Study On Detection Of Pathogenic Microorganism And Toxin By Protein Microarray

Some agents of disease have been used as weapons of terror for centuries. Bioterror agents are most often colorless, odorless microorganisms (bacteria, viruses) or toxins (usually protein toxins) that can be spread in air as aerosols or in food or drink to infect people. The outbreak of inhalation anthrax in Florida and cutaneous anthrax in New York in the first week of October 2001 might be an example of attack by bioterror agents. And some new disease have potent risk be used as terror agents. For example, Newcastle disease virus and Avian influenza virus have had ability to infect human. Most bioterror agents do not cause unique clinical signs and symptoms that are immediately recognizable in exposed individuals. Early detection of bioterror attack is of obvious importance.The traditional immunoassay platforms have limited multiplexing capability and high sample consumption, for example, enayme linked immunosorbent assay(ELISA). Protein microarray was development as an methods with many characteristics just like fast, low consumption of sample. The protein microarray is an array of protein on the surfaces of modified slide. There are many types of microarrays, and many ligands spot on (protein, peptides, receptors, some small molecule). Today, with the development of monoclonal antibody(McAb), it use in protein microarray.In this study, we development a protein microarray for detection of seven latent bioterror agents and latent threat to safe of public health. Such as abrin toxin, recin toxin, EHEC O157, Avian Influenza Virus, Newcastle Disease Virus , Brucella melitensis and Brucella abortus.Firstly, in above correlation research report foundation, use the hot phenol method to separately abstract LPS from B.abortus 544A, B.melitensis 16M and EHEC O157.The research recovers 6 hybridoma cell from the liquid nitrogen. After the expand-raising enables the cell quantity to achieve 5×10⁶, the abdominal cavity injects the Balb/C mouse to produce the monoclonal antibody ascites.Passes through the caprylic acid-ammonium sulfate to purify the antibodies thickly first, then HiTrap Protein G HP affinity column further purifies antibodies IgG, then the affinity, the IgG content, the titer carry on the appraisal. Abrin toxin McAb 4G1: the titer is 3.2×10⁵, the IgG content: 1.42 mg/mL,the affinity constants is 2.9×10⁹ M^-1; Recin toxin McAb 4D12: the titer is 1.2×10⁷, the IgG content: 2.12 mg/mL, the affinity constants is 5.5×10⁹ M^-1; Avian Influenza Virus McAb 3A13: the titer is 2×10⁶, the IgG content: 0.98 mg/mL, the affinity constants is 3.42×10⁸ M^-1; Newcastle Disease Virus McAb 1C10: the titer is 1.2×10⁶, the IgG content: 1.56 mg/mL, the affinity constants is 3.67×10⁸ M^-1; EHEC O157:H7 McAb 3D6 : the titer is 6.4×10⁵, the IgG content: 1.28 mg/mL, the affinity constants is 6.1×10⁸M^-1; Brucella melitensis McAb 4B8: the titer is 1×10⁵, the IgG content: 1.13 mg/mL, the affinity constants is 8.99×10⁸ M^-1.Spleen cells collected from BALB/c mice immunized with the whole cell antigen of Brucella abortus were fused with murine Sp2/0 myeloma cells. 3 hybridoma cell lines specific to Brucella abortus was established after screening and subcloning. McAbs were obtained, named D20. Brucella abortus 16M McAb D20: the titer is 1.6×10⁵, the IgG content: 2.33 mg/mL, the affinity constants is 5.22×10⁷ M^-1, its speciesspecific epitope located on LPS.We optimized many factors that could affect immobilization and hybridization of protein microarray. Such as slides, spotting solution dissolving protein, protein concentration immobilized on glass surface, spotting temperature and humidity, method of protein immobilized on glass surface, method of labeling sample, hybridization solution, hybridization temperature, hybridization time, etc. On the basis of results: Abrin toxin and recin toxin: 1μg/mL. EHEC O157, Brucella melitensis and Brucella abortus: 2.5 mg/mL. Avian Influenza Virus and Newcastle Disease Virus: 0.5 mg/mL. Probes resolved in 50% glycerine and spotting on glass slide modified with 1% agarose gel, guarantee time is 2 months at -20℃. Capture antibodies concentration is 5μg/mL, reacted with samples at 37℃1 h. The product added onto microarray, 25μL per block, 37℃1 h. Then hybridize with Cy3-labeled goat anti mouse IgG with3μg/mL, 37℃1 h。Standard sample was diluted to different concentration, which were used to test sensitivity of protein microarrays. Limit of detection of abrin toxin is 0.05μg/mL, recin toxin is 0.05μg/mL, EHEC O157 is 3.0×10³ CFU/mL, Brucella melitensis is 2.5×10³ CFU/mL, Brucella abortus is 2.5×10³CFU/mL, Avian Influenza Virus is 10μg/mL and Newcastle Disease Virus is 9.4μg/mL.