriuretic peptide (NT-proBNP) is an important marker for heart failure that reflects ventricular volume expansion, ventricular overload and the degree of cardiac injury. To establish a sensitive and simple detection method for it in serum, a regeneration-free immunosensor with novel Fab fragment monoclonal antibodies was prepared. The sensor detected NT-proBNP from 0.04 to 2.5 ng/ml with a limit of 0.03 ng/ml. The immunosensor is therefore a simple, cost-effective method to detect NT-proBNP and the proposed immunoassay system would enable other proteins to be detected and open new opportunities for protein diagnostics.

To prepare and apply monoclonal antibody (mAb) against human LOC339524 protein.The non-glycosylated antigenic gene sequence of the LOC339524 protein was expressed in triplicate to enhance immunogenicity. Then this synthetic gene was connected to pET28a plasmid. Recombinant LOC339524 protein was obtained by E.coli expression system and was administered intraperitoneally as an immunogen to BALB/c mice to obtain mAb. The specificity and titer of the mAb were characterized by ELISA. Recombinant LOC339524 protein was identified through Western blotting. The expression of the LOC339524 protein in human myocardial tissues and H9C2 cells were detected by immunohistochemistry, and its level in the sera of patients with different heart diseases was detected with the antibody.We obtained two hybridoma cell lines, 5-D3 and 4-F8, secreting specific mAbs against LOC339524 protein. The titer of 5-D3 was up to 2×10(6), higher than the titer of 4-F8. Western blotting and immunohistochemistry demonstrated that 5-D3 could specifically recognize LOC339524 protein of Homo sapiens and Rattus norvegicus. With the antibody we obtained, we successfully detected the serum level of LOC339524 protein in patients with different heart diseases.The mAb against human LOC339524 protein with good specificity and high titer has been successfully prepared.

To prepare and characterize the monoclonal antibody (mAb) against recombinant human heart-type fatty acid binding protein (H-FABP) and apply it to the clinical analysis.BALB/c mice were immunized with recombinant H-FABP (rH-FABP) from prokaryotic expression or synthesized peptide fragment. After common fusion and screening, the subtypes, titer and affinity of mAbs were detected respectively. After purification, the specificity of mAbs was tested by indirect ELISA and Western blotting. ELISA system was established by pair mapping and applied in the detection of clinical samples. Two epitope peptides were designed by bioinformatics and used to detect the epitope of 1-F10 through Western blotting.Four different hybridoma clones secreting anti-H-FABP antibodies were developed, with high titres of 1:51 200-1:1024 000. Immunoglobulin types of these mAbs were found to be IgG2a or IgG2b, respectively. The affinity of the mAb 1-F10 even reached 9.02×10(9); mol/L. ELISA and Western blotting showed that these mAbs could identify H-FABP specifically. In addition, the pair mapping of monoclonal antibodies 3-H5-1-F10 could recognize H-FABP in human serum samples. Furthermore, the specific target recognized by 1-F10 mAb was located within amino acid 86-133 of H-FABP.Four highly specific mAbs against H-FABP were successfully obtained and the established ELISA system could be used to detect the H-FABP in clinical serum samples, and the epitope of 1-F10 mAb was also verified.

Objectives The hepatitis B virus (HBV) preS1 protein is divided into an epitope region and a non-epitope region based on the respective antigenicities of these regions. Most of the antibodies that are currently used to detect the large surface protein of HBV (HBV LHB) are specific to the epitope region of preS1, which may contribute to the false negative results of HBV LHB detection assays. Here, we established a mouse monoclonal antibody (mAb) that could improve the efficiency of HBV LHB detection. Design and methods The HBV preS1 protein was expressed in E. coli strain BL21 and used to screen hybridoma clones. HBV preS1-specific mAb was produced by immunizing mice with a chemically synthesized peptide antigen derived from the non-epitope region of HBV preS1. The mAb was characterized by ELISA, Western blot, and immunocytochemistry and was subsequently used in serum sample tests. Results Based on in silico B cell epitope predictions, the HBV preS1 aa 91–117 peptide was synthesized as an antigen. Recombinant HBV preS1 was expressed in E. coli and identified by SDS-PAGE. The mAb D8 (IgG2b) recognized the recombinant preS1 protein in both ELISA and Western blot assays and also recognized the preS1 protein expressed in plasmid-transfected HepG2.2.15 cells by immunocytochemistry. Furthermore, the D8 mAb, which is specific for the non-epitope region of preS1, contributed to the improved sensitivity and specificity of HBV detection. Conclusions We established an mAb that is specific to the non-epitope region of HBV preS1 and improved the detection of HBV LHB in an ELISA assay. This mAb could help increase the accuracy of the clinical measurement of preS1.

Sepsis is characterized by uncontrolled inflammatory responses. Macrophage migration inhibitory factor (MIF) has been shown to play an important role in the progression of sepsis thus is a potential therapeutic target. The aim of this study is to produce IgG anti-MIF monoclonal antibodies (mAbs) with anti-septic abilities in vivo and to determine mechanisms of their function. We generated 8 IgG anti-MIF mAbs with high specificity and 3 of them showed potent protective abilities in murine lethal peritonitis induced by cecal ligation and puncture (CLP). One anti-MIF mAb, F11, showed 100% protection within 72 h after sepsis induction and 72% mice treated with this mAb survived up to 84 h with reduced lung and kidney pathology. F11 treatment also reduced tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels in septic mice. We further found that all 8 anti-MIF mAbs recognized the same epitope located in the amino acid residue 1-20 region of the N terminus of the MIF protein. Three of the mAbs, F11 in particular, inhibited tautomerase activity in association with their protective effect on CLP mice. Thus, we have produced anti-MIF mAbs that protected mice from CLP-induced sepsis by recognizing the same epitope domains in MIF. These mAbs are promising candidates for further development of therapeutics against inflammatory diseases.

Macrophage migration inhibitory factor (MIF) has a multitude of biological activity and is associated with a number of inflammatory and immune diseases, including rheumatoid arthritis (RA). The increased serum levels of MIF in patients not only suggest this protein as a marker for disease progression, but also as a potential therapeutic target. The aim of this study is to develop a novel electrochemical method to more precisely and conveniently measure MIF in patient sera. An IgM murine monoclonal antibody (mAb) against human MIF was prepared and used in the electrochemical immunosensor, with modified gold electrode coated with compounds of gold nanoparticles, titanium dioxide nanoparticles and thionine (NGP-NTiP-Thi) followed by adsorption of anti-MIF antibodies with IgM or IgG1 isotype. The IgM immunosensor recognized MIF in a linear relationship in the range of 0.03 and 230 ng/mL with the lower limit (S/N=3) of 0.02 ng/mL. The measurement showed considerable levels of sensitivity, selectivity, stability and long-term maintenance of bioactivity, as shown by testing with serum MIF in RA patients as compared to healthy donors. The performance of the IgM immunosensor was also superior to IgG1 sensor. Thus, we have developed a novel measurement approach for serum MIF, which may have great potential in the clinic for monitoring the course of diseases associated with increased MIF.