These data indicate that human anti-antibodies play a pivotal role in prevention of the bacterial colonization in the human epithelial layer. B subunit [3], and toxin-coregulated pilus (TCP) [4] have also been induced in cholera patients. Although specific antibodies against are known to play a role in protection in humans, there is no direct evidence as to which antibody isotype is the most important to provide immunity against cholera. Two isotypes, secretory IgA and serum IgG, have been shown to provide protective immunity against infection [5, 6], and LPS-specific IgA is increased in the sera and intestinal fluids from patients and vaccinated individuals [7C9]. Significant levels of LPS-specific serum IgG are also augmented in response to infection [10C12]. Given that IgG and IgM can trigger complement-mediated bacterial lysis through the classical complement pathway [1], IgM could be also responsible for protection against test that measures the ability of antibodies mediated by complements to kill virulent O1 [3]. Therefore, serum vibriocidal antibody titer has been used as a representative marker of immunity to O1 El Tor Inaba (strain “type”:”entrez-nucleotide”,”attrs”:”text”:”T19479″,”term_id”:”597224″,”term_text”:”T19479″T19479) was kindly provided by Prof. Jan Holmgren (Gothenburg University, Sweden) and was used as the target bacteria for the vibriocidal assay. Brain Heart Infusion (BHI) media and Guinea Almotriptan malate (Axert) pig complements were purchased from Difco (San Jose, CA, U.S.A.) and Rockland (Gilbertsville, PA, U.S.A.), respectively. Protein G-agarose (PGA) and rabbit anti-human IgM (-chain specific) or IgA (-chain specific) in rabbit IgG fraction were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.). Serum samples Convalescent sera with high vibriocidal antibody titers of 8,000 were pooled from cholera patients in India and used to Almotriptan malate (Axert) deplete target isotype of antibody. Bivalent oral cholera vaccine (Shanchol, Shantha Biotechnics), formulated with O1 Inaba, O1 Ogawa, and O139, was given with two doses at two week interval (days 0 and 14) through clinical trials [20C22]. Blood samples were obtained to use in this study from volunteers at 2 weeks (days 14 and 28) after first dose and second dose, respectively. Human sera were heated at 56C for 30 min before use to inactivate complements. After approval from the ethics committee of the National Institute of Cholera and Enteric Diseases, the Health Ministry Screening Committee of India, and the Institutional Review Board of the International Vaccine Institute, all serum samples were used. Depletion of antibody isotypes One milligram of PGA was initially mixed with 100 g of IgG fraction of rabbit anti-human IgM (-chain specific) or IgA (-chain specific) at room temperature for 1 h. Convalescent sera were then mixed with anti-human IgM- or IgA-bound PGA Almotriptan malate (Axert) and followed by precipitation of the antibody-agarose complex to deplete serum IgM or IgA. PGA was used to deplete IgG in the convalescent sera. The supernatants were then incubated again with fresh PGA, anti-IgM-, or anti-IgA-bound-PGA; this clearance procedure was repeated 7 consecutive times. Purification of LPS and recombinant OmpU LPS was purified from O1 Inaba (strain “type”:”entrez-nucleotide”,”attrs”:”text”:”T19479″,”term_id”:”597224″,”term_text”:”T19479″T19479) using LPS extraction kit (Intron, Seongnam, Korea) according to the manufacturers instruction as previously described [23]. The gene was amplified from O1 Inaba (strain “type”:”entrez-nucleotide”,”attrs”:”text”:”T19479″,”term_id”:”597224″,”term_text”:”T19479″T19479) by PCR and recombinant OmpU was expressed in the BL21 (DE3), and purified as previously described [24]. Quantification of antibody isotypes using ELISA To quantify total antibody isotypes in serum samples, a 96-well plate (Nunc, Roskilde, Denmark) was coated with anti-human and light chain antibodies (Bethyl Laboratories, Montgomery, TX, USA) overnight at 4C and blocked with BSA. Serially diluted sera and human IgM, IgG, or IgA (1 g/ml) (Jackson Immunoresearch, West Grove, PA, USA) were added. After incubation at 37C for 2 h, serum antibody isotypes were detected with alkaline phosphatase-conjugated anti-human IgM, IgG, or IgA (Jackson Immunoresearch) and developed with 4-nitrophenyl phosphate (Fluka Chemie, Buchs, Switzerland). Optical density was read at 405 nm using a microplate reader (Molecular Device, Sunnyvale, CA, USA). To measure LPS (2.5 g/ml), heat-killed (HKVC) (108 CFU/ml), or recombinant OmpU (rOmpU, 2.5 g/ml) at 4C overnight. Titers of total antibody and O1 (strain “type”:”entrez-nucleotide”,”attrs”:”text”:”T19479″,”term_id”:”597224″,”term_text”:”T19479″T19479) were cultured in 20 ml of BHI media for 2C3 h at 37C until they Rabbit polyclonal to RIPK3 reached mid-log phase. Bacteria were collected by centrifugation, washed with saline, and diluted with saline containing 10% guinea pig complements to adjust to 1106 cells/ml. An equal volume of diluted bacteria was added to the 96-well plate containing serially-diluted serum samples, and the plates were incubated at 37C for 1 h. Next, 150 l of fresh BHI broth was added, and plates were incubated for an additional 4 h. Bacterial growth was measured at OD600 using a microtiter-plate reader (Spectramax.