Generation of antibodies against COVID-19 virus for development of diagnostic tools

The COVID-19 China coronavirus started in Dec 2019 was challenged by the lack of accurate serological diagnostic tool for this deadly disease to quickly identify and isolate the infected patients. The generation of COVID-19-specific antibodies is essential for such tasks. Here we report that polyclonal and monoclonal antibodies were generated by immunizing animals with synthetic peptides corresponding to different areas of Nucleoprotein (N) of COVID-19. The specificities of the COVID-19 antibodies were assessed by Western Blot analysis against NPs from COVID-19, MERS and SARS. Antibodies were used for immunohistochemistry staining of the tissue sections from COVID-19 infected patient, as a potential diagnostic tool. A Sandwich ELISA kit was quickly assembled for quantitation of the virus/NP of COVID-19 concentrations in the vaccine preparations. Development of POCT is also aggressively undergoing.

Analysis of Distribution and Drug Resistance of Clinical Bacterial Strains Isolated from Children Patients in Our Hospital during 2014-2019 / 中国药房

OBJECTIVE：To pro vide reference for rational use of antibiotics in pediatric department. METHODS ：Clinical bacterial strains isolated from children outpatients and inpatients were collected from West China Guang ’an Hospital of Sichuan University（called“our hospital ”for short ）during Jan. 2014 to Jun. 2019. Distribution and drug resistance of bacteria were analyzed retrospectively. RESULTS ：During 2014-2019，total of 4 692 strains were detected ，accounting for 29.56％ of total ；those were mainly from sputum （3 749 strains，79.90％），blood（203 strains，4.33％）and secretion （137 strains，2.92％）specimen. Among them ，1 488 strains of Gram-positive bacteria （31.71％）were mainly Streptococcus pneumoniae （711 strains，15.15％） and Staphylococcus aureus （574 strains，12.23％）；3 204 strains of Gram-negative bacteria （68.29％）were mainly 2 466 strains of Haemophilus influenza （52.56％）. Totally 172 strains of methicillin-resistant S. aureus and 1 517 strains of β-lactamase producing H. influenzae were detected ；the detection rates were 29.97％ and 61.52％ ，respectively. Resistance rates of H. influenza to ampicillin，cefaclor and cefuroxime were higher than 50％，and the overall trend was on the rise ，resistance rates of cefotaxime ， rifampin and ofloxacin were all lower than 6％. Resistance rates of S. pneumoniae to erythromycin and tetracycline were more than 70％，and the resistance rate to erythromycin was increasing year by year. Resistance rates of S. pneumoniae to β-lactams and quinolones were generally lower than 20％. No resistant strains of linezolid and vancomycin were found. Resistance rate of S. aureus to penicillin G was more than 90％. S. aureus was relarively sensitive to aminoglycosides ，macrolides and tetracyclines ；no furantoin，linezolid and vancomycin-resistant strains were found. CONCLUSIONS ：Gram-negative bacteria are the main pathogens isolated from children in our hospital ，and most of them are H. influenza e，S. pneumon iae and other caustic bacteria. The detection rate of drug-resistant and enzyme producing strains is high ， and the resistance rate of several pathogens to commonly used 0826-2600251。E-mail：wenxiaozheng269@sina.com antibiotics is increasing year by year. Drug resistance is severe. In order to delay the emergence and spread of drug-resistant pathogens in real time and further standardize the use of pediatric antibiotics.

A PCR-lateral flow assay system based on gold magnetic nanoparticles for CYP2C19 genotyping and its clinical applications.

Presently, clopidogrel is the standard therapeutic drugs for antiplatelet therapy. Variants in the CYP2C19 gene influence the clinical response of clopidogrel. Thus, the US Food and Drug Administration suggested CYP2C19 genotyping needs to identify before taking medicine. Due to high cost, time consuming, and sophisticated instruments, current single nucleotide polymorphism detection methods are limited in clinical application. In the present study, we established a genotyping method for CYP2C19, which combines amplification refractory mutation system (ARMS)-PCR with a lateral flow assay used gold magnetic nanoparticles (GMNPs) named as PCR-gold magnetic lateral flow assay system (PCR-GoldMag LFA). The PCR products with specific genotype can be explained within 5 minutes, either through visual or by a magnetic reader automatically according to the captured GMNPs probes on the test lines of the LFA strips. The limit of detection of this method is 5 ng of genomic DNA. The PCR-GoldMag LFA system was applied in a clinical trial with 1356 samples of Han Chinese. The concordance rate between the LFA system and sequencing is 99.93%. The allele frequency of CYP2C19*2 and CYP2C19*3 are 30.38 and 7.08% in Han Chinese, respectively. This method provides a new way in the clinical application of CYP2C19 genotyping to guide the clopidogrel medication.

Relationship between the semi-quantitative index of 99 Tcm-3PRGD2 SPECT/ CT imaging and clinical pathological features of breast cancer / 中华核医学与分子影像杂志

Objective To investigate the tumor/ non-tumor (T/ NT) ratio during 99 Tcm-hydrazinon-icotinamide(HYNIC)-( poly-( ethylene glycol), PEG) 4-Glu( cyclo ( Arg-Gly-Asp-D-Phe-Lys ( PEG4 ))) 2 ( 99 Tcm-3PRGD2 ) SPECT/ CT imaging and clinical pathological features of breast cancer. Methods Forty-five female patients (age range: 39-76 (53.0±9.5) years) with suspected breast malignant nodules or mas-ses from October 2016 to June 2017 were prospectively enrolled. Patients underwent 99 Tcm-3PRGD2 SPECT/CT imaging before breast puncture and surgery. All subjects had pathological results, and estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2(HER-2), Ki-67 and mi-crovessel density ( MVD) were obtained by immunohistochemistry and fluorescence in situ hybridization(FISH). Two-sample t test and Kruskal-Wallis H test were used to analyze the data. Results Invasive duc-tal carcinoma was pathologically confirmed in 35 of 45 patients. There were 7 patients in stage Ⅰ, 11 pa-tients in stage ⅡA and 17 patients in stage ⅡB. The Luminal A subtype, Luminal B subtype, ERBB2+subtype, Basal-like subtype were found in 6, 9, 9 and 11 patients, respectively. The T/ NT ratio was signifi-cantly higher in the stageⅡB patients than that in stageⅠ+ⅡA patients (4.54±1.46 vs 3.32±1.72, t= -2.24, P<0.05). Patients with ERBB2+ subtype had higher T/ NT ratio compared to patients with Basal-like sub-type: 5.80(3.90, 6.70) vs 2.80(2.20,3.50), H= 11.06, χ2 = 15.31, both P<0.05. Besides, the T/ NT ra-tios in the HER-2 positive group and lymphatic metastasis group were significantly higher than those in the HER-2 negative group and group without lymph node metastasis (t values: -3.99, -2.51, both P<0.05). MVD of HER-2 positive group was higher than that of HER-2 negative group (t= 7.13, P<0.01). Conclu-sion The T/ NT ratio during 99 Tcm-3PRGD2 SPECT/ CT imaging has relations with TNM staging, lymph node infiltration and HER-2 in breast cancer.

Verification of analytical measurement range of serum creatinine detected by Roche Cobas 501 Biochemistry Analyzer / 国际检验医学杂志

Objective To investigate how the clinical laboratory conducting the verification of analytical measurement range (AM R) of quantitative items detected by the biochemical analyzer according to the requirements of the international standards by verifying the serum creatinine AMR for ensuring the accuracy and reliability of detection results .Methods The enzyme method was adopted to detect the 7‐concentration levels test specimens of CAP linear range proficiency test on the Roche Cobas 501 biochemical analyzer .These 7 specimens target values covered the low ,middle and high values of creatinine AMR marked by the manufacturer′s instructions .Each specimen was detected twice and the mean value was taken ,then the bias between the mean value and target value was calculated .In addition ,referring to the requirements of CLSI guiding document EP6‐P ,the patients′fresh serum contai‐ning high value creatinine was collected ,then mixed with certain proportion and centrifuged .The mixture concentration was calcu‐lated and served as the high value specimen(H) ,and the low value specimen was obtained by the same treatment .Then the high and low value specimens were dispensed with the relations of 5L ,4L+1H ,3L+2H ,2L+3H ,1L+4H and 5H and formed the series specimens .The creatinine levels in each specimen was detected on the Roche Cobas 501 biochemical analyzer ,each specimen was de‐tected 4 times .The obtained data were performed the regression analysis .Results The bias of 7‐level CAP specimen and target val‐ue was less than the allowable error ± 7 .5% [(1/2 × TE)% ] set by the clinical laboratory of the Beijing Sanfine Hopsital .The re‐gression equation of fresh mixed serums from patients was Y =0 .988 6X+16 .614 ,b=0 .988 6 ,between 0 .97 -1 .03 ,intercept a and 0 ,ta 0 .05 ,which showed no significant difference between intercept and 0 ,the regression line was through 0 point in fact .Conclusion The verification of creatinine AMR marked by the manufacturer′s instructions is passed ,which can be adopted by the clinical laboratory .

[GoldMag particle-based chemiluminescence immunoassay for human high sensitive C-reactive protein].

OBJECTIVE: To develop a sensitive, accurate detection method for high sensitive C-reactive protein (hsCRP). METHODS: With the GoldMag particle as the solid phase carrier, horseradish peroxidase (HRP)-lunimo-H2O2 as the chemiluminescence reaction system, we established a chemiluminescent immunoassay for hsCRP detection. Linear range, sensitivity, precision, accuracy and other indicators were evaluated. hsCRP level of 233 clinical human serum specimens were determined and compared by two different methods: the GoldMag particle-based magnetic chemiluminescence enzyme-linked immunoassay established in this study, and the commercialized scattering immunoturbidimetric assay from Germany SIEMENS. RESULTS: The chemiluminescent immunoassay for hsCRP based on GoldMag particle had a good linear relationship between 0.15 mg/L and 25 mg/L (R(2)=0.9937), with the detection limit of 0.076 mg/L. The intra-assay precision was less than 10.00% and the inter-assay precision was less than 15.00%. The average recovery rate for accuracy was 97.80%. In the contrast experiment of 233 clinical human serum specimens, the results obtained using the approach established in this study showed a high correlation and consistency with scattering immunoturbidimetric assay from Germany SIEMENS. CONCLUSION: GoldMag particle-based magnetic chemiluminescence enzyme-linked immunoassay for hsCRP has been successfully developed.

Application of dried blood spot in newborn screening for inherited metabolic diseases / 中华检验医学杂志

Neonatal inherited metabolic diseases are a group of metabolic disorders caused by singe gene defect to cause a series of clinical symptoms.Neonatal dried blood spots have the advantages of simple preparation,safety,good stability,and show strong practicability in different screening methods for inherited metabolic diseases.With the development of screening methods,more and more diseases could be diagnosed by screening.The emergence of tandem mass spectrometry and molecular biological techniques promote the newborn screening and automation for inherited metabolic disease effectively.Inherited metabolic diseases induce great harm to the newborn,which could cause not only system organs damage,but also lead to death.Therefore,early screening is important for patients' prognosis.

Study of the Correlation of Testing Results by Two Different D-dimer Reagents with Fibrin Degradation Products Result Values / 现代检验医学杂志

Objective To evaluate the clinical practicality of two different D-dimer assays by studying the correlation of the testing results by two different D-dimer reagents with fibrin degradation products (FDP)result values and analyzing the Clinical alignment.Methods A representative cross-sectional study of 762 clinical patients’plasma specimens were taken to measure the results of D-dimer and FDP by using Sysmex CA-1500 automated coagulation analyzer,D-DIMER detection rea-gent respectively Siemens Innovance D-DIMER and Dade Behring D-DIMER Plus reagents.Then rating frequency analysis and Spearman correlation test were applied to analyze the correlation of measurement results.Results Overall,to the same patient,the correlation of D-dimer tested by Siemens Innovance D-DIMER Reagent with FDP level was significant positive (r= 0.954,P 5 mg/L FEU,the correlation was the best (P5μg/ml)and FDP positive rate in both different cases when D-dimer results was also positive,FDP positive rate in Innovance D-DIMER Reagent group (about 65.76%)was lower than DD Plus Reagent group,it indicated that D-dimer results tested by Innovance D-DIMER Reagent could raise before FDP reacted,so D-dimer had a higher sensitivity than FDP.Conclusion It can provide a more precise clinical diagnosis and treatment infor-mation when using Siemens Innovance D-DIMER Reagent to detect D-dimer values and combined FDP values at the same time.

Irreversible airway obstruction assessed by high-resolution computed tomography (HRCT), exhaled nitric oxide (FENO), and biological markers in induced sputum in patients with asthma.

OBJECTIVE: The objective of this study was to explore the significance of assessing irreversible airway obstruction (IAO) in asthma patients by high-resolution computed tomography (HRCT), biological markers in induced sputum, and exhaled nitric oxide (FENO). METHODS: The study was conducted in 34 patients with IAO, 46 patients with reversible airway obstruction (RAO), 40 patients who did not have airway obstruction (NAO), and 40 healthy subjects serving as controls. These patients received a step therapy for at least 3 months based on the guidelines for the prevention and treatment of asthma. After achieving complete or partial control of asthma, HRCT, lung function, FENO, and chemokine levels in induced sputum were measured. RESULTS: The airway wall area (WA; %) correlated with forced expiratory volume-1 (FEV-1(L); r = -0.67, p < 0.0001), and significant differences in bronchial wall thickening (BWT) of the LEVEL E generation airways were observed between the asthma and control groups (p < 0.01). FENO levels correlated with FEV-1 (%) in the IAO group (r = 0.49, p = 0.01). The levels of matrix metalloproteases-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in asthma patients with IAO, RAO, and NAO were significantly higher than those in the controls (p < 0.05). The level of neutrophilia in the sputum from the IAO group was higher than that from the RAO, NAO and control groups. CONCLUSION: Asthma patients with IAO have an increased BWT. Airway measurements with HRCT scans appear to be valuable in the evaluation of airway remodeling in asthma patients with IAO.

Relationship between the CYP2C19 polymorphism and the efficacy of triple therapy with lansoprazole onHelicobacter pylori infection in elderly patients / 实用医学杂志

Objective To investigate the Relationship between the CYP2C19 polymorphism and the efficacy of triple therapy with lansoprazole on Helicobacter pylori infection. Methods 125 elderly patients diagnosed with H.pylori infection were treated with triple therapy. Polymorphism of CYP2C19 was measured by AS-PCR. 105 young patients were selected as control group. The relationship between the polymorphism and eradication rate were analyzed. Results Among the 125 patients,eradication rate of extensive metabolizer group,internal metabolizer group and poor metabolizer groups was 85.29%,76%and 89.39%, respectively. There was no significant difference between groups (P > 0.05). Eradication rate showed no difference between experimental and control group either (P>0.05)(P>0.05). Conclusion The results suggests that the CYP2C19 polymorphism has no correlation with the eradication rate of Helicobacter pylori infection by triple therapy with lansoprazole in elderly patients.

Purification of glutathione-S-transferase fusion protein by glutathione coupled magnetic particles / 生物工程学报

We established a purification system for glutathione-S-transferase (GST) fusion protein using glutathione coupled magnetic particle. Glutathione was coupled covalently to the surface of magnetic particles with isothiocyanate functional groups. Cell lysate, containing the fusion protein, was then incubated with these glutathione coupled magnetic particles at room temperature. Unbound and non-specifically bound proteins were removed by wash steps. Subsequently, the GST-fusion protein was eluted from the magnetic particles by the addition of reduced glutathione. The resulting fusion protein was tested for purity using SDS-PAGE and demonstrated by Western blotting. The concentration of the fusion protein was measured by Bradford method. Both the conditions for incubation and washing were optimized. The results showed that 150 microg glutathione could be bound on 1 mg of particle surface and 10 mg of the glutatione-coupled magnetic particles was suitable for 100 microL lysate, the optimal incubation time for reaction between particles and lysate was 40 min. The magnetic particles could help purify efficiently GST-fusion protein with a yield of around 516 microg fusion protein per 10 mg particles. Magnetic particles can be successfully used in a simple, rapid and reliable method for the purification of GST-fusion proteins.

Protective effects of Quercetin to 6 Gy irradiated rats / 中华放射医学与防护杂志

Objective To evaluate the radioprotective effects of Quercetin (QN) on 6 Gy X-ray irradiation-induced immune dysfunction and toxicity in hepatic tissue in rats. Methods 40 adult rats were randomly divided into 4 groups. Group Ⅰ was injected intraperitoneally with saline solution for 7 consecutive day sand served as control group. Group Ⅱ was daily injected with QN (40 mg/kg) for 7 consecutive days. Group Ⅲ was irradiated with a single dose of 6 Gy X-ray. Group Ⅳ received a daily injection of QN (40 mg/kg) for 7 consecutive days, and 1 h after the last injection rats were irradiated with a single dose (6 Gy) X-ray irradiation.The animals were sacrificed after 24 h. Lymphocyte transforming rate was measured with MTT method, and CD+4 T, CD+4 T and CD+8/CD+8 T were measured with flow cytometry method. Oxidative conditions in liver were measured with malondialdehyde (MDA), reduced glutathione hormone (GSH), supernxide dismutase (SOD) andglutathione peroxidase (GSH-Px) activities kits. HE staining was used to observe the general condition of rat's liver. Results Lymphocyte transforming rate, CD+4 T, CD+8 T and CD+8/CD+8 T in rats of Group Ⅳ were all higher than those in rats of Group Ⅲ ( F = 8.455,22.644, 18.911, P < 0.01 ). MDA content in the Group Ⅳ rat's liver was lower than that in the Group Ⅲ ( F = 10.059, P < 0.01 ) and antioxidant enzymes SOD, GSH-Px activities were higher than those in Group Ⅲ (F = 23.688,186.046,19.788, P < 0.01 ). The capillary of the hepatic lobules dilated and congested obviously in portal area, involving infiltration of polymorphonuclear leukocytes in the Group Ⅲ, while QN improved this change apparendy. Conclusions Pretreatment with Quercetin improved the irradiated rat's immune functions and protected the irradiated rats from oxidative stress to some extent.

Long-term results of chemotherapy alone or combined with radiation therapy for limited-stage small cell lung cancer / 中国肺癌杂志

<p><b>BACKGROUND</b>To evaluate therapeutic outcome of limited stage small cell lung cancer treated with chemotherapy alone or combined with radiation therapy with different doses.</p><p><b>METHODS</b>A retrospective analysis was performed in 128 limited-stage small cell lung cancer patients who were treated with three different ways of treatment, from February 1988 to March 1998 in Heilongjiang Cancer Hospital. All patients were pathologically proved. Forty-two patients received chemotherapy alone (C), 48 patients were treated by interdigitating chemoradiotherapy (IDG) and other 38 patients received concurrent chemoradiotherapy (CON). For thoracic radiation, 20 patients received a dose of ≤45 Gy, 23 ≥60 Gy, and 43 > 45 Gy but < 60 Gy .</p><p><b>RESULTS</b>The 3-year survival rates were 23.7%, 20.8% and 4.8% in the CON, the IDG and the C groups respectively. There was a significant difference between the CON and the C groups ( P < 0.05), as well as between the IDG and the C groups ( P < 0.05). There was no remarkable difference between the CON and the IDG groups ( P > 0.05). Loco-regional recurrence rate was significantly higher in ≤45 Gy group (55.0%) than that in ≥60 Gy group (8.7%)( P < 0.01).</p><p><b>CONCLUSIONS</b>The chemotherapy combined radiotherapy may improve the survival of patients with limited-stage small cell lung cancer. Dose of thoracic radiation might be related to the loco-regional recurrence.</p>

Development of visible protein microarrays based on GoldMag particles-labeling technique / 临床检验杂志

Objective To develop a visible detection system prepared by protein microarrays which was based on GoldMag particles-labeled technique and compare the results of detection using the protein which was labeled with GoldMag particles and colloidal gold.Methods Human IgG was printed on the glass slides modified with epoxy groups,and goat anti-human IgG conjugated with GoldMag particles and colloidal gold respectlively was then added on the slide.The glass slides were incubated,and then the black images of microarray spots were produced by immuno-gold-silver staining method and observed by naked eyes and recorded with common flatbed scanner.Results The high signal-to-noise ratio could be obtained when the optimized procedures of GoldMag particles-labeling probe were introduced to the protein chip.The conditions of optimum assay were as follows:the spotting concentration of human IgG was 0.2 mg/ml,the glass slides were incubated at 37 ℃ for 2 hour to immobilize human IgG,and the silver enhancement time was 10 min-15 min.Parallelly,the optimized conditions for colloidal gold were as follows:the spotting concentration of human IgG was 0.1 mg/ml,the slides were incubated at 37 ℃ for 1 hour to immobilize human IgG,and the silver enhancement time was 15 min-20 min.Conclusions GoldMag particles-labeled protein technique is comparable to colloidal gold in applying to protein microarrays.The method is considerably simple and practical,and the protein labeled by GoldMag particles could be quantitative.