Objective:To produce monoclonal antibodies(McAb) against aspergillus fumigatus and to establish rapid assay for the measurement of aspergillus fumigatus antigen.Methods:Recombinant galactomannoprotein of aspergillus fumigatus(AFMP1) was used to immune BALB/c mice.Monoclonal antibodies against AFMP1 were produced from hybridoma.Results:Three hybridomas producing antibodies against AFMP1 were obtained.IgG isotypes of three McAb were IgG1,IgG2a and IgG2b.The affinity constants(K) were 1.2?10 10 ,4.56?10 9 and 1.81?10 10 mol/L.The antibodies were proved to be specific for aspergillus fumigatus by Western blot and recognized different epitopes on AFMP1 by the additivity assay.An sandwich enzyme-linked immunosorbent assay(ELISA) to detect AFMP1 was established to produce standard curve which showed linearity between 0.1～60.0 ng/ml with a sensitivity of 0.1 ng/ml.Conclusion:These results show three hybridomas producing high specificity and affinity monoclonal antibodies against AFMP1 and can provide for rapid assay for the measurement of aspergillus fumigatus antigen.

Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.

Objective To compare the detection efficiency between multiplex RT-PCR method and liquichip technology for screening the viral etiological agents of diarrhea.Methods The development of the multiplex RT-PCR method.A total of 107 feces samples from patients who suffered from diarrhea and attended to Zhujiang Hospital of Southern University from September 2013 to February 2014 were collected and tested in parallel by both multiplex RT-PCR and xTAG Gastrointestinal Pathogen Panel ( xTAG GPP) for Adenovirus, Norovirus genogroupⅠandⅡ, as well as by both multiplex RT-PCR and monoplex RT-PCR for Astrovirus and Sapovirus.To evaluate the sensitivity and specificity of multiplex RT-PCR, xTAG GPP and monoplex RT-PCR were used as reference.Kappa coefficient test was used to evaluate the consistency among the methods.The detection limit and accuracy of multiplex RT-PCR were evaluated by detection of serial dilution of positive plasmids and products sequencing for the five viral agents.Results The multiplex RT-PCR showed high consistency with xTAG GPP and monoplex RT-PCR, in which Kappa value was 0.885 and 1.000 respectively( P=0.000 ).Compared to xTAG GPP, the sensitivity and specificity of the multiplex RT-PCR were at average of 80.8%( 21/26 ) and 100%( 295/295 ) respectively.The detection limit and accuracy of multiplex RT-PCR were 104 copies /μl-106 copies/μl.Conclusion The high consistency indicated that both the multiplex RT-PCR and xTAG GPP are useful as a special,sensitive, high throughput and rapid diagnostic tools for the detection of the major viral pathogens related to diarrhea in clinical laboratory.

OBJECTIVE To study the etiological diagnosis of invasive aspergillosis by the monoclonal antibodies against Aspergillus fumigatus. METHODS An animal model of rabbit invasive aspergillosis was established.The antigen of A.fumigatus in serum was detected by ELISA.The antigen of A.fumigatus in tissue was detected by immunochemistry. RESULTS ELISA assay showed positive 24,48 and 72 hours after infection.Immunochemistry was positive 72 hours after infection. CONCLUSIONS The monoclonal antibodies against A.fumigatus has great potency usage.

Objective To screen monoclonal antibodies (mAbs) for early diagnosis of invisive Aspergillus. Methods Monoclonal antibodies against different antigens of Aspergillus fumigatus were produced. The two pairs of combinations of monoclonal antibodies were selected accoring the distinct epitopes and double-antibody sandwich ELISA based on mAbs above were established. The sensitivity and specificity of the methods were analyzed by detecting culture supernatants of clinical isolates and environmental isolatesof Aspergillus. spp, Penicillium Marneffei, Candidas, and serum from animal models and patients. The epitopes recognized by mAbs were identified by immunobotting. Results A total of 32 hybridoma cell lines that stably produced MAbs were obtained. Two double- antibody sandwich ELISAs were established. One method was specific for 19 clinical isolates and environmental isolates of Aspergillus. spp, whereas the other one was specific for the clinical and environmental isolates of Aspergillus fumigatus without cross-reation with other Aspergillus. spp. For the same kind of medium of Aspergillus fumigatus, the sensitivity of the first method was 10 fold higher than the second method. Conclusions The specific mAbs for early diagnosis of invisive Aspergillus were obtained. Antigen recognized by the specific mAbs was mannoprotein with molecular weights of approximately 25 000-75 000. This antigen was potential early diagnostic marker for invasive Aspergillus.

OBJECTIVETo study the effects of Lactobacillus acidophilus (L. acidophilus) on the proliferation and cell cycle distribution of human tongue cancer cells (Tca8113 cells).

METHODSIn vitro cultivated human Tca8113 cells were treated by L. acidophilus supernatant, inactivated bacilli, cell free extracts and normal culture medium respectively, which were 1, 4, 16-fold(s) dilutelly, to investigate the proliferous effects of Tca8113 cells using of inverted microscope, cell counting, sulforhodamine B (SRB) and flow cytometry. The free radicals and Ca2+ in Tca8113 cells were also studied by confocal laser scanning microscope (CLSM).

RESULTSAt the 48th hour after adding different L. acidophilus components, the Tca8113 cells changed in shape from the diamond-like, polygonal and slabs into the elongated form. In the condition of different times and different culture concentrations, the proliferation of Tca8113 cells was significantly inhibited by L. acidophilus components, which enhanced as the time prolonged and the concentrations of each L. acidophilus components increased according to the cell counting and the SRB experimental analysis. The cell proliferation index (CPI) was significantly reduced (P<0.01). The free radicals and Ca2+ in Tca8113 cells under the effect of each L. acidophilus components for 48 h indicated an obviously rising (P<0.01).

CONCLUSIONL. acidophilus restrains the proliferation of Tca8113 cells, which might be due to the increase in quantity of free radicals and Ca2+ in Tca8113 cells, and might be resulted from the release of metabolic products of L. acidophilus.

Carcinoma, Squamous Cell ; Cell Proliferation ; Humans ; Lactobacillus acidophilus ; Tongue Neoplasms

OBJECTIVETo perform cytogenetic analysis for children, especially newborns suspected for chromosome abnormalities.

METHODSPeripheral blood or born marrow specimens were respectively cultured in proper media. Karyograms were analyzed following G-banding.

RESULTSOf 154 blood specimens, numerical chromosomal abnormalities were identified in 20 patients, which included 19 with trisomy 21. Structural aberrations were identified in 13 patients, among which chromosome 9 aberrations were seen in 6 cases. These included 3 inversions, 1 deletion, 1 insertion and 1 duplication. All aberrations were located in pericentromere region of chromosome 9 with clinical manifestations including congenital heart disease, peculiar facial appearance, paralysis, dysplasia and/or movement disorder. Chromosome polymorphisms were found in 20 patients, most of which had absence of satellites or variation of heterochromatin on chromosome 9. Of 10 bone marrow specimens from children suspected for acute leukemia, chromosome abnormalities were identified in 5 patients.

CONCLUSIONCytogenetic analysis is useful for children featuring multiple congenital abnormalities. Chromosome 9 abnormalities and their clinical relevance should attract more attention.

Adolescent ; Child ; Child, Preschool ; Chromosome Aberrations ; Chromosome Banding ; Chromosome Disorders ; epidemiology ; Chromosomes, Human, Pair 9 ; Humans ; Infant ; Infant, Newborn ; Physical Chromosome Mapping ; Prevalence

Objective:To investigate the clinical characteristics, influencing factors and prevention and treatment measures of nosocomial infection in patients with acute myeloid leukemia (AML) (non-acute promyelocytic leukemia) after applying intermediate-high dose cytarabine (Ara-C) chemotherapy.Methods:The clinical data of 80 patients with AML treated with intermediate-high dose Ara-C in the Second Hospital of Shanxi Medical University from March 2013 to January 2020 were analyzed retrospectively. The clinical features of nosocomial infection were summarized and the influencing factors of infection were analyzed by using multivariate logistic regression.Results:A total of 80 patients received 198 times of chemotherapy, and the infection rate was 72.7% (144/198). Infection sites mainly included respiratory tract infection, pulmonary infection, gastrointestinal infection. A total of 45 strains of pathogenic bacterias were detected, among which Gram negative bacilli accounted for 55.6% (25/45), Gram positive cocci accounted for 24.4% (11/45), fungi accounted for 8.9% (4/45) and viruses accounted for 11.1% (5/45). There were no significant differences in infection rate, hospitalization time, neutrophils recovery time and hospitalization expenses between the sterile laminar flow ward and the general ward (all P > 0.05). Multivariate logistic regression analysis showed that infection during induction chemotherapy was independent risk factor of infection ( OR = 5.076, 95% CI 1.978-13.022, P =0.001), and antibiotic prevention was independent protective factor of nosocomial infection ( OR = 0.332, 95% CI 0.136-0.803, P = 0.014). Conclusions:The infection rate of AML patients receiving intermediate-high dose Ara-C chemotherapy is high. During the treatment, we should be alert to the infection during induction chemotherapy and use antibiotics to prevent it in time. For patients undergoing intermediate-high dose Ara-C chemotherapy, strengthening the environmental cleanliness of general wards may achieve the same preventive effect as that of sterile laminar flow wards.

OBJECTIVETo clone and express VP1-VP4 genes encoding the structural proteins of Coxsackie virus A16 and analyze the antigenicity of the expressed recombinant proteins.

METHODSThe VP1-VP4 cDNAs were amplified with RT-PCR from the extracted viral RNA and cloned into pMD19-T vectors. The VP1-VP4 genes were inserted to the multi-cloning sites of the plasmid pQE30a, and the protein expressions in E. coli M15 were induced by IPTG. After purification by washing with 8 mol/L urea under denaturing condition, the recombinant proteins were identified by Western blotting and ELISA for their immunogenicity against rabbit antisera of Coxsackie virus A16 and enterovirus 71, respectively.

RESULTSThe recombinant VP1-VP4 proteins were highly expressed in E. coli M15. The purified proteins could be recognized by rabbit antiserum of Coxsackie virus A16 and showed cross reactivity with the rabbit antiserum of Enterovirus 71.

CONCLUSIONThe recombinant Coxsackie virus A16 VP1-VP4 proteins obtained possess good antigenicity.

Antigens, Viral ; genetics ; immunology ; Capsid Proteins ; classification ; genetics ; immunology ; Cloning, Molecular ; Enterovirus A, Human ; genetics ; Gene Expression ; Genetic Vectors ; Plasmids ; Recombinant Proteins ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the preventive effects of multi-glycoside of Tripterygium wilfordii (GTW) on glomerular lesions in experimental diabetic nephropathy (DN).

METHODThe DN model of rats was established with streptozotocin (STZ) and intervened with GTW. In the same time, normal, benazepril, and vehicle control groups were set up. After 8 weeks of oral treatment with GTW (50 mg x kg(-1) BW), benazepril (6 mg x kg(-1) BW), and vehicle (physiological saline), the changes of body weight, urine albumin (UA1b), blood glucose (BG), serum creatinine (Scr), blood urea nitrogen (BUN) and glomerular morphology were examined. In addition, the level of protein expression of alpha-smooth muscle actin (alpha-SMA) and collagen type I in glomeruli was determined by immunofluorescence.

RESULTBoth GTW and benazepril reduced UA1b. GTW ameliorated glomerular injury, such as mesangial cell proliferation, alpha-SMA and collagen type I over-expression, in DN model. Compared with benazepril, beneficial effects of GTW on glomerulusclerosis were more significant (total cell number: GTW group 54.44 +/- 2.41, benazepril group microg/67.83 +/- 4.41, P < 0.05; alpha-SMA score: GTW group 1.98 +/- 0.52, benazepril group 2.27 +/- 0.46, P < 0.05; collagen type I score: GTW group 2.11 +/- 0.37, benazepril group 2.88 +/- 0.58, P < 0.05).

CONCLUSIONPreventive effects of GTW on glomerular lesion in DN model are related to decreasing UA1b and ameliorating glomerulusclerosis.

Animals ; Diabetic Nephropathies ; drug therapy ; metabolism ; prevention & control ; Disease Models, Animal ; Glycosides ; administration & dosage ; Humans ; Kidney Glomerulus ; drug effects ; injuries ; metabolism ; Male ; Plant Extracts ; administration & dosage ; Random Allocation ; Rats ; Tripterygium ; chemistry