Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.
Adenosine Triphosphate ; chemistry ; Chromatography, Affinity ; Escherichia coli ; Luminescent Measurements ; methods ; Lysostaphin ; chemistry ; Recombinant Proteins ; chemistry ; Staphylococcus aureus ; isolation & purification

OBJECTIVETo perform a systematic comparative analysis of two different commercial automated systems using chemiluminescence immunoassay to quantitatively detect hepatitis B virus surface antigen (HBsAg) in patient sera.

METHODSThe Elecsys2010 electrical chemiluminescence immunoassay (ECLIA; manufactured by Roche) and the ARCHITECT il000 chemiluminescence magnetic microparticle immunoassay (CMIA; manufactured by Abbott) were used to detect HBsAg in 100 serum samples of individuals who presented at our department with suspected hepatitis infection between January and May 2012. The manufacturer's protocols were strictly followed. The categorical data was analyzed by Chi-squared test, and linear regression analysis was used to compare the results of the two assay systems.

RESULTSThe HBsAg detection results from the two different assay systems showed good correlation (r >or= 0.95), and had good correlation at a low (r = 0.966), medium (r = 0.974) and high (r = 0.984) cutoff values. However, the positive detection rate of CMIA was significantly higher than that of ECLIA(94% vs. 88%, P < 0.05). When the HBsAg content was below 0.10 IU/ml, the ECLIA detection rate and sensitivity were slightly higher than those of CMIA.

CONCLUSIONThe ARCHITECT i1000 and Elecsys 2010 immunoassay systems have good correlation in quantitative detection of HBsAg, but the former may be more sensitive.

Hepatitis B Surface Antigens ; blood ; Humans ; Immunoassay ; methods ; Luminescent Measurements ; Sensitivity and Specificity

This paper is aimed to evaluatethe thyroglobulin autoantibody (TgAb) interference in measurement of thyroglobulin (Tg) between electrochemiluminescent assay (ECLIA) and radioimmunoassay (RIA). Tg and TgAb of 84 sera, including 22 Graves' hyperthyroidism(GD), 24 Hashimoto thyroiditis (HT) and 38 differentiated thyroid carcinomas (DTC), were measured by RIA and ECLIA, respectively. Recovery tests were carried out in 3 groups. The sera samples of the first group were added 3 different amount of Tg calibrator; the sera samples of the second group were diluted 5 times, then 100 ng/ml Tg calibrator was added; the sera samples of the third group were divided into different subgroups depending on TgAb concentration with adding 100 ng/ml Tg calibrator,Tg and TgAb were measured in each dilution by ECLIA and RIA. Recovery rate was calculated. The Tg and TgAb values measured by ECLIA were correlated with that measured by RIA (r = 0.676, P = 0.000; r = 0.677, P = 0.000, respectively). When TgAb concentration increased, the Tg values decreased by ECLIA and increased by RIA. The TgAb values were decreased when sera were diluted, and the Tg values also reduced by RIA and increased by ECLIA. The added different amount of Tg calibrator had not significant influence on Tg recovery rates. When TgAb concentration increased, recovery rates of Tg were decreased by ECLIA and increased by RIA. When sera were diluted, the recovery rates of Tg were increased by ECLIA while decreased by RIA. RIA and ECLIA have good correlation with Tg measurement in 10-400 ng/ml. ECLIA has wider measuring range and higher sensitivity than RIA. RIA and ECLIA have good correlation with TgAb measurement. When TgAb is positive, Tg values are underestimated by ECLIA and overestimated by RIA. When sera are diluted, Tg value and the recovery rate are increasing by ECLIA and decreased by RIA. Recovery test can not efficiently rectify Tg value when TgAb is positive.
Adolescent ; Adult ; Aged ; Autoantibodies ; blood ; Child ; Electrochemical Techniques ; Female ; Graves Disease ; blood ; Hashimoto Disease ; blood ; Humans ; Luminescent Measurements ; methods ; Male ; Middle Aged ; Radioimmunoassay ; methods ; Thyroglobulin ; blood ; immunology ; Thyroid Neoplasms ; blood ; Young Adult

OBJECTIVE: To explore the forensic application value of detection of matrix metalloproteinase-11 (MMP-11) in menstrual blood by enhanced chemiluminescence method. METHODS: Menstrual blood, vaginal swab, peripheral blood, saliva stain, urine stain and semen stain were collected to detect whether or not there were MMP-11 using enhanced chemiluminescence method. The specificity and reliability of the MMP-11 assay along with its sensitivity were evaluated. RESULTS: The positive detection rate of MMP-11 in menstrual blood was 89.47%, whereas no MMP-11 was found in vaginal swab, peripheral blood, saliva stain, urine stain and semen stain. When 25 microL sample was added, the mass concentration of protein was 1.329 microg/microL, then MMP-11 could be detected. A positive detection rate of 89.58% was observed in MMP-11 positive menstrual blood samples after stored at 4 degrees C for 20 months. CONCLUSION Enhanced chemiluminescence method is sensitive and specific for detecting MMP-11, and can be applied to distinguish menstrual blood from common stain such as peripheral blood, vaginal fluid.
Biomarkers/blood* ; Blood Stains ; Blotting, Western ; Female ; Forensic Medicine/methods* ; Humans ; Luminescent Measurements/methods* ; Matrix Metalloproteinase 11/blood* ; Menstruation ; Reproducibility of Results ; Saliva/chemistry* ; Sensitivity and Specificity ; Urine/chemistry* ; Vagina/chemistry*

Serum thyroglobulin (Tg) is primarily used as a tumor marker to detect the recurrent or persistent disease in patients with differentiated thyroid carcinomas. Unfortunately, the serum Tg measurement is technically challenging and the thyroglobulin autoantibody (TgAb) interference remain the most serious problem limiting the clinical value of serum Tg. The direction and magnitude of the interference are related to the method and the concentration and affinity of the TgAb in the specimen. The objective of this study was to evaluate TgAb's interference with assay of Tg by electrochemiluminescent assay (ECLIA). The Tg and TgAb of 84 sera were measured by the ECLIA. Recovery tests were carried out in 3 groups. 3 different Tg calibrators, 50, 100, 200 ng/ml, respectively, were added into the sera of the first group. The sera of second group were doubly diluted 5 times, and meawhile the Tg and TgAb were measured after each dilution, and then 100 ng/ml Tg calibrator was added into the sera. The sera of third group were divided into different subgroups according to TgAb concentration and then 100 ng/ml Tg calibrators were added. Recovery rate (%) was calculated. Tg value decreased when TgAb concentration increased by ECLIA, TgAb value became lower when sera were diluted, Tg value increased. The added Tg calibrator had not significant influence on Tg value and Recovery rate. Recovery rate was lower when TgAb concentration increased. When sera were diluted, the recovery rate was increased. Tg value were underestimated by TgAb interference. After sera were diluted, Tg value became increasing by ECLIA. The added Tg calibrator had not significantly influence on Tg value and Recovery rate. When sera were diluted, the recovery rate was increased. TgAb concentration had significant influence on Recovery rate. Recovery test can not efficiently rectify Tg value when TgAb was positive.
Adolescent ; Adult ; Aged ; Autoantibodies ; blood ; immunology ; Child ; Electrochemical Techniques ; False Positive Reactions ; Female ; Graves Disease ; blood ; Hashimoto Disease ; blood ; Humans ; Luminescent Measurements ; methods ; Male ; Middle Aged ; Thyroglobulin ; blood ; immunology ; Young Adult

OBJECTIVETo evaluate chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA) and ELISA in determination of low level HBsAg.

METHODSAccording to the standard of CLIA Architect i2000, 70 samples were divided into three groups by HBsAg concentration : <1 ng/ml, 1-5 ng/ml and >4 ng/ml. The samples were also determined by ECLIA MODULAR170 and ELISA, and the results were compared with those measured by CLIA Architect i2000.

RESULTSThe concordance rates of ECLIA MODULAR170 with Architect i2000 was 79.2% for <1 ng/ml group, 100% for 1-5 ng/ml group, 100% for >4 ng/ml group. And the concordance rates of ELISA with Architect i2000 was 0 % for <1 ng/ml group, 45.5% for 1-5 ng/ml group and 100 % for >4 ng/ml group.

CONCLUSIONFor determination of low level HBsAg,CLIA Architect i2000 and ECLIA MODULAR170 have high credibility. ELISA is also credible when HBsAg >5 ng/ml, but not for low level HBsAg, especially for HBsAg <1 ng/ml.

Enzyme-Linked Immunosorbent Assay ; Hepatitis B Surface Antigens ; blood ; Humans ; Immunoassay ; methods ; Luminescent Measurements ; methods

In this review we summarize the progress made towards understanding the role of protein-protein interactions in the function of various bioluminescence systems of marine organisms, including bacteria, jellyfish and soft corals, with particular focus on methodology used to detect and characterize these interactions. In some bioluminescence systems, protein-protein interactions involve an "accessory protein" whereby a stored substrate is efficiently delivered to the bioluminescent enzyme luciferase. Other types of complexation mediate energy transfer to an "antenna protein" altering the color and quantum yield of a bioluminescence reaction. Spatial structures of the complexes reveal an important role of electrostatic forces in governing the corresponding weak interactions and define the nature of the interaction surfaces. The most reliable structural model is available for the protein-protein complex of the Ca(2+)-regulated photoprotein clytin and green-fluorescent protein (GFP) from the jellyfish Clytia gregaria, solved by means of Xray crystallography, NMR mapping and molecular docking. This provides an example of the potential strategies in studying the transient complexes involved in bioluminescence. It is emphasized that structural studies such as these can provide valuable insight into the detailed mechanism of bioluminescence.
Animals ; Anthozoa ; physiology ; Aquatic Organisms ; physiology ; Bacteria ; metabolism ; Binding Sites ; Calcium ; metabolism ; Crystallography, X-Ray ; Fluorescence Resonance Energy Transfer ; Green Fluorescent Proteins ; metabolism ; Hydrozoa ; physiology ; Imidazoles ; metabolism ; Luciferases ; metabolism ; Luminescent Measurements ; instrumentation ; methods ; Luminescent Proteins ; metabolism ; Models, Molecular ; Protein Binding ; Pteridines ; metabolism ; Pyrazines ; metabolism ; Scyphozoa ; physiology ; Spectrometry, Fluorescence

We developed a high-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay (hs-CRP CLIA). The high-purity native CRP was purified from hepatic cirrhosis patient ascetic fluid by affinity and ion exchange chromatography and used as an immunogen to develop the monoclonal antibodies (mAbs) against CRP. Twenty-two mAbs were identified reactive with CRP in ELISA and 13 of them were reactive in the phosphorycholine ligand capture ELISA. The mAbs 10C5 and 10C11 were selected to develop the hs-CRP CLIA. The linearity and performance of the hs-CRP CLIA was characterized. It was showed not reactive when testing against other serum materials (IgG, hemoglobin and triglyceride). The reliable correlation (R2 > 0.993) was obtained between testing value (RLU/S) and the concentration of human serum CRP calibrator. The linearity fell in the range of 0.04-20.38 mg/L. The assay has good accuracy and reproducibility, the mean recovery was 99% and the precision of the intra- and inter assay was CVs (4.2%-5.8%) and (9.0%-11.5%), respectively. In testing of 90 human sera, this assay performed well and correlated comparably with a commercial hs-CRP ELISA kit. Thus, hs-CRP CLIA is an accurate, reliable, quantifiable assay for detection of high-sensitive C-reactive protein in serum, it may be useful to improve the risk assessment of cardiovascular disease and the prognosis of inflammatory bowel disease.
C-Reactive Protein ; analysis ; chemistry ; Humans ; Immunoassay ; methods ; Luminescent Measurements ; methods ; Sensitivity and Specificity

OBJECTIVETo explore the value of adenosine triphosphate-tumor chemosensitivity assay (ATP-TCA) in individualized treatment of recurrent epithelial ovarian cancer (REOC), and to evaluate the correlation between the in vitro chemosensitivity assay and clinical drug sensitivity.

METHODSSixty-nine REOC specimens were tested by ATP-TCA assay retrospectively. The patients were divided into strong sensitive, moderate sensitively and resistant groups according to the ATP-TCA assay results. The clinical results were evaluated according to imaging and serum CA125 analysis. The correlation between in vitro ATP-TCA assay and clinical outcome was statistically analyzed by χ(2) test. The progression free survival (PFS) and overall survival (OS) of each group were analyzed using Kaplan-Meier method.

RESULTSThe results of ATP-TCA assay had significant correlation with clinical outcome. The clinical chemotherapy outcome became better with increased drug sensitivity in vitro (χ(2) = 9.066, P = 0.004). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy rate for ATP-TCA method to predict the clinical chemotherapy sensitivity of REOC were 87.5%, 45.9%, 58.3%, 80.9% and 65.2%, respectively. The mean PFS of strong sensitive group, moderately sensitive group and resistant group were 187.1 days, 195.0 days and 60.3 days, respectively. The mean OS were 476.7, 335.7 and 237.5 days, respectively, following the start of TCA-directed therapy. The PFS and OS of the two sensitivity groups in vitro were significantly longer than that of the in vitro-resistant group (P < 0.01).

CONCLUSIONThe results of ATP-TCA assay are well correlated with clinical treatment responses. The assay may be an important and useful method for individualized chemotherapy for recurrent ovarian cancer.

Adenosine Triphosphate ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; CA-125 Antigen ; blood ; Carcinoma, Endometrioid ; blood ; drug therapy ; metabolism ; Cystadenocarcinoma, Serous ; blood ; drug therapy ; metabolism ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; Disease-Free Survival ; Doxorubicin ; administration & dosage ; Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; methods ; Etoposide ; administration & dosage ; Female ; Follow-Up Studies ; Humans ; Luminescent Measurements ; Neoplasm Recurrence, Local ; Ovarian Neoplasms ; blood ; drug therapy ; metabolism ; Paclitaxel ; administration & dosage ; Predictive Value of Tests ; Retrospective Studies ; Sensitivity and Specificity ; Survival Rate ; Topotecan ; administration & dosage

Mesenchymal stem cell (MSC)-based cell therapy has shifted into clinical trials to repair the damage of various tissues. In this setting, the survival of the transplanted cells contributes critically to the therapeutic effectiveness. To investigate the in vivo tracing of MSCs, a recombinant retroviral vector carrying firefly-luciferase reporter gene [pL (FLUC) SN] was constructed and several GPE+86 cell clones that stably expressed fluc were selected. The retroviral supernatants were collected and used to transfect MSC derived from C57 mice. The cells were then screened with G418 and the expression of the exogenous gene was identified by luciferase enzyme activity analysis. Labeled mouse MSCs (2x10(6)) were injected into skeletal muscles, and the in situ expression was noninvasively tracked by in vivo bioluminescence imaging for 1, 3 and 6 days after transplantation. The results showed that the survival rates of the grafted cells dropped sharply with time, they were 57.2+/-11.7%, 8.6+/-2.5% and 5.4+/-3.1% on day 1, 3 and 6 after transplantation, and no fluorescent signals above background were detected on day 10. It is concluded that the method described above could be used for in vivo tracing of grafted cells. Furthermore, MSCs could not survive even transplanted into the none-ischemic skeletal muscles.
Animals ; Bone Marrow Cells ; cytology ; Bone Marrow Transplantation ; methods ; Cell Survival ; Female ; Genetic Vectors ; Green Fluorescent Proteins ; Luminescent Measurements ; methods ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL