Association of PRM1-190C- > A polymorphism with teratozoospermia / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the association of single nucleotide polymorphism (SNP) of the Protamine 1 (PRM1) gene in infertile men with teratozoospermia.</p><p><b>METHODS</b>We collected semen samples from 157 infertile men with teratozoospermia (case group) and 37 age-matched male volunteers (control group), and subjected them to morphological analysis. We extracted genome DNA, genotyped the polymorphism of the PRM1-190C- > A SNP (rs2301365) using the Sequenom MassARRAY system, compared the genotype frequencies between the case and control groups, and analyzed the sperm morphological parameters of different genotypes in the infertile males with teratozoospermia.</p><p><b>RESULTS</b>The frequencies of the genotypes CC, CA and AA were 38.9% (61), 44.6% (70) and 16.6% (26) in the case group, as compared with 45.9% (17), 51.4% (19) and 2.7% (1) in the control, with that of AA significantly higher in the patients than in the volunteers (P<0.05). The frequencies of the alleles C and A were 57.6% and 42.4% in the former, with no significant differences from 71.6% and 28.4% in the latter (P>0.05). Nor were any statistically significant differences observed in sperm morphology parameters between the genotype CC and CA, AA and CA + AA in the male patients (P>0.05).</p><p><b>CONCLUSION</b>The SNP of PRM1-190C- > A might be associated with teratozoospermia-induced male infertility in the Han Chinese. Although this SNP may attribute to abnormal sperm morphology, the targeted part of sperm remains unclear.</p>

Development of a time-resolved fluoroimmunoassay for detecting S1 subunit of pertussis toxin and its application / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine.</p><p><b>METHODS</b>A double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine.</p><p><b>RESULTS</b>The sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine.</p><p><b>CONCLUSION</b>The TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.</p>

Development of an amplified luminescent proximity homogeneous immunoassay kit for detecting human hepatitis B virus e antibody / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop an amplified luminescent proximity homogeneous immunoassay (AlphaLISA) kit for the detection of human hepatitis B virus e antibody (HBeAb).</p><p><b>METHODS</b>The neutralizing and competitive inhibition method was used to develop the AlphaLISA kit for detection of serum HBeAb.</p><p><b>RESULTS</b>The working range of the kit was 0.003-16 NCU/ml with a sensitivity up to 0.003 NCU/ml. The intra- and inter-assay coefficient of variation was 5.3% and 6.8%, respectively. The kit showed no cross-reaction with HBcAb, and comparison of the detection results with those of a commercially available Elecsys HBeAb kit (Roche) for 136 samples showed a correlation coefficient of 0.961.</p><p><b>CONCLUSION</b>The AlphaLISA kit for HBeAb detection meets the clinical requirements for detection HBeAb in human serum.</p>

Preparation and characterization of reference samples of Mycobacterium tuberculosis culture filtrate protein-10 for time-resolved fluoroimmunoassay / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare reference samples of Mycobacterium tuberculosis culture filtrate protein-10 (CFP-10) and CFP10-streptavidin fusion proteins (CFP10/SA) for time-resolved fluoroimmunoassay (TRFIA).</p><p><b>METHODS</b>The CFP10 gene was amplified by PCR from Mycobacterium tuberculosis strain H37Rv and cloned into pET24b, pET24b-streptavidin (SA) or pET21a-SA expression vectors. The recombinant proteins CFP10, CFP10-SA and SA-CFP10 were expressed in Rosetta cells, purified via nickel affinity chromatography and refolded by dialysis. The sensitivity and stability of the resultant proteins as reference samples were evaluated by double-antibody sandwich TRFIA.</p><p><b>RESULTS</b>CFP10-SA and SA-CFP10 fusion proteins were expressed as inclusion bodies, whereas CFP10 was expressed in a soluble form. The resultant purity of the 3 recombinant proteins all exceeded 95%. TRFIA results showed that CFP-SA fusion protein possessed the best sensitivity (0.02 µg/L) and stability.</p><p><b>CONCLUSION</b>The reference samples of CFP10 for TRFIA detection have been successfully prepared and can be used in the development of a diagnostic kit for Mycobacterium tuberculosis.</p>

Relationship between clinical stages of nasopharyngeal carcinoma and Epstein-Barr virus antibodies Rta/IgG, EBNA1/IgA, VCA/IgA and EA/IgA / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the relationship between the clinical stages of nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) antibodies Rta/IgG, EBNA1/IgA, VCA/IgA and EA/IgA.</p><p><b>METHODS</b>Serum samples obtained from 211 untreated patients with NPC categorized by the project of 92' stage were examined for the presence of the EBV antibodies Rta/IgG and EBNA1/IgA by enzyme-linked immnunosorbent assay (ELISA) and for VCA/IgA and EA/IgA by immunoenzymatic assay. The positive rates and antibody levels in the NPC patients in different TNM stages and clinical stages were analyzed statistically.</p><p><b>RESULTS</b>No significant difference in Rta/IgG rA value was found in the NPC patients in different TNM or clinical stages (P>0.05). The EBNA1/IgA rA value was significantly lower in stage T1, N0, and clinical stage I than in the other corresponding T stages, N stages and other clinical stage (P<0.05). The antibody titers of VCA/IgA and EA/IgA differed significantly between the N stages and the clinical stages (P<0.05).</p><p><b>CONCLUSION</b>The expression of EBV Rta/IgG is not associated with NPC stage. The expression of EBNA1/IgA is relatively low in early NPC. The antibody level of VCA/IgA and EA/IgA are significantly correlated to the degree of neck lymph node metastasis, and might be helpful to classify the clinical stages of NPC.</p>

Combined detection of Epstein-Barr virus antibodies for serodiagnosis of nasopharyngeal carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the value of combined detection of Epstein-Barr virus (EBV) VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA in serodiagnosis of nasopharyngeal carcinoma (NPC).</p><p><b>METHODS</b>Serum samples obtained from 211 untreated patients with NPC and 203 non-NPC ENT patients were examined for the presence of VCA/IgA and EA/IgA by immunoenzymatic assay and for Rta/IgG and EBNA1/IgA by enzyme-linked immunosorbent assay (ELISA). The receiver operating characteristic (ROC) curve was generated to confirm the cutoff values of different antibodies. The evaluation indexes of combined detection of multiple antibodies used for serodiagnosis of NPC were calculated with compounded positive judgment method.</p><p><b>RESULTS</b>Compared to a single antibody, combined detection achieved a higher sensitivity and specificity. The sensitivity of VCA/IgA + Rta/IgG + EBNA1/IgA (98.1%) was higher than the other 3 combinations with a specificity, accuracy, Youden index and positive predictive value (PPV) of 88.7%, 93.5%, 0.868 and 90.0%, respectively. The combination of EA/IgA+Rta/IgG+EBNA1/IgA had the highest specificity (95.1%), accuracy (94.9%), Youden index (0.899) and PPV (95.2%), with a sensitivity of 94.8%, suggesting its higher accuracy in the serodiagnosis of NPC. Combined detection of the 4 antibodies had the highest sensitivity (98.6%) with a specificity, accuracy, Youden index and PPV of 88.2%, 93.5%, 0.868 and 89.7%, respectively.</p><p><b>CONCLUSIONS</b>Combined detection of Rta/IgG against immediate early antigens, EA/IgA against early antigens, VCA/IgA against late antigens, and EBNA1/IgA against latent antigens provides better understanding of the expression profiles of EBV lytic and latent antigens with excellent complementarity, and may serve as an optimal combination for NPC serodiagnosis.</p>

A novel method for hepatitis C virus genotyping using RT-PCR reverse dot blot hybridization technique / 南方医科大学学报

<p><b>OBJECTIVE</b>To develop a rapid and specific method for hepatitis C virus ( HCV) genotyping using reverse dot blot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong.</p><p><b>METHODS</b>The primers and the probes targeting the 5'untranslated region (5'UTR) and core region of HCV genotypes 1b, 2a, 3a, 3b and 6a were designed, and the RT-PCR reverse dot blot hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C were genotyped using this method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method.</p><p><b>RESULTS</b>Of the 115 patients, 111 were successfully genotyped to be 1b, 2a, 3a, 3b, 6a and mix-infection of 1b/2a at frequencies of 56.8%, 8.1 %, 3.6%, 5.4%, 25.2% and 0.9% respectively, and all the 15 healthy control samples showed negative results. The accuracy and reliability of the genotyping method of PCR-RDH was confirmed in 38 cases by amplification of HCV core and NS5B regions followed by DNA sequencing and phylogenetic analysis.</p><p><b>CONCLUSION</b>This method for HCV genotyping, with high reliability and specificity, is suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a decreases while 1b remains the dominant genotype in Guangdong, where the prevalence of 6a significantly increases as compared with that 10 years ago.</p>

Development of time-resolved fluoroimmunoassay kit for detection of IgM antibodies against hepatitis B core antigen / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare a time-resolved fluoroimmunoassay (TRFIA) kit for clinical detection of IgM antibodies to hepatitis B core antigen (HBc).</p><p><b>METHODS</b>Immunocapture method was used to develop the TRFIA kit for detection of the anti-HBc IgM antibodies, and the precision, cross-reactivity and sensitivity of the kit were tested with the clinical serum samples.</p><p><b>RESULTS</b>The intra- and inter-assay coefficients of variation of the TRFIA kit were 4.8%-7.2% and 7.5%-8.6%, respectively, and no cross-reactivity with anti-HBs, anti-HBc-IgG or anti-HBe was found. Comparison of the results of the TRFIA kit and enzyme-linked immunosorbent assay (ELISA) demonstrated greater sensitivity of the kit than ELISA in detecting the anti-HBc IgM antibodies in 584 serum samples. According to the detection results in 300 serum samples from healthy donors, the cutoff value of the TRFIA kit was 4.5 times of the fluorescence value of the negative control.</p><p><b>CONCLUSION</b>This TRFIA kit for detecting anti-HBc IgM antibodies meets the demand for clinical application and can replace the ELISA kits.</p>

A novel system for producing lentiviral vectors / 生物工程学报

The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamine2000. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT-PCR and the Western blot, the results showed that lentiviral vectors were found in the culture supernatant; approximately (11.71 +/- 0.80) x 10(11) copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real-time PCR; the vector stocks with titers was up to (1.3 +/- 0.18) x 10(8) tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.

Comparison of effectiveness of whole viral, N and N199 proteins by ELISA for the rapid diagnosis of severe acute respiratory syndrome coronavirus / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although severe acute respiratory syndrome (SARS) has been controlled, the subsequently emerging sporadic cases in 2004 emphasize the necessity of developing a rapid diagnostic method, which would be of great help in clinical diagnosis and also wild host screening. This study aims to establish an effective and rapid serological tool for the diagnosis of SARS-CoV by comparison among whole viral, N and N199 proteins by ELISA.</p><p><b>METHODS</b>SARS-CoV N and N199 (a truncated nucleocapsid gene) genes were cloned, expressed, identified by Western blotting, and applied in screening of human and swine samples. Sera of SARS convalescent-phase patients, normal human sera, sera of patients with other respiratory diseases, and swine sera were screened by ELISA, with whole SARS-CoV F69, N and N199 proteins as antigens.</p><p><b>RESULTS</b>The sensitivity and specificity of N and N199 proteins in human sera diagnosis were approximate (P = 0.743), which was higher than whole viral protein but the difference was not significant (P = 0.234). The N199 protein proved to be more specific in swine sera screening than whole viral and N protein (P < 0.001).</p><p><b>CONCLUSION</b>N199 protein is feasible in both clinical diagnosis and SARS-CoV reservoir screening.</p>

Preparation and evaluation of a time-resolved fluoroimmunoassay kit for CA50 determination / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To prepare a rapid and sensitive diagnostic kit for detection of CA50 based on time-resolved fluoroimmunoassay.</p><p><b>METHODS</b>A sandwich-TRFIA diagmostic kit was developed using anti-CA50 monoclonal antibody and all parameters of the kit were evaluated.</p><p><b>RESULTS</b>The linear measurement range of the kit was (5 - 300) U/ml. The sensitivity was 0.2 U/ml. The intra- and inter-assay coefficients of variation were 4.3% - 8.2% and 7.7% - 11.2%, respectively. There was no cross-reaction with CEA, CA12-5, CA15-3 and AFP. The cross reactivity with CA19-9 was 0.7 U/ml. The correlation coefficient of detection results of 107 blood samples between this newly developed kit and commercially available CA50 RIA kit was 0.901.</p><p><b>CONCLUSION</b>This newly developed CA50-TRFIA kit is a valuable test tool for clinical application with even better sensitivity, specificity and accuracy.</p>