Rapid And Quantitative Detection Of Procalcitonin (PCT) Reagents Based On Time-Resolved Immunochromatography

Background:Infectious diseases,the most common disease in daily clinical practice,characterize with a gradually higher incidence and septic shock in serious infection patients.Different subtypes of infectious diseases should be dealed with each case on its merits,such as bacterial infections needed treatment with antibiotics in time,but there has no effect on antibiotic treatment for non-bacterial infections.As a disease with high mortality rate,Sepsis is the main cause of death in non-cardiac ICU patients.There are about 3 million cases per year,with the mortality of 40%-50%in China.The deficiency of effective antibiotic treatment leads to increasing in death risk.The death rate will increase 7.6%with an hour's delay.Therefore,the importance of early diagnosis and appropriated therapeutic schedule for these infectious diseases is obvious.As a useful serological marker for sepsis and inflammation,Procalcitonin(PCT)detecting contributes to the differential diagnosis,antibiotic-usage guiding and prognostic evaluation for clinical infectious diseases.So far,the main methods for PCT test include quantitative methods(Chemiluminescence method,enzyme-linked immunofluorescence assay)and semi-quantitative method(colloidal gold method)are still not sufficient for clinical use.Chemiluminescence and enzyme-linked immunosorbent assay can provide accurate quantification,but the requirement of the special large-scale instruments brings the shortcomings such astexpensive and inconvenient.The enzyme-linked immunofluorescence assay is confined to scope of use mainly because of its instability.The Colloidal gold method has the advantage of quick and convenient,but no accuracy.Since the clinical value of dynamic monitoring and early detection of PCT,to establish a rapid?easy-operated and accurate method for PCT test is urgently needed.Objective:Developing a real time detection kit(also known as Point of care testing)facilitates rapid quantify and on-site monitoring of PCT.To develop the new PCT test kit,a double-antibody sandwich immunofluorescent assay was employed based on time-resolved immunofluorescent assay(TRFIA)and lateral flow immunoassay(LFIA).Methods:1.Two pairs of PCT-specific and packaged antibodies were assembled.To acquire the best labeling and packaged antibodies by time-resolved immunofluorescence assay(TRFIA).2.Double antibody sandwich technology is used to establish a method for rapid and quantitative detection of procalcitonin(PCT)reagent by time-resolved fluorescence immunochromatography.The optimization of each reagent's parameters are as follows:nitrocellulose membrane(NC membrane),detection line(T)and quality control line(C)for coated antibody dose,the optimal crossed dose and labeled antibody's dose,the best reaction time.3.To evaluate the indicators of reagent performance,the following experiments were made:Draw standard curve,analysis of sensitivity,accuracy,precision and specificity,clinical assessment experiment.Results:1.?The optimal labeling antibody(MJ03)and coating antibody(16B5)have been screened out according to the results of the synthesis dose-response curve correlation coefficient(R),Signal-to-noise ratio,background and so on,by TRFIA.2.A reacting model based on time-resolved technology and immunoassay technology has been established.HF135 is choosed as the best nitrocellulose membrane.;The optimal coating dose of 16B5 for detection line(T),goat anti-rabbit IgG for quality control line(C)and labeled antibody MJG03 are 2.0mg/mL,1.0mg/mL and 0.1 mg/mL respectively.The optimal sprayed speed onto the line was 0.08?L/mm.The best dosage of rabbit IgG is 0.1 mg/mL.The best reaction time is 15min.3.The evaluation results of reagent performance are as follow:Standard curve regression equation:log(y)=1.1143+0.8529 log(x),r = 0.9994;Analysis sensitivity was 0.08 ng/mL;Accuracy(recovery)arranged from 93%to 105%;The inter-assay and intra-assay analysis precision(CV)were between 5.4%?13.4%;No interference were detected with human calcitonin,CRP,human anti-calcitonin and IL-6;The results of 234 clinical samples showed that there was a good agreement(Kappa = 0.875)and correlation(R = 0.977,P<0.01)between self-made and commercially available Roche Elecsys BRAHMS PCT Kit.Conclusion:A rapid quantitative detection kit of PCT based on time-resolved fluorescence immunochromatography has been developed.The various parameters and performance indicators of reagent are satisfying,all of them met the requirements of diagnostic reagents in vitro.Meanwhile,the question of clinical insufficiency for rapid quantitative PCT test has been resolved.