J. unique immunoglobulin M (IgM) or IgG in sera from patients with leptospirosis or other febrile illnesses and healthy subjects. The results showed that this r-LMP protein acknowledged IgG and IgM in all the sera that were microscope agglutination test positive, and there were no cross-reactions with other D-glutamine patient sera. This approach of creating customized antigens coupled to overexpression and simple purification offers a promising alternate option for leptospirosis diagnosis, with the potential to circumvent the drawbacks of whole-leptospirosis-antigen-based assays. Leptospirosis is an important infectious disease; the mortality rate in the severe form can be as high as 15% (9). Leptospirosis exhibits a broad spectrum of clinical manifestations, ranging in severity from acute to chronic (with multiorgan syndromes) and fatal (13). Although leptospirosis can be treated with antibiotics, its broad clinical presentation and similarities with other febrile illnesses complicate the diagnosis (1, 8). Misdiagnosis has become a significant problem, as diseases with comparable early symptoms Mouse monoclonal to CD31.COB31 monoclonal reacts with human CD31, a 130-140kD glycoprotein, which is also known as platelet endothelial cell adhesion molecule-1 (PECAM-1). The CD31 antigen is expressed on platelets and endothelial cells at high levels, as well as on T-lymphocyte subsets, monocytes, and granulocytes. The CD31 molecule has also been found in metastatic colon carcinoma. CD31 (PECAM-1) is an adhesion receptor with signaling function that is implicated in vascular wound healing, angiogenesis and transendothelial migration of leukocyte inflammatory responses.
This clone is cross reactive with non-human primate
occur (4, 10). Obviously, improving the index of clinical suspicion and developing a quick and specific test are essential for the identification of leptospirosis. The standard D-glutamine method for the diagnosis of leptospirosis, the microscopic agglutination test (MAT), is not only technically complex but also time-consuming (6). The sensitivities of other quick and less complicated antibody-based alternatives, such as standard enzyme-linked immunosorbent assays (ELISAs) and immunofluorescence assays, are very low during the early phase of the contamination (3, 13). In recent years, several attempts have been made to overcome these diagnostic hurdles, including the development of an antigen-based test (12, 15) and molecular methods, such as PCR and real-time PCR (16). Although their rapidity and diagnostic efficacy at the acute phase of the illness may be appreciable, their use is restricted in developing countries due to the gear cost (5). It is necessary to develop a cost-effective, safe, and efficacious diagnostic test that combines D-glutamine sensitivity, specificity, and laboratory as well as field applicability. Previously, we examined the potential B-cell epitope-containing peptides of OmpL1, LipL21, and LipL32 (11, 18). In the present work, we designed a recombinant leptospirosis multiepitope gene, recombinant host strains DH10B and BL21(DE3) plysS and plasmids pBacPAK8 and pET-28a(+) were managed in the laboratory. The secondary antibody-enzyme conjugates (goat anti-human immunoglobulin M [IgM]- and IgG-horseradish peroxidase [HRP]) were from Jackson ImmunoResearch, and the goat anti-rabbit IgG-HRP conjugate was from Santa Cruz. Sera from patients with fever, myalgia, headache, vomiting, jaundice, conjunctival suffusion, and abdominal symptoms were collected during the patients’ visits to hospitals in the Guangdong, Sichuan, and Zhejiang provinces and managed in our laboratory. The acute and convalescent phases were defined as previously reported (7). Briefly, serum samples collected at a median of 7 days (range, 2 to 23 days) after the reported onset of the symptoms were defined as acute phase, and serum samples collected at a median of 29.5 days (range, 17 to 113 days) were defined as convalescent phase. The case definition for MAT confirmation was a fourfold rise in MAT titer between paired sera or a MAT titer of 1:80 for a single serum sample (17). Sera from patients with other febrile illnesses (18 with hemorrhagic fever and 6 with dengue) and 10 healthy counterparts were used as patient and normal controls, respectively. This study was approved by the Institutional Review D-glutamine Table at our institution, and informed consent was obtained from each participant. In silico identification and characterization of epitopes. The B-cell epitopes from outer membrane proteins OmpL1, LipL21, and LipL32 were predicted by the ANTIGENIC program in EMBOSS (http://bioinfo.hku.hk/EMBOSS/). The predicted epitopes were cloned in phage vector M13KE for phage surface display. Epitopes with strong immunogenicity were selected based on the results of Western blot analysis (11, 18) and utilized for the synthesis of the recombinant gene. Construction of the recombinant gene expression vector. A synthetic gene, recombinant expression, was first generated by ligation oligonucleotides encoding five linear immunodominant epitopes of leptospire OmpL1, LipL21, and LipL32 proteins. These epitopes contained 11 to 19 amino acid residues, and adjacent epitopes were joined together by tetraglycyl linkers. In recombinant was inserted into the linearized pBacPAK8 vector to construct p8-lmp and sequenced. Then, recombinant was inserted into BglII- and EcoRI-digested p8-lmp to construct p8-2lmp. After that, recombinant was doubled and 10 epitopes were combined together. The 2lmp fragments were inserted into the BamHI and EcoRI sites of bacterial expression vector pET-28a(+) to generate the plasmid pET28a-2lmp. Recombinant clones were.