Objective To develop and verify a double antibody sandwich ELISA method for quantitative detection of TrypLE.Methods The optimal concentration of capture antibody and detection antibody were determined by orthogonal experiments to develop TrypLE double antibody sandwich ELISA quantitative detection method,which was verified for linear range,specificity,limit of detection(LOD),limit of quantitation(LOQ),accuracy and reproducibility.A variety of biological products were detected by the developed method to verify the applicability.Results The TrypLE double antibody sandwich ELISA quantitative detection method was established by using 3 μg/mL capture antibody and 15 000 times dilution of detection antibody,with a linear range of 0.41 ～ 40.00 ng/mL,a LOD of 0.258 ng/mL,a LOQ of 0.5 ng/mL.The measurement deviation was less than 5% and the CV of reproducibility verification was less than 5% when detecting standards and samples.The recovery rates of different types of samples were within 80% ～ 120%.Conclusion The established TrypLE double antibody sandwich ELISA quantitative detection method accurately,effectively and quickly detected residual amount of TrypLE in various types of biological products with good specificity,accuracy and reproducibility.

Objective To perform quantirative study of HEK293 cell genomic DNA by microfluidic chip-based digital PCR technology, and to provide new ideas for more accurate gene quantification and genomic DNA related detection.Methods Firstly, the HEK293 cell DNA was obtained by Spin Column, identified and analyzed for the purity by agarose gel electrophoresis. Then, the genomic DNA of HEK293 cells was quantified by traditional ultraviolet spectrophotometry, Qubit fluorescence quantification and microfluidic chip-based digital PCR technology and statistically analyzed.Results The concentration of genomic DNA in HEK293 cells was detected as 100. 08 ng/μL by ultraviolet spectrophotometry, 93. 98 ng/μL by Qubit fluorescence quantification and the copy number was determined as 29 722. 81 copies/μL by digital PCR. Roughly calculated based on human single-copy genome of about 3. 3 pg, the converted copy numbers of spectrophotometry and Qubit fluorescence quantification were about 30 327 and 28 479 copies/μL, respectively. The deviation of the digital PCR detection value from the spectrophotometry detection value was only 2%, and the deviation from the Qubit detection value was only4%.Conclusion In this study, HEK293 genomic DNA was detected and compared by digital PCR, spectrophotometry and Qubit fluorescence, which provides a new detection method and idea for DNA quantification, and also provides a certain reference for the quantification of other nucleic acids.

Objective To construct a plasmid reference for the detection of residual DNA from early region 1A(E1A) protein and simian virus 40 large T antigen(SV40LTA), and to explore a digital PCR(dPCR)-based analytical technique for copy number calibration of plasmid reference to quantify plasmid reference accurately, thereby providing a new idea for host cell DNA residual detection in biological products.Methods The plasmid pUC19-E1A-SV40LTA was constructed, and the whole gene was synthesized and then amplified to obtain sufficient reference plasmid. The digital PCR system was employed to determine the copy numbers using double fluorescence channels of E1A and SV40LTA primer-probe sets, respectively.The reference material calibrated by digital PCR was subsequently applied to qPCR to establish a method for detecting the specific E1A and SV40LTA sequences. The developed qPCR assay was systematically validated for plasmid reference linearity, accuracy, limit of quantification(LOQ), precision, specificity, applicability, and stability. Furthermore, the established qPCR system was utilized for the detection of recombinant adenovirus(rAdV) sample and recombinant adeno-associated virus(rAAV) sample.Results The copy numbers of E1A and SV40LTA targets in the plasmid reference were found to be essentially consistent through digital PCR using dual fluorescence channels. The detection values detected by E1A and SV40LTA primer-probe sets were 3. 55 × 109and 3. 48 × 109copies/μL, respectively. The coefficient of variation(CV) of these two values was 1. 42%, and the mean value of 3. 51 × 109copies/μL was taken as the calibration value of digital PCR.The qPCR system established by using digital PCR to calibrate copy number exhibited good linearity, accuracy, LOQ, precision, specificity, applicability and stability, and the recovery rates for rAdV and rAAV samples were between 70% and130%.Conclusion A reference plasmid for detecting E1A and SV40LTA residues in biological products was established,and a more sensitive and accurate digital PCR method was introduced for quantification, which can be used to detect the residual DNA of E1A and SV40LTA in gene therapy products produced with HEK293 and HEK293T as host cells.

Objective To evaluate the nutrition status and the prognosis of acute stroke patients with hy-pothyroidism during hospitalization. Methods The clinical data of 28 acute stroke patients with hypothyroidism (study group) and 28 stroke patients with normal thyroid function (control group) were retrospectively analyzed.Results After (10±4) days of hospitalization, hemoglobin and serum albumin levels in study group significantlydecreased ( P < 0. 05 ). The requirement of calories and protein, incidence of pulmonary infections, alimentarytract hemorrhage and diarrhea, and hospital stays were significantly higher in study group than in control group (P < 0. 05 ). Conclusion The nutrition status is poor in acute stroke patients with hypothyroidism, who were more easier to be suffered from clinical complications and worse prognosis.

BACKGROUND:For evaluation of risks of periprosthetic fractures in elderly patients aged>75 years old after hemiarthroplasty, we should perform dynamic observation of postoperative physical health status, quality of life, hip function and bone mineral density. Presently, there is lack of general investigation.
OBJECTIVE:To provide references for clinical diagnosis and prediction of periprosthetic fractures after hemiarthroplasty in elderly patients.
METHODS:On the basis of arranging the exploration results of recent studies on risk factors for periprosthetic fractures of hip joint, we analyzed the monitoring method of scholars concerning fracture-associated risk factors. Simultaneously, in combination of the development of modern inspection sciences, the method was applied in the clinic. Thus, we summarized general evaluation methods with clinical significance for risk factors of prosthesis fracture in elderly patients after hemiarthroplasty.
RESULTS AND CONCLUSION:For elderly patients with femoral neck or intertrochanteric fracture combined with various medical il ness, hemiarthroplasty is an effective manner presently. Fractures surrounding the prosthesis in elderly patients postoperatively gradual y increased. Once fracture appeared, it would bring a great attack on patients’ spirit, economy and even life. Therefore, early evaluation on the risk factors for fractures surrounding the prosthesis is a necessary measure for preventing and saving this disastrous consequence by selecting general correct prevention and treatment strategies. This wil greatly improve patients’ prognosis and elevated patient’s quality of life and survival rate. Present short-term smal-sample prospective fol ow-up studies suggested that comprehensive dynamic evaluation possibly has a certain clinical significance for the evaluation of risks of fractures after hemiarthroplasty in elderly patients, and deserves further investigations.

Two hour after LAK—like cells were co—cultured with gastric cancer cells,the mixed sus-pension was spread on slids and dyed with histochemical method.Through analysing the dyedcells,we directly confirmed what subset the LAK—like cells belonged to.The featrue of LAK—like cells adhering to cancer cell was observed under electron micrscope in our experiment.A-mong the lymphoctes which were adherent to cancer cells,the number of CD8~+ cells was in-creased.Most of adherent to cancer cells,the number of CD8~+ cells was increased.Most of ad-herent cells were TAC~+,suggesting that the expression of TAC antigen plays an important rolein cell mediated cytotoxity.

OBJECTIVE: To analyze the varieties, pharmaceutical utilization, adverse drug reaction(ADR) and majority problems in clinical use of poisonous traditional Chinese medicines in Beijing hospital, so as to provide references for their clinical rational use. METHODS: The varieties of Chinese patent medicines contain poisonous ingredients in Beijing hospital were surveyed, and their poisonous ingredients, drug categories, consumption sums, defined daily dose system(DDDs), adverse reactions and rational uses were analyzed. RESULTS: There were more than 300 Chinese patent medicines in Beijing hospital, in which 105 contained poisonous ingredients. Pharmaceutical utilization research indicated that most of the poisonous Chinese patent medicines with much higher consumption sums and DDDs were used for orthopedics and cardio-cerebrovascular diseases. In the statistics of ADR for 5 years in our hospital, 185 ADR reports of Chinese patent medicines were collected, among which poisonous Chinese patent medicines were involved in 49 ADR reports. CONCLUSION: The poisonous traditional Chinese medicines play important roles in clinical treatment, however, the adverse reaction rates of these medicines were much higher and may cause serious outcome. Therefore, their poisonous constituents and action mechanisms should be researched in depth constantly and the research should be linked with their clinical practices, so as to ensure the safe and rational use of poisonous traditional Chinese medicines.

Objective To evaluate a blood test,UniCAP system(screening IgE to food or inhalant allergens,eosinophil cationic protein,ECP)in differentiation the capability of lgE-mediated allergic diseases in children. Methods 26 atopic dermatitis,33 asthma/bronchiolitis,20 allergic purpura, 15 Kawasaki disease patients and 20 normal children were tested total lgE,lgE to food or inhalant allergens,ECP by UniCAP system and compared.Resuits Fx5E positive rate are 73.1%,24.2%,25.0%,13.3%in atopic dermatitis,asthma,allergic purpura,Kawasaki disease respectively,and the Phadiatop positive rate are 23.1%、69.7%、10.0%、6.6%.The serum levels of ECP、TIgE of patients were higher than the mormal controls,with the highest level in asthma, and lowest in Kawasaki disease,the differences were significant(P<0.05).Conclusions Food orinhalant allergens plays an important role in the inflammatory process of asthma,atopic dermatitis,and some role in allergic purpura,Kawasaki disease.Food allergens are more important to atopic dermatitis.and inhalant allergens are more important to asthma.

Ohjective To approach the therapeutic methods of urinary fistula after hypospadias urethroplas- ty.Methods Forteen post-hypospadia repair urethral fistula managed between 2000 to 2005 were reviewed.Differ- ent procedures were carried out according to the size and location of orificium fistulae.Results Eleven repairs were successful after one attempt.The success rate was 78% (11/14).Conclusion The procedure of fistula repair should be chosen according to the size,location and regional condition of orificium fistulae.

Objective To appraise two kinds of treatment plan of treating recurrent candidal vaginitis with fluconazole capsule. Methods The control group( also called the single medication) :husband and wife take flucon-azole capsule 150mg and add terbinafine hydrochloride vasinal effervescent tablets into the vagina,one time a day and one tablet a tlme,taking 7 days continually is a treatment course;the treatment group( also called taking drugs continu-onsly) :after a treatment course,at the first day of the menstrual period,husband and wife take fluconazole capsule 150mg at the same time, after the clean of menstruation, add terbinafine hydrochloride vaginal effervescent tablets into the vagina,one time a day and one tablet a time,taking 7 days continuously,three months in total. Appraise the two kinds of treatment plans from long-term,short-term curative effect and the recurrent rate. Resu/ts The curative effect of the two kinds of treatment plan about the treatment of recurrent candidal vaginitis,there is no difference between the short-term( one week) curative effect( P>0.05) ,but the long-term curative effect in the group of taking tablets con-tinuously is obviously better than the single medication( P<0.01 ) ,and the recurrent rate also obviously decreases( P<0.01 ). Conclusion The treatment pLan of taking the tablets continuously is the safey, effective treatment plan for the recurrent candidal vaginitis,no adverse reaction had been discovered.