Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.
COVID-19/diagnosis* ; Humans ; Microfluidics ; Nucleic Acid Amplification Techniques ; Polymerase Chain Reaction ; SARS-CoV-2/genetics*

Objective To discuss the protective effect of creatine phosphate (CP)on isolated rat liver against cold preservation.Methods Isolated perfused rat liver model under simple cold preservation was established.The liver of the control group was perfused with pure University of Wisconsin (UW)solution. With UW solution as the base fluid,the liver of the low-dose group was perfused with 1 g/100 ml CP in UW solution;the liver of the middle-dose group was perfused with 2 g/100 ml CP in UW solution;the liver of the high-dose group was perfused with 3 g/100 ml CP in UW solution.The livers of each group were cold preserved in the corresponding perfusion fluid at 4 ℃.The content of alanine aminotransferase (ALT)and lactate dehydrogenase (LDH)in preservation solution in infrahepatic vena cava were determined.The content of malondialdehyde (MDA)and activity of myeloperoxidase (MPO)in liver tissues were detected.The apoptosis index (AI)of liver cells in liver tissues and positive expression rate of NF-κB in liver tissues were observed. Pathologic changes of liver tissues were observed under optical microscope.Results At 12 h after the cold preservation,the content of ALT and LDH in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 18 h after the cold preservation,the content of MDA and MPO in the liver tissues of low-,middle-and high-dose groups were lower than those of the control group (all in P <0.05).At 12 h and 18 h after the cold preservation,AI and positive expression rate of NF-κB in liver cells in the rat livers of low-,middle-and high-dose groups were lower than those of the control group (all in P<0.05).At 24 h after the cold preservation,the content of ALT and MDA in preservation solution of the high-dose group was obviously higher than that of the control group as well as the low-and middle-dose groups (all in P <0.05).The results of pathological examination indicated that the injuries to the livers of the high-,middle-and low-dose groups were obviously lighter than that of the control group.There was no obvious difference among each dose group.Conclusions CP in UW solution may well protect the isolated rat liver against cold preservation,which is better than pure UW solution.