[Establishment of measurement threshold and comparison of results for detection of Treponema pallidum specific antibody by Siemens ADVIACentaur XP chemiluminescent immunoanalyzer].

To investigate the detection threshold of Treponema pallidum specific antibody method by chemiluminescent immunoassay (CLIA) in Siemens ADVIA Centaur XP for Syphilis serological test, and compare with the results derived from CMIA, TP-WB and TPPA method. The result can serve as reference for the application of CLIA. In total 30 887 samples screened by Treponema pallidum specific antibody method were collected by Abbott architect i2000 CMIA from July 2018 to July 2019 in Yanda Hospital of Hebei Province. We selected 153 patients with the ratio of sample absorbance to critical value (S/CO) of 1-9 by CMIA screening of Treponema pallidum specific antibody as the research objects. The reverse sequence of syphilis serological detection was adopted, and TP-WB and TPPA were used as the confirmation methods respectively. MedCalc software was used to analyze the results of ROC curve, and the cut-off value was obtained. Chi square test was used to test the difference significance of counting data. The detection results of Treponema pallidum specific antibody in the same batch of serum samples were unequal by different methods. There was no significant difference between CLIA method and TPPA method, but significant difference between CLIA method with TP-WB method and CMIA method was found. TPPA test results and TP-WB test results were taken as gold standards, ROC curve analysis showed that the best diagnostic cutoff value of CLIA method was 4.01 and 16.06, respectively, and the area under the curve was 0.961 and 0.838. The suggested cutoff value of CLIA method is quite different when using different syphilis serological test methods as the gold standard, Therefore, when the S/CO value determined by CLIA is between 1.00 to 16.06, TP-WB method should be recommended as the first choice in laboratory serological test for recheck and confirmation to avoid clinical misdiagnosis.

Establishment of measurement threshold and comparison of results for detection of Treponema pallidum specific antibody by Siemens ADVIACentaur XP chemiluminescent immunoanalyzer / 生物工程学报

To investigate the detection threshold of Treponema pallidum specific antibody method by chemiluminescent immunoassay (CLIA) in Siemens ADVIA Centaur XP for Syphilis serological test, and compare with the results derived from CMIA, TP-WB and TPPA method. The result can serve as reference for the application of CLIA. In total 30 887 samples screened by Treponema pallidum specific antibody method were collected by Abbott architect i2000 CMIA from July 2018 to July 2019 in Yanda Hospital of Hebei Province. We selected 153 patients with the ratio of sample absorbance to critical value (S/CO) of 1-9 by CMIA screening of Treponema pallidum specific antibody as the research objects. The reverse sequence of syphilis serological detection was adopted, and TP-WB and TPPA were used as the confirmation methods respectively. MedCalc software was used to analyze the results of ROC curve, and the cut-off value was obtained. Chi square test was used to test the difference significance of counting data. The detection results of Treponema pallidum specific antibody in the same batch of serum samples were unequal by different methods. There was no significant difference between CLIA method and TPPA method, but significant difference between CLIA method with TP-WB method and CMIA method was found. TPPA test results and TP-WB test results were taken as gold standards, ROC curve analysis showed that the best diagnostic cutoff value of CLIA method was 4.01 and 16.06, respectively, and the area under the curve was 0.961 and 0.838. The suggested cutoff value of CLIA method is quite different when using different syphilis serological test methods as the gold standard, Therefore, when the S/CO value determined by CLIA is between 1.00 to 16.06, TP-WB method should be recommended as the first choice in laboratory serological test for recheck and confirmation to avoid clinical misdiagnosis.

Differential Immune Reactivity Pattern of SW48 and SW1116 Colorectal Cancer Cell Lines with Colorectal Cancer Patients Sera.

BACKGROUND: Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. It is also known as the second leading cause of deaths as the early stage detection is not yet available by current methods. So identification of biomarkers can also be functional in early diagnosis and prognosis. MATERIALS AND METHODS: We examined sera from 60 CRC patients of different stages as a source of auto-antibody as well as two human CRC cell lines with different invasive capacities (SW48 and SW1116) as the source of antigens. The pattern of immune reactivity in immuneblotting tests between mentioned cell lines and CRC patients' sera were evaluated by ImageJ software. RESULTS: The Immune reactivity pattern of two cell lines (SW48 and SW1116) with CRC patients' sera were different in band intensities and the most immune reactivity intensity was observed in SW48 cell lysate with sera from Stage III CRC patients. CONCLUSION: Due to the humoral immune response, sera from Stage III CRC patients contained autoantibodies that demonstrated higher immune reactivity. Moreover, SW48 cell line with high aggressive behavior reacted to CRC patients' sera with greater intensity compared with less aggressive behavior cell line (SW1116). Therefore, it is required to use other techniques such as two-dimensional electrophoresis and mass spectrometry.

Immunological characterization of diphtheria toxin recovered from Corynebacterium pseudotuberculosis.

Diphtheria toxin (DT) is a potent toxin produced by the so-called diphtheria group which includes Corynebacterium diphtheriae (C. diphtheriae), Corynebacterium ulcerans (C. ulcerans), and Corynebacterium pseudotuberculosis (C. pseudotuberculosis). The present investigation is aimed to study in detail the production of DT by C. pseudotuberculosis. Twenty isolates were obtained from sheep diseased with caseous lymphadenitis (CLA) and twenty-six isolates were obtained from 26 buffaloes diseased with oedematous skin disease (OSD). All isolates were identified by standard microbiological and DT production was assayed serologically by modified Elek test and immunoblotting. All sheep isolates were nitrate negative, failed to hydrolyze starch and could not produce DT, while all buffalo isolates (biotype II) revealed positive results and a specific band of 62 kDa, specific to DT, was resulted in all concentrated cell fractions (CF), but was absent from non-toxigenic biotype I isolates. At the same time, another band of 31 kDa specific to the PLD gene was obtained with all isolates of biotype I and II. Moreover, all isolates showed positive synergistic hemolytic activity and antagonistic hemolysis with ß-hemolytic Staphylococci. The obtained results also indicated that C. pseudotuberculosis could be classified into two strains; non-toxigenic biotype I strain, which failed to produce DT as well as being negative to nitrate and starch hydrolysis, and toxigenic biotype II strain, which can reduce nitrate, hydrolyze starch as well as produce DT.

Cross-priming amplification targeting the coagulase gene for rapid detection of coagulase-positive Staphylococci.

AIMS: To develop and evaluate cross-priming amplification (CPA) combined with immuno-blotting for the detection of coagulase-positive Staphylococci including Staphylococcus aureus. METHODS AND RESULTS: Twenty-four sets of cross and detection primers were designed according to four sequences of coagulase gene in Staph. aureus. The most specific primer pair was screened out for the next amplification and interaction. The specificity was evaluated in a total of 53 species of Staph. aureus and non-Staph. aureus. Two red lines indicating positive were always observed on the BioHelix Express strip for 12 subspecies of Staph. aureus. In contrast, only one signal line showing negative results was detected in all of non-Staph. aureus samples. The limit of detection (LOD) of CPA was 3·6 ± 2·7 fg for the genomic DNA, which is about 100 and 10 times sensitive than those of PCR and loop-mediated isothermal amplification respectively. For the pure culture of Staph. aureus and milk powders, the LODs of CPA were about 1·34 CFU per reaction and 5·2 ± 3·7 CFU per 100 g of milk powder respectively. The CPA method was also successfully applied to evaluate the contamination of Staph. aureus in 318 samples of daily food. CONCLUSIONS: CPA is a very sensitive and rapid method to detect Staph. aureus by simple laboratory instrument. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report on the application of the CPA with immuno-blotting for detection of coagulase-positive Staphylococci including Staph. aureus.

Anti-HBV activity of the different extracts from Phyllanthus rheedei Wight in cell culture based assay systems.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthusrheedei Wight is a plant used by Muthuvan tribes of Kerala for treating liver related diseases. MATERIALS AND METHODS: The different extracts of Phyllanthus rheedei were analysed on cell lines were viz, PLC/PRF, Hep3B, FLCII10 and HepG2215 for its anti-HBV property. The analysis was done through ELISA, SQRT-PCR and immuno blotting. The most active extract was then divided in to fractions using HPTLC and the most active fraction was further identified. RESULTS: From the screening experiments it was shown that the ethanol extract of this plant has the maximum activity in lowering the viral markers like HBsAg, HBV Core and HBV X protein and whole virions with comparatively lesser cytotoxicity. The dose responses of this particular extract were further established. CONCLUSIONS: This study concluded that the ethanol extract of Phyllanthusrheedei is very much effective in preventing the multiplication of HBV at the cellular level. This study scientifically validated the tribal claim of the use of this plant for severe liver disorders.

Modification in the expression of Mre11/Rad50/Nbs1 complex in low dose irradiated human lymphocytes.

Despite the fact that high doses of radiation are detrimental, low dose radiation (LDR) often protects the organism against a subsequent exposure of lethal doses of radiation. Present study was undertaken to understand the role of Mre11, Rad50 and Nbs1 genes in the low dose radio-adapted human peripheral blood mononuclear cells (PBMCs). Optimum time interval between low dose (0.07 Gy) and high dose (5.0 Gy) of (60)Co-gamma-radiation was observed to be 5.0 hours, at which PBMCs showed maximum LDR induced resistance (RIR). At cytogenetic level, micronuclei frequency was found to be reduced in LDR pre-irradiated PBMCs subsequently exposed to high dose radiation (HDR) as compared to controls. At transcriptional level, with reference to sham-irradiated cells significantly (p< or =0.05) altered expression of Mre11, Rad50 and Nbs1 genes was observed in low dose irradiated cells. At protein level, Mre11, Rad50 and Nbs1 were enhanced significantly (p< or =0.05) in low dose pre-irradiated cells subsequently exposed to high dose of radiation as compared to only high dose irradiated cells. Transcriptional as well as translational modulation in the expression of MRN complex components upon low dose irradiation may confer its participation in repair pathways, resulting in induced resistance.

Determinación de anticuerpos al virus de la inmunodeficiencia humana tipo 1 (VIH-1) en muestras de sangre seca en papel de filtro

Se estudia la posibilidad de detectar anticuerpos al VIH-1 mediante un estuche de Immunoblotting, en un papel de 125 muestras conocidas de sangre seca colectadas en papel de filtro y su correspondiente muestra de suero. No se apreciaron diferencias en los patrones de bandas con ambos tipos de muestras, así como en la sensibilidad y especificidad, donde se alcanzaron cifras del 100 %, lo que permitió concluir que la muestra de sangre tomada en papel de filtro puede ser empleada para la detección de anticuerpos al VIH-1 por el sistema DAVIH-BLOT y puede ser conservada a 4 ºC por 30 semanas.

The possibility of detecting HIV-1 antibodies by an inmunoblotting kit is studied in a panel of 125 known specimens of dried blood spotted on filter paper and their corresponding serum samples. No differences were observed in the patterns of bands with both types of samples or in the sensitivity and specificity, where 100 % figures were attained, allowing to conclude that the blood specimen taken on filter paper may be used for the detection of HIV-1 antibodies by the DAVIH-BLOT system and may be kept at 4 ºC during 30 weeks.