Study On The Diagnostic Value And Clinical Application Of AlphaLISA In Infectious Diseases

Part Ⅰ Development and Derformance Evaluation of Alpha LISA for Detecting Influenza B VirusBackground The principle of amplified luminescent proximity homogeneous assaylinked immunosorbent assay(Alpha LISA)is to make the donor beads and the recipient beads close to each other by the intermolecular interaction,so as to generate amplified signals.This method has the advantages of high sensitivity and simple operation.Influenza B virus(IBV)can cause seasonal epidemic respiratory disease in the human population,resulting in a large burden on public health.However,the commonly used diagnostic methods for IBV currently have issues such as low sensitivity or timeconsuming and laborious performance.Therefore,there is an urgent need for new sensitive and rapid detection methods for the screening and diagnosis of IBV.Objective To develop an Alpha LISA rapid detection method for IBV,and evaluate the diagnostic performance of this method.Methods(1)A total of 228 throat swab samples were collected from The Fifth Medical Center of Chinese PLA General Hospital from February to July 2019.(2)The Alpha LISA rapid detection method for IBV was developed by screening the optimal proportion of IBV labeled acceptor beads,streptavidin-conjugated donor beads and biotinylated IBV antibody,as well as the incubation time and temperature.(3)Alpha LISA was evaluated by inactivated IBV,IBV antigen and throat swab samples,and compared with chemiluminescent enzyme immunoassay(CLEIA)and lateral flow colloidal-gold immunoassay(LFIA)Results(1)The detection effect of Alpha LISA were the best when IBV labeled acceptor beads were 50 μg/m L,biotinylated IBV antibody was 0.5 μg/m L,and the streptavidinconjugated donor beads were 40 μg/ m L.(2)Alpha LISA,used for detecting IBV,did not cross-react with other respiratory viruses.The inter assay coefficient of variation(CV)and intra-assay CV were both <5%.(3)The results of 228 clinical throat swab samples showed good agreement between Alpha LISA and LFIA(Kappa=0.982)and good agreement between CLEIA and LFIA(Kappa=0.813).And Alpha LISA showed the highest sensitivity in detecting inactivated IBV.Conclusions Alpha LISA had good sensitivity,speciffcity and repeatability in the detection of IBV and had the advantages of easy operation and rapid reaction which can be applied to the screening of IBV.Part Ⅱ Application of Alpha LISA Detection of Serum Human Neutrophil Lipocalin in Patients with SepsisBackground As an emerging biomarker,human neutrophil lipocalin(HNL)has been proved to have high sensitivity and specificity in the diagnosis of bacterial infection.At present,Currently,ELISA is the main method used to detect HNL,which has the disadvantage of time-consuming and laborious.Sepsis as a common cause of death in ICU patients is insidious and associated with high mortality.However,the role of commonly used biomarkers in diagnosing septic shock and predicting the prognosis of patients with sepsis is limited,so there is an urgent need for new biomarkers to assist clinical diagnosis.Objective To develop an Alpha LISA method for detecting the concentration of HNL,and to explore the role of HNL in the diagnosis of septic shock and the prognosis of patients with sepsis.Methods(1)A total of 146 serum samples were collected from the Fifth Medical Center of the Chinese PLA General Hospital from September 2021 to April 2022.(2)The Alpha LISA method for HNL was developed by screening the optimal set of monoclonal antibodies of HNL and serum dilution ratio.(3)The HNL test results of Alpha LISA were compared with the commercial ELISA kits.(4)78 patients with sepsis were studied.The receiver operating characteristic(ROC)curve was used to evaluate the performance of HNL in the diagnosis of septic shock and the prognosis of patients with sepsis.Results(1)The optimal set of monoclonal antibody and dilution ratio of serum was1F2-1G2 and 1:20,respectively.(2)The Alpha LISA detection range of serum HNL was1.5 ng/m L ~ 1000 ng/m L,with a limit of detection of 1 ng/m L and a detection time of 25 minutes.(3)There was high concordance between Alpha LISA and ELISA results(R2=0.9413).(4)The level of HNL in septic shock group was significantly higher than that the sepsis group(median 356.47 ng/m L vs 158.93 ng/m L,P <0.0001),and that in28-d non-survival group was significantly higher than that in 28-d survival group(median331.83 ng/m L vs 175.17 ng/m L,P<0.0001).(5)ROC curve analysis showed that the AUC of HNL was 0.857 and 0.784 in the diagnosis of septic shock and prognosis of patients with sepsis,respectively.(6)HNL was highly correlated with SOFA score and APACHE II score.Conclusions(1)Alpha LISA had good consistency with commercial ELISA,and had shorter operation time and wider detection range compared with ELISA.(2)Serum HNL had potential value in diagnosing septic shock patients and predicting the prognosis of sepsis patients.