| With the rapid development of science and technology,advanced technologies play a more and more important roles in the medical field.There are two main developmental trends for laboratory medicine.One is that various large-scale,high-efficiency automation platforms are continuously being developed,greatly improve the efficiency of clinical laboratories with modular combinations and streamlined "automaton".The other is that miniature laboratory equipments with simple process and easy operation become increasingly popular among clinical staffs,patients and their families,besides accurate reports of result could be provided timely.Point of Care Testing(POCT)technology was first proposed in the AST2-P guidelines in March,1995(Later in AST2-A,1999)by the National Committee for Clinical Laboratory Standards(NCCLS,CLSI since 2005)has become another booming direction of Laboratory medicine.In the modem world,a fast pace has become the way of life and in the mainstream workplace.POCT is well-adapted to the time requirements of people and the needs of modern medicine,especially regarding the time needs for treating the critically ill,such that patients are diagnosed and treated as early as possible.The advancement of medical care raises the quality of life of the worldwide population and enhances the concept of health.People are obviously concerned about the state of their health and disease progression,and wish to get their health care information anytime and anywhere.POCT has become an important direction in the development of modem laboratory medicine,providing portable,convenient operation and timely and reliable resultsto improve people's understanding of their health while satisfying the needs of healthcare professionals regarding quick and accurate patient diagnosis and treatment.Immunochromatography,dry chemical technique,biosensors,biochips and infrared and far-infrared spectrophotometry techniques each have advantages and continue to expand in POCT development,resulting in first-generation qualitative detection products(paper strips),second generation semi-quantitative products(read colorimetric by color card or semi-quantitative instruments),the third generation fully quantitative sy stem products(manual)and fourth-generation technology platforms(automation,Informatization and Intellectualization).The series of product groups available make the use of POCT more convenient,with a wider range of applications and detection capabilities,from blood glucose and pregnancy,for example,extending to complex testing such as monitoring coagulation status,myocardial damage,acid-base balance,infectious diseases and Therapeutic Drug Monitoring(TMD).With portable equipment,diagnostics can be performed anywhere,from the home or scene of an accident extending to the emergency room,intensive care unit,operating room and even into,community health centers,private clinics and Customs offices.The types of applications have also extended from the clinical into the food,environmental testing,anti-narcotics and forensic fields.In these types of on-site,high-speed applications,immunochromatographic techniques have a distinct advantage because they are homogenous and do not require additional markers for detection.Immunochromatographic assay(ICA)is a simple,rapid detection and analysis technique developed in the early 1980s which combines highly-sensitive labeled tracer technologies,highly-specific antigen-antibody reactions and fast and easy chromatographic techniques.Homogenous immunochromatographie assays provide for simple performance and can achieve results in a relatively short time(<30min inside).Immunochromatographic assays may be divided into two categories,according to the principles by which a detection signal is acquired.One class uses colored markers such as latex particles,colloidal gold and liposomes.Color is rendered because the marker and analyte complex are enriched within a specific detection range.Qualitative results may be determined by the presence or absence of color.Semi-quantitative analysis may be performed by the depth or severity of color.The second class of immunochromatographic assays uses markers such as fluorescein,radioisotopes or rare-earth elements,and quantitative results are determined by signal intensity.Since September 2010,severe fever with thrombocytopenia syndrome virus(SFTSV),a bunya virus,has resulted in the deaths of 36 patients in the Hubei,Henan,Shandong,Jiangsu,Anhui and Liaoning provinces of China.Patients are mainly distributed in the mountainous and hilly areas in rural areas and infection is highly sporadic.The population at highest risk for infection are those residents and workers in the hills,mountains and forests,as well astourists going to such area for outdoor activities.Disease mortality has decresed from the initial 30%,but still remains at a high le-vel of about 10%.In response,the Ministry of Health formulated the "Severe Fever with Thrombocytopenia Syndrome Prevention Guide"(2010 edition),and identified laboratory diagnostic methods for the detection of SFTSV infection.Determinations are done mainly by real-time quantitative PCR detection of viral nucleic acid,virus isolation or detection using ELISA,IFAT,and neutralization tests to detect specific IgG antibodies.These tests provide a very effective means for timely SFTSV diagnosis and treatment,but these methods require professional and technical personnel and the disease often occurs in poorly equipped medical conditions in the mountains where the availability of the necessary laboratory equipment is limited.In addition to the cumbersome processes involved with these types of detection methodologies,these technologies require a relatively long period of time and are thus unable to meet the needs of early diagnosis.As such,the development of rapid detection reagents for SFTSV is an urgent concern.Based on the infectious characteristics of SFTSV,the demand in remote or poorly equipped locations is to be able to detect infection qualitatively,without complex equipment in a simple to use,portable and fast manner.Gold immunochromatography assays meet the needs of this market.The development of this reagent obtained great support from one of the projects of the National High-Tech&Science(863)" Development of the immuno-diagnosis reagents for major infectious diseases".With the increase of invasion of diagnostic procedures,bacterial resistance,the incidence of severe bum trauma,organ transplant patients and radiation and chemotherapy patients,the hospital infections,septicemia,sepsis,septic shock and multiple organ dysfunction syndrome(MODS)are rising.Septic shock is a common illness in ICU patients with a high mortality rate.There is a high risk for patients in ICU wards who have recently undergone surgery,due to infections caused by septicemia and sepsis,and for new born babies.Septicemia and acute organ dysfunction(severe septicemia)are the leading causes of death in non-coronary ICU.Sepsis is one of the most common serious diseases in the ICU,having a high rate of disability and mortality,and is a common cause of systemic inflammatory response syndrome and MODS.Septic shock is common in critically ill patients and is the leading cause of death in ICU ward patients.The key to effective treatment is a fast and accurate diagnosis,and,thus,early detection is important for this infection complication.Procalcitonin(PCT)is a non-hormonal precursor of the propeptide calcitonin(CT).Detectable levels of PCT rise in bacterial infections,but do not increase in most other inflammatory reactions,such as viral infections,autoimmune disease and trauma,making PCT a good marker for sepsis with good specificity and sensitivity.In severe systemic bacterial,fungal and parasitic infections,PCT levels are abnormally elevated and the degree of elevationis associated to infection severity and inversely proportional to prognosis.Additionally,PCT reflects the effectiveness of antibiotics and may be used to guide antibiotic treatment,reduce the use of antibiotics,reduce the cost of treatment and avoid bacterial drug resistance.Currently,there are a variety of PCT detection kits on the market,such as PCT detection kits based on chemiluminescence and PCT semi-quantitative detection kit based on colloidal gold.The former can accurately quantify,but requires large testing equipment which limits the availability of the PCT test in under-equipped locales and therefore cannot be used as a first-line method to monitor the PCT indicators.The latter kit can be quickly and easily used on site,but the existing deficiency prevents effective quantitation.Therefore,there is a pressing need for the development of a detection reagent which is fast and simple but provides accurate quantitation.Time-resolved fluorescence chromatographic immunoassays can avoid the mutual interference caused by the partial overlap of excitation and emission spectra by virtue of large Stokes shift,it allows a high signal-to-noise ratio based on a long decay time of the fluorescence and has the advantages of chromatographic techniques.As such,these techniques have a higher value in the development of PCT rapid quantitative detection reagent for clinical applications.Methods:?.Development of a detection reagent for IgM/IgG antibody to SFTSV by gold immunochromatography assay1.The optimum pH and amount of labeled SFTSV recombinant antigen and the optimum pH and amount of labeled streptavidin.It was in the context of an excess of the labeled antibody to ensure the aggregation of colloidal gold was not because of the amount of antibody,the aggregation of colloidal gold was only because of the change of pH,in this context,the optimum pH of SFTSV recombinant antigen and streptavidin were determined.In the optimum pH,the optimum amount of labeled SFTSV recombinant antigen and streptavidin was the minimum amount of antibody which can maintain the colloidal gold stable plus 10%.2.The screening of coating antibody:Different antibodies were coated under the same conditions for coating antibody screening.The high rate of negative and positive was selected as the selecting standard.·3.Determination of the dilution of gold-labeled streptavidin and the amount of coated biotin-BSA for control line:Determination of the dilution of gold-labeled streptavidin and the amount of coated biotin-BSA for control line using chessboard titration method.The conditions with best and the most clear of color was selected as the optimum dilution of gold-labeled streptavidin and the optimum amount of coated biotin-BSA for control line.4.Determination of the dilution of gold-labeled SFTSV recombinant antigen and the amount of coated test line M:Determination of the dilution of gold-labeled SFTSV recombinant antigen and the amount of coated test line M using chessboard titration method.The conditions with the high rate of negative and positive was selected as the optimum dilution of gold-labeled SFTSV recombinant antigen and the optimum amount of coated test line M.5.Determination of the optimum amount of coated nAb A37420 for test line G:According to the established conditions,the A37420coated on the G line was diluted to 3mg/ml,2mg/ml,lmg/ml with coating buffer of 10mM pH7.8 PBS.The conditions with the high rate of negative and positive was selected as the optimum amount of coated mAb A37420 for test line G.6.Selection of the reaction buffer:Buffer helps chromatography,maintains the reaction environment stability and shields non-specific binding.We formulated different reaction buffer,10 copies of the negative samples and the positive samples were detected simultaneous.The buffer with the high rate of negative and positive was selected as the final reaction buffer.7.Determination of the volume of the sample:To decide the optimal sample volume through observation of the quality of chromatography phenomenon using different sample volumes.8.Determination of the optimal dilution of the sample:To select the optimal dilution by a gradient experiment(1:100,1:200,1:400).9.Performance evaluation of the reagent:9.1 Evaluation of the minimum detection limit:To evaluate the minimum detection limit using a series of samples which serial 2-fold dilution from the IgM antibody positive serum sample to SFTSV and the IgG antibody positive serum sample to SFTSV.The minimum detectable value of positive samplewas selected as the minimum detection limit.9.2 Evaluation of the repeatability:Five portions reagents were extracted from the same batch of reagents.The reagents were measured by the precision reference.To assess the intra-assay repeatability by whether the sane of reaction results and uniform of the purple stripe.Five portions reagents were extracted from three consecutive batches of reagents.The reagents were measured by the precision reference materials.To assess the inter-assay reproducibility by whether the same of reaction results and uniform of the purple stripe.9.3 Evaluation of the specificity:Influenza virus antibody-positive serum,hantavirus infectionpositive serum,hepatitis B virus surface antigen positive serum,hepatitis C virus antibody-positive serum,HIV-positive serum,antinuclear antibodies and rheumatoid factor positive serum with the negative IgM/IgG to SFTSV were collected,these samples were tested by the reagent.To evaluation of the cross-reactivity of the reagent by whether produce false positive results.9.4 Evaluation of the interference:SFTSV-IgM and SFTSV?IgG weak positive serum adding cholesterol,bilirubin and triglycerides respectively were measured to determine the interference of cholesterol,bilirubin and triglycerides to the reagent.Cholesterol,bilirubin and triglycerides were gradually increased until impact on the test result or testing process.9.5 Evaluation of the stability:The maximum time of the reagent saved at 37?whileit was tested without afifecting their performance.10.Evaluation of the clinical trials:Clinical trial was designedusing blind and control method.The "gold standard" method of SFTSV laboratory validation method was used as the comparison method:"SFTSV nucleic acids was detected from patient specimens,using the real-time quantitative PCR nucleic acid detection reagent for SFTS approved by CFDA and/or "The convalescent serum specific IgG antibody titers was four times higher than the acute phase" by IFAT test were as the ultimate basis for the determination of the sample infected with SFTSV and presence of IgM antibody to SFTSV in the sample;The determination of whether presence of IgG antibody in the sample by IFAT was as the comparisonto detect IgG antibody.The data was analysed by SPSS 13.0 ofstatistical software.?.Development of a detection reagent for procalcitonin(PCT)by Time-resolved Immunochromatography1.Collection and concentration measurement of Eu-labeled antibody:The concentration of each Eu-labeled antibody was measured by BCA.2.The choice of nitrocellulose membrane:The nitrocellulose membrane of HF 125 and HF 150(Millipore)and 70CNPH-N-SS40(MDI)were as alternative membranes.Their performance was studied through a series of experiments.The final nitrocellulose membrane was determined considering the fluorescence of the positive controls,the background in the negative controls and the uniformity of the test line and the control line.3.Screening of the raw materials by time-resolved immunofluorescence:The combination of the purchase draw materials was screened by TRPIA.The best combination was determined considering the correlation coefficient(r)of the dose-response curve and supplemented by the criteria of TRFIA.4.Determination of the amount of coated test line:The coated antibody was diluted to the same concentration respective with the coating buffer.The coated antibody was coated on NC membrane in differing amounts.It was assembled with conjugate pad with Time-resolved fluorescence microspheres labeled antibody after drying and other accessories,and these were cut into strips.The amount of coated test line was determined by the next performance testing.5.The choice of the concentration of labeled antibody:The microsphere-labeled antibody was diluted to 0.01mg/ml,0.1mg/ml and lmg/ml with dilution buffer.The amount of labeled antibody was 0.1?l/mm.Those were assembled with nitrocellulose membranes coated with 16B5(4.2mg/ml and the amount of 0.07?l/mm)and cut into strips.The best concentration of labeled antibody was selected considering the background and fluorescence signals by testing the standard.6.Performance evaluation of the reagent:6.1 Drawing of the dose-response curves:The logarithmic of the concentration of self-reference standard was as the independent variable(X),the logarithmic of the signal value was as the dependent variable(Y),the dose-response curves was drawn with the double logarithmic Log-Log of mathematical model.6.2 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated.The signal value,which was obtained by the mean signal value of zero calibrations plus double standard deviation,was substituted into the standard curve equation to acquire the corresponding concentration.This concentration was defined as assay sensitivity.6.3 Accuracy:A high concentration of 62.8ng/ml of PCT sample was measured by Procalcitonin assay kit(ECLIA)produced by German Roche Diagnstics Gmbu company.Serial double dilutions of the high concentration of PCT by Negative Serum of PCT were tested by the homemade kit.The ratio of real concentration to its theoretical value was calibrated.6.4 Precision:The three control samples of low,medium,high concentration(control samples ?,?,?)were measured.The experimental of intra-assay precision was set up with five strips from the same batch,the experimental of inter-assay precision was set up with the same batch of reagents which was operated three different people independently.Each mean concentration,standard deviation and the coefficients of variation were calculated.6.5 Specific:CRP,IL-6,human calcitonin and human anti-calcium were diluent to 50ng/ml,1IU/ml,60ng/ml and 30ng/ml respectively.The results should be measured less than 0.08ng/ml with ancillary equipment.Result:1.Development of a detection reagent for IgM/IgG antibody to SFTSV by gold immunochromatography assay1.The optimum pH and amount of labeled SFTSV recombinant antigen and the optimum pH and amount of labeled streptavidin:In the context of an excess of the labeled antibody to ensure aggregation of colloidal gold was not because of the amount of antibody,the aggregation of colloidal gold was only because of the change of pH.In this context,the optimum pH of labeled SFTSV recombinant antigen was added 20?l 0.1 M KZCO3 into lml gold,the optimum pH of labeled streptavidin was added 5?l 0.1 M K2CO3 into 1ml gold.Under the optimum pH,the optimum amount of labeled SFTSV recombinant antigen and streptavidin both were added 23.1?g which was the minimum amount of antibody to maintain a stable colloidal gold plus 10%into 1ml gold.2.The screening results of coating antibody:The A37420 and A37130 from Biospacific company and streptavidin from Sigma and biotin-BSA from You Di Bio Technology Co.Ltd.of Guangzhou with the highest compliance rate of negative and positive were selected as the raw materials for this reagent.3.Determination of the dilution of gold-labeled streptavidin and the amount of coated biotin-BSA for control line:According to the results,when the dilution of gold-labeled streptavidin was ODs35?10,the concentration of control line was 0.05mg/ml and the amount of line was 0.1?l/mm,the color effect was good and clear.Therefore,the optimum dilution of gold-labeled streptavidin was OD535?10,the optimum concentration of control line was 0.05mg/ml and the optimum amount of line was 0.1?l/mm.4.Determination of the dilution of gold-labeled SFTSV recombinant antigen and the amount of coated test line M:According to the results,when the dilution of gold-labeled SFTSV recombinant antigen was OD535?45,the amount of gold-labeled SFTSV recombinant antigen was 0.8?l/mm,the concentration of test line M was 2mg/ml and the amount of test line M was 0.1 ?l/mm,the compliance rate of negative and positive were the highest.So the optimal dilution of SFTSV recombinant antigen was OD535?45,the amount of gold-labeled SFTSV recombinant antigen was 0.8?l/mm,the optimum concentration of test line M was 2mg/ml and the optimum amount of test line M was 0.1?l/mm.5.Determination of the optimum amount of coated mAb A37420 for test line G:According to the results,when the concentration of test line G was 2mg/ml and the amount of test line G was 0.1?l/mmn the compliance rate of negative and positive were the highest,so the optimum concentration of test line G was 2mg/m land the optimum amount of test line G was 0.1?l/mm.6.Selection of the reaction buffer:Experiments showed that Buffer4 had the highest compliance rate of negative and positive,so the optimal reaction buffer was Buffer 4.7.Determination of the volume of the sample:Experiments showed that 60 ul sample volume had the best chromatographic process,therefore,the optimum volume was 60 ? 1.8.Determination of the optimal dilution of the sample:Experiments showed that the sample with dilution of 1:100 had the best results,therefore,the optimum dilution of the sample was 1:100 ?l.9.Performance evaluation of the reagent:9.1 Evaluation of the minimum detection limit:The minimum detection limit of the reagent was the lowest detection value of positive result.The test results showed that the minimum detection limit for IgM antibody to SFTSV was the dilution not higher than 1:128 and the minimum detection limit for IgG antibody to SFTSV was the dilution not higher than 1:512.9.2 Evaluation of the repeatability:The results showed that intra and inter-assay reaction results were consistent with a uniform purple bands.The intra and inter-assay reproducibility were good.9.3 Evaluation of the specificity:According to the test results,the reagent were not cross-react with Influenza virms antibody-positive serum,hantavirus infection positive serum,hepatitis B virus surface antigen positive serum,hepatitis C virus antibody-positive serum,HIV-positive serum,antinuclear antibodies and rheumatoid factor positive serum.The specificity was good.9.4 Evaluation of the interference:According to the test,when the blood cholesterol level was less than 230g/L,it had no effect on the test result;when the bilirubin concentration was less than 1700mg/L,it had no effect on the test result;the concentration of triglycerides was less than 290g/L,it had no effect on the test result.9.5 Evaluation of the stability:The thermal stability assessment found that it had not effect on the performance of the reagents when the reagent was placed at 37?for 22 days.The stability was good.10.Evaluation of the clinical trials:A total of 1082 cases of samples,including 245 cases of clinical diagnosis of SFTS samples,112 cases of influenza samples,58 cases of HFRS sample,38 cases of the common cold population sample,589 cases of the general population,10 cases of HIV,HBV,HCV and RF sample respectively.Compared with the test results of comparison method,1062 cases were consistent with the comparison method,20 cases were inconsistent with the comparison method.After statistical analysis,the positive coincidence rate of the reagent was 96.7%,the negative coincidence rate was 98.6%,the total coincidence rate was 98.2%,the total Youden index was 0.953,the Kappa value was 0.948(P<0.001)compared with the test results of comparison method.Among them,the positive coincidence rate of IgM was 98.4%,the negative coincidence rate was 100%,the total coincidence rate was 99.6%,the Youden index was 0.984,the Kappa value was 0.989(P<0.001);the positive coincidence rate of IgG was 97.7%,the negative coincidence rate was 98.7%,the total coincidence rate was 98.5%,the Youden index was 0.964,the Kappa value was 0.945(P<0.001).The result of clinical trials indicated that the pending evaluation reagent was reliable,accurate,safe,simple and stable.It had a high clinical value.?.Development of a detection reagent for procalcitonin(PCT)by Time-resolved Immunochromatography1.Collection and concentration measurement of Eu-labeled antibody:Each Eu-labeled antibody were gathered about 2.5ml of eluent after purification,the concentration of Eu-labeled antibody were 90-110?g/ml by BC A assay.2.The choice of nitrocellulose membrane:MDI's 70CNPH-N-SS40 was the final choice considering the fluorescence of the positive,the background of the negative and the uniformity of the test line and the control line.3.Screening of the raw materials by Time-resolved immunofluorescence:MJG03 was the final choice for marking and 16B5 was the final choice for coating considering the correlation coefficient(r)of the dose-response curve and supplemented by the criteria of TRFIA.4.Determination of the amount of coated test line:The final choice of the amount ofcoated test line was 0.08?l/mm considering the fluorescence of the positive,the background of the negative and the uniformity of the test line and the control line.5.The choice of the concentration of labeled antibody:The final choice of the concentration of labeled antibody was 0.1mg/ml considering the background and fluorescence effect.6.Performance evaluation of the reagent:6.1 Drawing of the dose-response curves:The linear regression equation was Y ?0.872X + 3.088treated with the double logarithmic Log-Log of mathematical model.The linear correlation coefficient of the dose-response curve was r = 0.9994.6.2 Sensitivity:The zero calibration was detected for 20 times repeatedly and the mean signal value and the standard deviation were calculated.The corresponding concentration was acquired by the mean signal value of zero calibrations plus double standard deviation then substituted into the standard curve equation.The minimum detection limit of the regent was 0.08ng/ml?namely the sensitivity was 0.08ng/ml.6.3 Accuracy:A high concentration of 62.8ng/ml of PCT sample was measured by Procalcitonin assay kit(ECLIA)produced by German Roche Diagnstics Gmbu company.Serial double dilutions of the high concentration of PCT by Negative Serum of PCT were tested by the homemade kit.The ratio of real concentration to its theoretical value was calibrated.The recovery rate was calculated with three parallel experiments.The ratio of real concentration to its theoretical value was between 0.90-1.13,the recovery rate was between 90%-113%.6.4 Precision:The three control samples of low,medium,high concentration(control samples ?,?,?)were measured.The experimental of intra-assay precision was set up with five strips from the same batch,the experimental of inter-assay precision was set up with the same batch of reagents which was operated three different people independently.Each mean concentration,standard deviation and the coefficients of variation were calculated.The results of intra-assay coefficient of variation was between5.4%-7.7%,inter-assay coefficient of variation was between 5.7%-13.4%.6.5 Specific:CRP,IL-6,human calcitonin,human anti-calcium were diluent to 50ng/ml,1IU/ml,60ng/ml and 30ng/ml respectively,the results should be less than 0.08ng/ml measured with ancillary equipment.The results showed that the kit did not cross-reaction with CRP,IL-6,human calcitonin and human anti-calcium.Conclusion:The results of this study indicated that various indicators(accuracy,sensitivity,precision,specificity,etc.)of the development of a detection reagent for IgM/IgG antibody to SFTSV by gold iumunochromatography assay and the development of a detection reagent for procalcitonin(PCT)by Time-resolved Immunochromatography were satisfied the registration requirements of in vitro diagnostic reagent.The reagents were expected to be applied to production after further optimization. |
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