Development Of Immunochromatographic Colloidal Gold Test Strips For The Detection Of Human Haemophilus Influenzae

Haemophilus influenzae is one of the major pathogens causing a variety of upper respiratory diseases and lower respiratory diseases,including acute otitis media,pneumonia,bronchitis,chronic obstructive pulmonary disease and conjunctivitis.In addition,the incidence of non-typeable Haemophilus influenzae(NTHi)is increasing year by year,and its clinical isolation rate has reached more than 50%.Therefore,rapid and accurate detection of Haemophilus influenzae is more clinically meaningful in the early stage of infection.At present,there are three main types of detection methods:serological detection,nucleic acid detection and direct detection of pathogens.These three methods have certain limitations,and it is difficult to meet the needs of clinical rapid,simple and accurate detection.Therefore,there is an urgent need to establish a rapid,highly sensitive and highly specific detection method.The study found that P6 protein is a surface-exposed peptidoglycan-related outer membrane lipoprotein with high conservation and strong antigenicity.In this study,P6protein was used as a detection marker of Haemophilus influenzae,and monoclonal antibody was prepared by using P6 recombinant protein as antigen.A colloidal gold immunochromatographic technique was used to establish a rapid and accurate method for detecting Haemophilus influenzae.In this paper,through the bioinformatics analysis of P6 protein,the DNA coding sequence corresponding to the peptide with the most abundant epitope was found.The whole gene sequence after chemical synthesis was cloned into the expression vector and then induced to express at low temperature.The purity of the protein was more than90%.The monoclonal antibody was prepared.Finally,11 hybridoma cells(Hi-1#~Hi-11#)stably secreting the target antibody were obtained,and the titer reached1:256000 by indirect ELISA..The two kinds of antibodies were identified by Western blotting,immunofluoresce-nce and biofilm interferometry.The results showed that the prepared monoclonal antibodies can specifically recognize Haemophilus influenzae surface antigen and have higher affinity.The prepared four monoclonal antibodies were subjected to colloidal gold labeling and nitrocellulose membrane coating,the best combination is that Hi-1#coated on the NC membrane as the location of the capture test line and Hi-7#as a gold standard antibody.The prepared test strip has high sensitivity(1×10~6 CFU/mL)and strong specificity.The test time of the sample is within 15 min,and it does not cross-react with other nine common respiratory pathogens such as Streptococcus pneumoniae,Moraxella catarrhalis,Mycoplasma pneumoniae,Legionella pneumophila.Test strips are well preserved at 45?.In addition,using the culture method as a control,288 clinical samples were tested with the Haemophilus influenzae colloidal gold test strip prepared in this experiment,and the positive coincidence rate of the two was 90.7%,indicating that the prepared test strips has a good clinical application prospect.