Rapid Detection Of Helicobacter Pylori Based On Immunomagnetic Beads And Quantum Dots Binding Specific Antibodies

Helicobacter pylori（H.pylori）is a spiral-shaped Gram-negative bacterium,a human pathogen that has strict requirements for the growth environment and is slightly aerobic.H.pylori infection is the main cause of atrophic gastritis,precancerous lesions,and gastric cancer.Generally,H.pylori is infected in early childhood and will remain infected without antibiotic treatment.However,it’s impractical to conduct epidemiological analysis or upper gastrointestinal endoscopy of both asymptomatic children and young people infected with H.pylori.In addition,because younger children are less sensitive,they are not suitable for serum antibodies testing.The ¹³C-urea breath test（UBT）,although it can be performed in older children,is not suitable for young children.The fecal sample has a complicated matrix and a long pretreatment time.The traditional detection technology of enzyme-linked immunosorbent assay（ELISA）cannot meet the requirements of stool antigen detection.Therefore,some new technologies for stool antigen detection have attracted much attention.In this paper,H.pylori outer membrane protein and KLH-synthetic peptide were used as antigens to immunize New Zealand white rabbits and Balb/C mice,respectively,to obtain polyclonal antibodies and monoclonal antibodies.Then,the specific Ig G was obtained by a saturated ammonium sulfate gradient salting-out method and Protein G affinity purification.SDS-PAGE gel electrophoresis analysis was used to determine the purity and relative molecular mass of the polyclonal and monoclonal antibodies;the enzyme-linked immunosorbent assay（ELISA）was used to detect the titer of the purified polyclonal antibodies and monoclonal antibodies.Polyclonal antibodies are combined with fluorescent quantum dots to form quantum dot probes;monoclonal antibodies are combined with immunomagnetic beads to form immunomagnetic bead probes,and immunomagnetic bead probes efficiently and specifically bind to target bacteria in complex matrices to form antigen-antibodies-immunomagnetic The bead complex is eluted to form an antigen-antibodies complex to specifically bind to the quantum dot probe.Finally,this complex is detected under a fluorescence spectrometer.The results of this study indicate that the purity of the monoclonal antibodies and polyclonal antibodies that have passed through the saturated ammonium sulfate gradient salting-out method and the Protein G affinity purification are all above 90%.The titer of the purified monoclonal antibodies reached 1:900,the titer of the polyclonal antibodies before purification was 1:32000,and the titer of the purified antibodies was 1:128000,which increased the titer by 4 times.The prepared monoclonal antibodies and polyclonal antibodies have the characteristics of high titer,good specificity and stable properties.By optimizing the detection method,the total detection time for the detection method of H.pylori by immunomagnetic beads and quantum dot binding antibodies is less than or equal to120 minutes,the detection limit is 10²CFU/m L,and the linear range is 10 to 10⁶CFU/m L.The regression linear equation is y=53.0342x+15.2080,and the correlation coefficient R²=0.9962,showing a good linear relationship.The detection did not cross-react with 10 other common bacterial species in feces,indicating that the established method has strong specificity.This method was used for the preliminary qualitative detection of H.pylori with different concentrations in the feces of healthy humans.The spiked recoveries of the samples were 89.1%-107.2%,indicating that the established method has good accuracy.Compared with the results of literature detection time and detection limit of other immunomagnetic beads and quantum dot-bound antibodies,the research method in this paper has the advantages of short time-consuming and high sensitivity.In summary,the method for detecting H.pylori in human feces established in this paper meets the detection requirements specified by immunoassay,thereby laying a foundation for the diagnosis of H.pylori.