Fusion expression, purification, and characterization of cytokeratin 19 fragments in E. coli for enhanced stability in diagnostic applications.

Cytokeratin 19 fragment (CYFRA21-1) serves as a crucial tumor marker in the context of lung cancer patients, playing a pivotal role as a calibrator in the realm of in vitro diagnostics. Nevertheless, during practical application, it has come to light that the recombinantly synthesized full-length CYFRA21-1 antigen exhibits suboptimal stability at the requisite concentration, while the utilization of natural antigens incurs a substantial cost. To address this issue, our investigation harnessed a strategic approach whereby the soluble fragment of cytokeratin 19 (Aa244-400) was integrated into the pET32a vector, subsequently being expressed within E. coli through a fusion with the TrxA protein. This process involved induction of protein expression through 0.2 mM IPTG at 16 °C for a duration of 16 h. After induction, the target protein was purified through Ni affinity and ion exchange chromatography. Subsequent characterization of the targeted protein was executed through the SEC-HPLC technique. The attained CYFRA21-1 antigen, as generated within this study, was effectively incorporated into a chemiluminescence-based in vitro diagnostic detection kit. The results indicate that the fusion protein exhibited commendable reactivity and stability, manifesting a deviation of less than 10 % following incubation at 37 °C for 7 days. Importantly, the production yield achieved a notable magnitude of 300 mg/L, thus rendering it a cost-effective and scalable alternative to natural antigens for clinical diagnostic applications.

Ultrasensitive quantitation of Paraquat based on a small molecule-induced dual-cycle amplification strategy.

Paraquat (PQ) is a typical biotoxic small molecule. Knowledge of how to directly introduce it into cyclic amplification rather than transform it into a secondary target is lacking in current analytical methods. Considering the urgent need for trace pesticide residue detection and the inherent defects of small molecule analysis, a CRISPR/Cas12a-driven small molecule-induced dual-cycle strategy was developed based on the immune competition method. The key to signal amplification is the mutual activation and acceleration between Cycle 1 triggered by the small molecule and Cycle 2 driven by CRISPR/Cas12a. Impressively, small molecules have been successfully incorporated into the dual-cycle strategy, which achieves a low detection limit (3.1 pg/mL) and a wide linear range (from 10 pg/mL to 50 µg/mL). Moreover, the designed biosensor was successfully employed to evaluate the PQ residual level in real samples and showed effective implementation for the bioanalysis of small molecule targets and pesticide residue-related food safety.

A rapid and reliable immunochromatographic strip for detecting paraquat poinsoning in domestic water and real human samples.

Paraquat (PQ) is one of the most commonly used herbicides, but it has polluted the environment and threatened human health through extensive and improper usage. Here, a new naked-eye PQ immunochromatographic strip was developed to recognize PQ in domestic water and real human samples within 10 min based on a novel custom-designed anti-PQ antibody. The PQ test strip could recognize PQ at a concentration as low as 10 ng/ml, reaching the high-efficiency time-of-flight mass spectrometry detection level and identifying trace amounts of PQ in samples treated with a diquat (DQ) and PQ mixture. Notably, both the performance evaluation and clinical trial of the proposed PQ strips were validated in multiple hospitals and public health agencies. Taken together, our study firstly provide the clinical PQ-targeted colloidal gold immunochromatographic test strip designed both for environment water and human sample detection with multiple advantages, which are ready for environmental monitoring and clinical practice.

A novel photoelectrochemical strategy based on an integrative photoactive heterojunction nanomaterial and a redox cycling amplification system for ultrasensitive determination of microRNA in cells.

An ultrasensitive photoelectrochemical (PEC) bioassay for determination of microRNA was proposed based on an integrative photoactive heterojunction nanomaterial to provide the basis of excellent PEC responses and an efficient redox cycling amplification system to improve the detection performances. To establish the bioassay system, the biosensor was firstly modified with Bi2WO6@Bi2S3 and alkaline phosphatase (ALP). The detection solution was composed of ascorbic acid phosphate (AAP) and ferrocenecarboxylic acid (FcA), where ALP converted AAP into ascorbic acid (AA) to trigger a process of redox cycling amplification by reducing FcA+ to FcA, resulting in enhanced photocurrent responses of Bi2WO6@Bi2S3. In the presence of microRNA 21, it could trigger a hybridization chain reaction via the special designed hairpin DNA to produce a long repeated DNA sequences to inhibit ALP activity. Thus the reduced ALP activity and consequently decreased photocurrent signal could be obtained for detection of microRNA 21. As expected, this bioassay system performed the satisfactory performances for the ultrasensitive detection of microRNA 21 in the range from 1 fM to 1 nM with an experimental detection limit of 0.26 fM and acceptable practical applicability. Collectively, an efficient PEC bioassay for microRNA 21 is established and this strategy can be expanded to detect other microRNAs, even other molecules in cells.

Capillary morphogenesis gene 2 maintains gastric cancer stem-like cell phenotype by activating a Wnt/ß-catenin pathway.

A growing body of evidence shows that the development and progression of gastric cancer (GC) is mainly associated to the presence of gastric cancer stem-like cells (GCSLCs). However, it is unclear how GCSLC population is maintained. This study aimed to explore the role of capillary morphogenesis gene 2 (CMG2) in GCSLC maintenance and the relevance to GC progression. We found that CMG2 was highly expressed in GC tissues and the expression levels were associated with the invasion depth and lymph node metastasis of GC, and inversely correlated with the survival of GC patients. Sorted CMG2High GC cells preferentially clustered in CD44High stem-like cell population, which expressed high levels of stemness-related genes with increased capabilities of self-renewal and tumorigenicity. Depletion of CMG2 gene resulted in reduction of GCSLC population with attenuated stemness and decrease of invasive and metastatic capabilities with subdued epithelial-mesenchymal transition phenotype in GC cells. Mechanistically, CMG2 interacted with LRP6 in GCSLCs to activate a Wnt/ß-catenin pathway. Thus, our results demonstrate that CMG2 promotes GC progression by maintaining GCSLCs and can serve as a new prognostic indicator and a target for human GC therapy.

Pre-exposure to 50 Hz-electromagnetic fields enhanced the antiproliferative efficacy of 5-fluorouracil in breast cancer MCF-7 cells.

Resistance to 5-fluorouracil (5-FU) and its induced immune suppression have prevented its extensive application in the clinical treatment of breast cancer. In this study, the combined effect of 50 Hz-EMFs and 5-FU in the treatment of breast cancer was explored. MCF-7 and MCF10A cells were pre-exposed to 50 Hz-EMFs for 0, 2, 4, 8 and 12 h and then treated with different concentrations of 5-FU for 24 h; cell viability was analyzed by MTT assay and flow cytometry. After pre-exposure to 50 Hz-EMFs for 12 h, apoptosis and cell cycle distribution in MCF-7 and MCF10A cells were detected via flow cytometry and DNA synthesis was measured by EdU incorporation assay. Apoptosis-related and cell cycle-related gene and protein expression levels were monitored by qPCR and western blotting. Pre-exposure to 50 Hz-EMFs for 12 h enhanced the antiproliferative effect of 5-FU in breast cancer cell line MCF-7 in a dose-dependent manner but not in normal human breast epithelial cell line MCF10A. Exposure to 50 Hz-EMFs had no effect on apoptosis and P53 expression of MCF-7 and MCF10A cells, whereas it promoted DNA synthesis, induced entry of MCF-7 cells into the S phase of cell cycle, and upregulated the expression levels of cell cycle-related proteins Cyclin D1 and Cyclin E. Considering the pharmacological mechanisms of 5-FU in specifically disrupting DNA synthesis, this enhanced inhibitory effect might have resulted from the specific sensitivity of MCF7 cells in active S phase to 5-FU. Our findings demonstrate the enhanced cytotoxic activity of 5-FU on MCF7 cells through promoting entry into the S phase of the cell cycle via exposure to 50 Hz-EMFs, which provides a novel method of cancer treatment based on the combinatorial use of 50 Hz-EMFs and chemotherapy.

[Preparation and application of monoclonal antibody against human LOC339524 protein].

OBJECTIVE: To prepare and apply monoclonal antibody (mAb) against human LOC339524 protein. METHODS: 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. RESULTS: 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. CONCLUSION: The mAb against human LOC339524 protein with good specificity and high titer has been successfully prepared.

Identification of broadly conserved cross-species protective Leishmania antigen and its responding CD4+ T cells.

There is currently no clinically effective vaccine against leishmaniasis because of poor understanding of the antigens that elicit dominant T cell immunity. Using proteomics and cellular immunology, we identified a dominant naturally processed peptide (PEPCK335-351) derived from Leishmania glycosomal phosphoenolpyruvate carboxykinase (PEPCK). PEPCK was conserved in all pathogenic Leishmania, expressed in glycosomes of promastigotes and amastigotes, and elicited strong CD4(+) T cell responses in infected mice and humans. I-A(b)-PEPCK335-351 tetramer identified protective Leishmania-specific CD4(+) T cells at a clonal level, which comprised ~20% of all Leishmania-reactive CD4(+) T cells at the peak of infection. PEPCK335-351-specific CD4(+) T cells were oligoclonal in their T cell receptor usage, produced polyfunctional cytokines (interleukin-2, interferon-γ, and tumor necrosis factor), and underwent expansion, effector activities, contraction, and stable maintenance after lesion resolution. Vaccination with PEPCK peptide, DNA expressing full-length PEPCK, or rPEPCK induced strong durable cross-species protection in both resistant and susceptible mice. The effectiveness and durability of protection in vaccinated mice support the development of a broadly cross-species protective vaccine against different forms of leishmaniasis by targeting PEPCK.

[Preparation, epitope analysis and clinical application of monoclonal antibodies against heart-type fatty acid binding protein].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection.

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.

A novel microfluidic immunoassay system based on electrochemical immunosensors: an application for the detection of NT-proBNP in whole blood.

Electrochemical immunosensors have attracted great interest in the search for a selective, simple and reliable system for molecular recognition. Presently, electrochemical immunosensors have been widely studied for biomedical molecular's detection, but the regeneration of these immunosensors has restricted their wide application. To prepare a regeneration-free immunosensor, which may be more suitable for clinical determination, a repeatable immunoassay system was developed based on an electrochemical immunosensor with magnetic nanoparticles, biotin-avidin system (BAS) and Fab antibodies for the heart failure markers aminoterminal pro-brain natriuretic peptides (NT-proBNP). At the same time, a microfluidic system was combined into the proposed system, which enabled continuous determination. Using NT-proBNP as a model system, the proposed immunosensor exhibited rapid and sensitive amperometric response to NT-proBNP with good selectivity, stability, and a wide linear range (0.005-1.67 ng/mL and 1.67-4 ng/mL with a detection limit of 0.003 ng/mL under optimal conditions). Importantly, the proposed immunosensor was also suitable for the detection of other proteins and provided new opportunities for disease diagnosis.

Elevation of plasma soluble CD26 levels during pregnancy.

AIM: CD26 is a type II transmembrane protein with dipeptidyl peptidase IV (DPPIV) activity expressed on a variety of cell types. Recent studies have indicated that CD26 or enzymatic activity levels were previously associated with immune-mediated disorders. As immunoregulation is very important for a successful pregnancy, we hypothesize that CD26 may play an important role during pregnancy, and herein, we sought to determine the association between circulating levels of soluble CD26 (sCD26) and pregnancy outcome. MATERIAL AND METHODS: In this study, a stable hybridoma cell line 1F1 was produced by fusion of murine splenocytes and myeloma cells. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for the detection of the maternal plasma sCD26. We measured the plasma levels of sCD26 in 80 normal pregnant women and 45 non-pregnant women. RESULTS: Our results indicated that the plasma level of sCD26 was significantly higher in the pregnant group (P < 0.001) than that in the non-pregnant group. CONCLUSION: These findings hinted that CD26 may play a role in successful pregnancy and it is not an absolute surrogate marker for the Th1-type immunity as the dominant Th2-type immunity during pregnancy.

Characterization, epitope identification and mechanisms of the anti-septic capacity of monoclonal antibodies against macrophage migration inhibitory factor.

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.

Application of a Fab fragment of monoclonal antibody specific to N-terminal pro-brain natriuretic peptide for the detection based on regeneration-free electrochemical immunosensor.

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.

Ultrasensitive electrochemical strategy for NT-proBNP detection with gold nanochains and horseradish peroxidase complex amplification.

A novel electrochemical immunosensor for sensitive detection of cardiac biomarker N-terminal pro-B-type natriuretic peptide (NT-proBNP) is fabricated based on the nanostructural gold and carbon nanotubes composite as desirable platform for the capture antibodies immobilization and gold nanochains (AuNCs) and horseradish peroxidase (HRP) complex labeled secondary antibodies (AuNCs-HRP-Ab(2)) for signal amplification. The gold nanochains were prepared by the employment of L-ascorbic acid (AA) as a mediator and template. With the surface area enhancement by nanostructural gold functionalized carbon nanotubes composite, the amount of immobilized primary antibodies (Ab(1)) can be enhanced. More importantly, enhanced sensitivity can be achieved by introducing the multibioconjugates of AuNCs-HRP-Ab(2) onto the electrode surface through "sandwich" immunoreactions. The linear range extended from 0.02 to 100 ng/mL with a correlation coefficient of R=0.997 and a limit of detection reaching 6 pg/mL at a signal-to-noise ratio of 3:1, which is well below the commonly accepted concentration threshold (0.1 ng/mL) used in clinical diagnosis. The specificity, regeneration and stability test demonstrated the feasibility of the developed immunoassay, which gives the attractive characteristics to be a candidate for the detection of NT-proBNP and other proteins of interest in both fundamental and applied research.

Overexpression of the Interferon regulatory factor 4-binding protein in human colorectal cancer and its clinical significance.

BACKGROUND: IFN regulatory factor 4-binding protein (IBP) is a novel type of activator of Rho GTPases. It has been linked with differentiation and apoptosis of lymphocytes, but its function in oncogenesis remains unclear. Here we studied the expression of endogenous IBP in four human colorectal cancer cell lines, normal, adenoma and tumor colorectal tissues. METHODS: Molecular (Western blot and RT-PCR), and confocal analyses were used to investigate IBP expression in human colorectal cancer cell lines. Matched normal and tumor tissue sections of 63 patients and 15 adenoma tissue sections were analyzed for IBP expression by immunohistochemistry (IHC). RESULTS: IBP was ubiquitely expressed in human colorectal cancer cell lines. The expression of IBP can be detected at both the mRNA and protein level in SW480, SW620 and HT29 cells. Clinically, IBP were elevated in human colorectal cancer specimens in comparison to normal colorectal tissues. Substantial high expression of IBP was observed in colorectal cancer tissues (67%), whereas corresponding normal tissues and 15 adenoma tissues showed consistently absent immunoreactivity of IBP. Moreover, IBP expression is correlated with the differentiation level of colorectal cancer cells (p<0.05) and clinical stage of patients (p<0.01). CONCLUSIONS: Our data show, for the first time, a dysregulated expression of IBP in human colorectal cancer, offering new perspectives for its role in cancer development and progression. IBP may be a novel tumor marker and a therapeutic target for colorectal cancer.

A novel, label-free immunosensor for the detection of alpha-fetoprotein using functionalised gold nanoparticles.

BACKGROUND AND OBJECTIVES: Novel immunoassay methods based on electrochemical sensors have been developed, but most of these immunosensors are unsuitable for clinical detection because their preparation requires complicated chemical procedures and because their detection sensitivity is restricted. In order to develop a highly sensitive, label-free amperometric sensor for immunoassays, we synthesised novel, functionalized gold nanoparticles (SV-GNP) by covalently capping the surface of gold nanoparticles (GNP) with 1,1'-bis-(2-mercapto)-4,4'-bipyridinium dibromide, a kind of sulfhyrdryl viologen (SV). DESIGN AND METHODS: We fabricated an immunosensor in a multi-step fashion, by first coating the SV-GNP onto a glassy carbon electrode surface; the resulting electrode core could then adsorb a suitable antibody in a second step to afford the desired immunosensor. alpha-fetoprotein (AFP) was used as a model analyte in this work. RESULTS: The anti-AFP/SV-GNP-modified electrode was sensitive to AFP with a linear relationship between 1.25 and 200 ng/mL and a correlation coefficient of 0.9983; the detection limit at a signal to noise ratio of 3 was 0.23 ng/mL under optimal conditions. In addition, the proposed immunosensor exhibited good sensitivity, selectivity, stability and long-term maintenance of bioactivity. CONCLUSION: The described immunosensor preparation and immunoassay methods offer promise for label-free, simple, and cost-effective analysis of biological samples.

[Preparation and characterization of rabbit anti-human apurinic/apyrimidinic endonuclease(APE1) polyclonal antibody].

AIM: To prepare a rabbit anti-human apurinic/apyrimidinic endonuclease polyclonal antibody and then have its characterization analyzed. METHODS: A rabbit polyclonal antibody was prepared through a modified rapid immune procedure and then it was purified using a specific avidity column. The efficacy of this antibody was detected by ELISA, Western blot and immunohistochemistry. RESULTS: The titer of the antiserum determined by ELISA was up to 1:128,000. The kaff value of the antibody was 8.96 x 10(-6) mol/L. Western blot analysis showed the antibody was of high specificity. The final purified antibody was highly specific to native form of APE1 protein in mice and rats. CONCLUSION: The rabbit anti-human APE1 polyclonal antibody with high titer and specificity has been successfully prepared. It can be used not only to elucidate the roles of APE1 protein in many important cellular procedures, but also to detect the expression of the APE1 protein in mice and rats.

Development of a novel method to measure macrophage migration inhibitory factor (MIF) in sera of patients with rheumatoid arthritis by combined electrochemical immunosensor.

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.