The Preliminary Application Of Nephelometric Immunoassay And Immunomagnetic Separation Mediated By Staphylococcus Aureus And SPA In The Rapid Automatic Quantitative Detection Of Shigella.flexneri

Background:Shigella, which was also known as dysenteriae bacteria, could invade colon epithelial cells and cause human bacillary dysentery. Based on the antigen structure and biochemical reactions it was divided into Sh. dysentery, Sh. flexneri, Sh. sonnei and Sh. boydii. The clinical symptoms caused by Shigella included: diarrhea, dysentery and grume stool with pyoid blood, spasmodic abdominal pain and tenesmus, which were even life-threatening [1,2,3]. In recent years, Shigella flexneri was the most important pathogen of bacillary dysentery which was the common gastro-intestinal disease in summer and autumn, and there was a high incidence and serious hazards. Recently, it was reported that there was a growing antibiotic resistance of Shigella, among which Shigella flexneri was the most significant [4]. There are many different types of gastrointestinal pathogenic bacteria, which were hided in the normal flora with relatively low concentration and be easily missed as undetected pathogen. But traditional method of pure culture isolation and identification of bacteria is time-consuming and cumbersome operating [5]. Therefore the new methods for bacteria detection need to be developed.Immunomagnetic beads (IMB) have the capability of capturing, separating and enriching the specific pathogen, which demonstrated special advantages in the detection of gastrointestinal pathogen: specificly capturing and isolating the pathogen from a complex flora, the enrichment of IMB could concentrate a small proportion of pathogen even the absolute amount was small. Thereby it enhanced the detection sensitivity and accuracy [6]. A protein of Staphylococcus aureus (Staphylococcal protein A, SPA) could bind with the Fc fragment of people and a wide range of mammalian IgG molecules in serum, and the IgG retains its antigen binding capacity. The features of SPA and Staphylococcus aureus were useful in the research of the rapid automatic quantitative detection of Shigella flexneri. Objective:To establish a nephelometric immunoassay combined with immunomagnetic separation mediated by Staphylococcus aureus and SPA for the rapid automatic quantitative detection of Shigella.flexneri.Methods:.1.Culturing Shigella flexneri 1a confirmed by biochemical and serological identification and preparing immunogen. Then preparing the specific antiserum by rabbits immunized with inactivated Shigella flexneri 1a.2.The magnetic beads coated with SPA binding to the specific IgG from the antiserum were used to capture the Sh.flexneri from the sample. Then the bacteria captured by magnetic beads was inoculated on MH plate and cultivated at 37℃for 18-24h, and the enrichment efficiency of the immunomagnetic beads was calculated after counting the colony.3.The control experiment was done with commercialized magnetic beads at the same time and the comparison was also done with the traditional method of counting colony.4.SPA-rich Staphylococcus aureus killed by formalin was combined with the specific IgG from the specific antiserum made previously then used as nephelometric immunoassay reagent.5.The rapid automatic quantitative detection of Shigella flexneri was explored and performed on the Hitachi 7060 biochemistry analyzer.6.The control experiment was done with SYSMEX UF-50 automatic urine analyzer which has the function of bacteria quantitative analysis by flow cytometry(FCM).7.Shigella flexneri 1a from the simulated samples was enriched by immunomagnetic separation, then after amplified in Luria-Bertanin broth it was detected by nephelometric immunoassay on the Hitachi 7060 biochemistry analyzer. Through which we want to establish a nephelometric immunoassay combined with immunomagnetic separation mediated by Staphylococcus aureus and SPA for the rapid automatic quantitative detection of Shigella.flexneri.Results:1.The specific antiserum with high quality of agglutination titer reached 1:320 was prepared from rabbits immunized with inactivated Shigella flexneri 1a.2. The immunomagnetic beads have shown some enrichment effect of Shigella flexneri 1a, and the colony counting of plate culture after enriched by immunomagnetic beads indicated that the capture efficiency was about 30% and the bacterial concentration multiplied, which have the same effect with the commercialized magnetic beads.3. SPA-rich Staphylococcus aureus combined with the specific IgG from the antiserum was able to agglutinate with the corresponding antigen,which was called coordinated agglutination reaction [7]. This reagent has a significant agglutination phenomenon with the Shigella on the slide and performed well on the automatic biochemistry analyzer.4.The method of automatic quantitative detection of Shigella flexneri was established successfully on the automatic analyzer, which has a detection limit of 1000 cfu and relatively satisfied specificity and linear range. Combined with immunomagnetic separation, this method could accomplish the identification of bacteria from clinical samples in hours,while the routine methods need 2-3 days.Conclusion:The magnetic beads coated with SPA binding to the specific IgG from the antiserum have shown some specific capture and enrichment effect of Shigella flexneri 1a . The SPA-rich Staphylococcus aureus coated with the specific IgG was used as nephelometric immunoassay reagent and could lead to good results. On the basis of nephelometric immunoassay combined with immunomagnetic separation, the rapid automatic quantitative immunoassay for the detection of Shigella flexneri was established, which provides a new way for the rapid diagnosis and automatic quantitative analysis of pathogenic bacteria.