Study on the location of membrane and detection of antibody in the sera of genus-specific antigen LipL41s in patients with Leptospira interrogans / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine the location on outer envelope and natural antibody response and types of genus-specific lipoprotein antigen LipL41s in patients with Leptospira interrogans.</p><p><b>METHODS</b>Microscope agglutination test (MAT) was used to examine leptospirosis patients' serum samples from Sichuan area, China. Ni-NTA affinity chromatography was performed to extract the target recombinant rLipL41/1 and rLipL41/2 products that expressed under inducement of IPTG. Western blot assay was performed to detect the immunoreactivity between the sera from the patients infected with different serogroups of L. interrogans and rLipL41s. Immune aurosol electron microscopy was selected to locate the position of LipL41s on leptospiral envelope. ELISA based on rLipL41s was established to confirm the level and types of specific antibody.</p><p><b>RESULTS</b>L. interrogans serogroup icterohaemorrhagiae remained to be the most dominant leptospiral serogroup in Sichuan area. All the sera from patients infected with different serogroups of L. interrogans could efficiently recognize the LipL41s which were the protein molecular that located on the external surface of leptospiral envelope. In the 156 serum samples from MAT positive leptospirosis patients, the positive rates for rLipL41/1 or rLipL41/2 specific IgM appeared to be 84.6%-87.8% and 78.2%-83.3%, respectively, while for rLipL41/1 or rLipL41/2 specific IgG they were 69.2%-81.4% and 75.0%-80.1%, respectively.</p><p><b>CONCLUSION</b>LipL41s were the leptospiral superficial protein antigen of L. interrogans. Both the LipL41/1 and LipL41/2 could induce serum antibodies IgM and IgG with extensive antigenic-cross reaction during natural infection of L. interrogans in general populations. Hence, rLipL41/1 or rLipL41/2 could be used as the antigen candidate for developing universal genetic engineering vaccine and detection kit.</p>

Study on ELISAs with rTpN17, rTpN47 and its fusion protein as antigens in the detection of serum samples from syphilis patients / 中华流行病学杂志

<p><b>OBJECTIVE</b>To construct the prokaryotic expression systems of tpnl7 and tpn47 genes and tpn17-tpn47 fusion of Treponema pallidum, and to establish ELISAs based on rTpN17, rTpN47 and rTpN17-TpN47 as antigens to evaluate the sensitivity and specificity of the ELISAs for detection of serological diagnosed syphilis.</p><p><b>METHODS</b>tpn17 and tpn47 genes were amplified and cloned by routine molecular biological methods. PCR with linking primers was used to construct artificial fusion gene tpn17-tpn47. The prokaryotic expression systems of the genes were then constructed. SDS-PAGE was used to measure the expression of the target recombinant proteins rTpN17, rTpN47 and rTpN17-TpN47. Ni-NTA affinity chromatography was applied to extract the three recombinant proteins, while Western blot was performed to determine their immunity. Using rTpN17, rTpN47 and rTpN17-TpN47 as the coated antigens, ELISAs (rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47- ELISA) were established to detect serum samples from 200 healthy individuals, 25 RA patients, 17 SLE patients and 419 syphilis patients. Results of the ELISAs were compared to those with TRUST and TPHA.</p><p><b>RESULTS</b>The sequence similarities of the cloned tpnl7 and tpn47 genes and the constructed tpn17-tpn47 fusion gene were 100%, compared with the corresponding sequences in GenBank. The expression outputs of rTpN17, rTpN47 and rTpN17-TpN47 were 37.2%, 23.3% and 29.8% of the total bacterial proteins, respectively. Each of the three purified recombinant proteins showed a single fragment in gel after electrophoresis, and could take place remarkable conjugation reactions to the positive sera from syphilis patients. The detection results of rTpN17-ELISA, rTpN47-ELISA and rTpN17-TpN47-ELISA were negative for the serum samples from healthy individuals, RA and SLE patients, while presented 84.4%, 82.3% and 98.1% positive detection rates for the serum samples from syphilis patients. The positive detection rates of rTpN17-ELISA and rTpN47-ELISA were lower than that of TPHA (P<0.01), while the positive detection rate of rTpN17-TpN47-ELISA was similar to that of TPHA (P>0.05). All the positive detection rates from ELISA tests were higher than that of TRUST (71.4%).</p><p><b>CONCLUSION</b>rTpN17-ELISA, rTpN47-ELISA and especially rTpN17-TpN47-ELISA established in this study were of great hope as it was rapid, simple, convenient, safe, with high sensitivity and specificity for serological screening and detection of syphilis.</p>

Study on the immunogenicity of major leptospiral genus-specific protein antigens and the distribution of antigens in different serogroups of Leptospira interrogans / 中华流行病学杂志

<p><b>OBJECTIVE</b>The determination of antigenicity and immunogenicity of Leptospira interrogans genus-specific outer envelope proteins (OEPs) will offer evidence for developing universal leptospiral genetic engineering vaccine and detection kit.</p><p><b>METHODS</b>In this study, Ni-NTA affinity chromatography is used to purify the recombinant products rLipL21, rOmpL1/1, rOmpL1/2, rLipL32/1, rLipL32/2, rLipL41/1 and rLipL41/2 expressed by the major genotypes of four leptospiral OEPs of 15 serogroups. SDS-PAGE is applied to examine the expression and purity of the recombinant proteins. Rabbits are intracutaneously immunized with the recombinant proteins to obtain antisera. Microscope agglutination test (MAT) is used to measure the cross inmmunoagglutination titers of antisera. The OMPs of the reference standard strains belonging to 15 serogroups of L. interrogans in China and L. biflexa strain Patoc I are prepared using salt-denature method. By each of the antisera as the first antibody, Western blot assay is established to detect the natural expressions and immunoreactivity of the four OEPs.</p><p><b>RESULTS</b>The outputs of rLipL21, rLipL32/1, rLipL32/2, rLipL41/1l, rLipL41/2, rOmpL1/1 and rOmpL1/2 are 10%, 40%, 35%, 15%, 10%, 30% and 15%, respectively. Each the purified recombinant proteins shows a single fragment after SDS-PAGE. Each the rabbit antisera displays extensive cross immunoreactivity between the products expressed by different genotypes of the same gene and the MAT titers ranging from 1:2-1:128. All the four OEPs can be detectable in the OEPs preparations. However, LipL21 is found to exist only in L. interrrogans.</p><p><b>CONCLUSION</b>The results of this study indicate that all the four OEPs are superficial genus-specific antigens of Leptospira which can be used as the candidate antigens of leptospiral universal vaccine and detection kit.</p>