Establishment And Preliminary Application Of Rapid Quantitative Detection Method For Bacterial Infection Markers

BackgroundSepsis is defined as life-threatening organ dysfunction resulting from dysregulated host responses to infection.Every year,more than 19 million people suffer from sepsis globally,among which 6 million die with a mortality rate of more than 1/4.Among the surviving patients,about 3 million have cognitive dysfunction.The reason for such a high mortality rate is that sepsis is misdiagnosed or missed diagnosed.Therefore,timely and accurate detection of bacterial infection markers can assist diagnosis and treatment of sepsis and reduce the mortality rate.Procalcitonin（PCT）and interleukin 6（IL-6）have been widely recognized as markers of bacterial infection.Both of them are positively correlated with the severity of bacterial infection.PCT is more specific in the diagnosis of bacterial and non-bacterial infections,and dynamic monitoring of IL-6 is useful for judging the treatment and the prognosis.The concentrations of PCT and IL-6 in healthy people were less than 0.05 ng/mL and about 6 pg/mL,respectively.And in the case of severe bacterial infection,they can reach up to 100 ng/mL and 1000 pg/mL,respectively.The current detection methods are mainly enzyme-linked immunosorbent assay（ELISA）and chemiluminescent immunoassay（CLIA）.ELISA is complicated,time-consuming,multi-step,expensive and batch operation.The intermediate process is affected by many factors.CLIA operations are relatively complex,time-consuming,require large and specialized equipment,and slightly more expensive.At present,the government and the health care commission are vigorously promoting graded diagnosis and treatment,and developing community out-patient clinics and other primary health care institutions.However,primary medical institutions often rely on experience in diagnosis and treatment of infectious diseases,which can easily lead to misdiagnosis,missed diagnosis and overuse of antibiotics.Therefore,appropriate PCT and IL-6 detection methods are needed,and ELISA and CLIA are not applicable to such primary medical institutions.Therefore,there is a requirement for a rapid,simple,easy-to-use and beneficial to the promotion of primary medical institutions to detect PCT and IL-6,which is conducive to the timely and effective clinical diagnosis and treatment for sepsis.Immunochromatography assay（ICA）is also known as lateral immunoassay（LFIA）,is a combination of thin layer chromatography and immune recognition reaction of solid phase immunoassay,which provides a good platform for point of care testing（POCT）.It has the advantages of rapidity,simplicity,easy operation,wide use in the detection of materials such as metal ions,nucleic acid,protein and bacterial.The core of LFIA is the label.The labels applied to LFIA include colloidal gold,carbon nanomaterials,up-converting phosphor nanoparticles（UCP）,quantum dots（QDs）,europium nanoparticles（Eu-np）and magnetic nanomaterials,of which the Eu-np have unique optical properties:long fluorescence lifetimes,outstanding photostabilities,large stokes shift（over 150nm）,no cross between excitation spectrum and emission spectrum,sharp characteristic peak,small volume of the marker,and the marker will not affect the spatial structure of the marker（especially the protein）,which ensures the stability of the detected substance.Due to the unique optical properties of Eu-np,non-specific fluorescent signals can be greatly reduced to obtain high sensitivity.PCT and IL-6 can be quantitatively detected by the test/control（T/C）ratio.ObjectvesIn this study,based on the principle of time-resolved technology and immunochromatographic technology,a time-resolved immunochromatographic test strip,which can be used for rapid,simple and quantitative detection of PCT and IL-6,was developed with the test strip as the carrier and the portable immunofluorescent analyzer as the detection platform.It lays the foundation for the rapid and accurate diagnosis of sepsis for primary medical institutions.Methods1.Development of time-resolved immunochromatographic test strips for PCT.After Eu-np were activated,they were labeled with chicken IgY and PCT capture antibody MJG03 to obtain Eu-np-CIgY and Eu-np-MJG03 complexes.The complexes were diluted into respective proportions to spray onto a conjugate pad and dried at 37℃.The nitrocellulose（NC）membrane was coated with two T-lines with a diluted PCT detection antibody MJG05 and a C-line with goat anti-chicken IgY（anti CIgY）,and then dried at 37℃.After the assembling was completed,the serum sample was diluted twice with the sample dilution buffer and added to the sample well.After 15 minutes of reaction,it was detected by a portable fluorescence strip reader to obtain the（T₁+T₂）/C ratio.According to the standard curve set in the strip reader,we could obtain PCT concentration directly.2.Optimization and preliminary application of time-resolved immunochromatographic strip for PCT.The PCT strip was optimized from three aspects,including T-line concentration,conjugated pad sprayed concentration and detection time.Its performance evaluation was evaluated from six aspects,including linearity,sensitivity,precision,specificity,recovery rate and comparison of clinical specimens.3.Development of time-resolved immunochromatographic test strips for IL-6.After Eu-np were activated,they were labeled with chicken IgY and IL-6 capture antibody MIS02 to obtain Eu-np-CIgY and Eu-np-MIS02 complexes.The complexes were diluted into respective proportions to spray onto a conjugate pad and dried at 37℃.The NC membrane was coated with a T-line with a diluted IL-6 detection antibody MIS01 and a C-line with goat anti CIgY,and then dried at 37℃.After the assembling was completed,the serum sample was added to the sample well directly.After 15 minutes of reaction,it was detected by a portable fluorescent strip reader to obtain the T/C ratio.According to the standard curve set in the strip reader,we could obtain IL-6 concentration directly.4.Optimization and preliminary application of IL-6 time-resolved immunochro matographic strip.The IL-6 strip was optimized with the detection time.Its performance evaluation was evaluated from the aspects of parallel test,linearity,sensitivity,recovery,precision,specificity and comparison of clinical specimens.Results1.The detection method for PCT has good linearity with the linear range 0.05-100 ng/mL and a sensitivity of 0.03 ng/mL.The specificity is good.The intra-assay coefficient of variation（CV）and the inter-assay CV are 1.39%-5.42%and 2.00%-6.86,respectively.The recovery rate is between 95%and 101%,which is in good consistent with the AE-180 automatic chemiluminescence immunoassay:y=0.9009x+0.4252（n=95,r=0.9936,p<0.01）.The positive coincidence rate was 96.61%,the negative coincidence rate was 94.44%,the false positive rate was 5.56%,the false negative rate was 3.39%,and the diagnostic coincidence rate was 95.53%.2.The detection method for IL-6 has good linearity with a linear range of 2-500 pg/mL and a sensitivity of 0.37 pg/mL.The specificity is good.The intra-assay CV and the inter-assay CV are 5.92%-8.87%and 7.59%-9.04%,respectively.The recovery rate is between 102%and106.This method has a good consistency with SEIMENS automatic chemiluminescence immunoassay:y=0.9502 x+6.1397（n=214,r=0.9757,p<0.01）.Conclusions1.We successfully activated Eu-np when we use EDC alone without NHS,which can reduce operational steps and save reagent.2.The PCT test and IL-6 test are rapid,and the test results can be obtained only after 15minutes of adding samples,which is better than the CLIA of about 1 h and the ELISA of about5 h.3.PCT test only requires the diluted solution of 50μL of serum and 50μL of sample dilution buffer to be mixed and added to the sample pad.IL-6 test requires only 70μL of serum and can be detected when the reaction completes.The two detection methods are two-step,easy to operate,and need a small amount of sample.4.The PCT test strip had a strongerability to capture PCT antigen due to double T lines which helped to improve the sensitivity and linearity.With the linear range of 0.05-100 ng/mL,the l linearity was good（Y=0.0747X+0.5246,R²=0.9904）.The sensitivity was 0.03 ng/mL,the intra-assay precision CV≤8.76%,the inter-assay precision CV≤6.86%.The specificity was good,and the correlation with the AE-180 automatic chemiluminescence immunoanalyzer was good（Y=0.9011X+0.4137,R²=0.9873,p<0.01）.It can meet the clinical requirements.5.The IL-6 test strip had the linear range of 2-500 pg/mL,the linearity was good（Y=0.0020 X+0.0051,R ²=0.9988）.The sensitivity was 0.37 pg/mL,the intra-assay precision CV≤8.87%,the inter-assay precision CV≤9.04%,the specificity was good,and the correlation with SIEMENS IMMULITE 1000 automatic chemiluminescence immunoanalyzer was good（Y=0.9502X+6.1397,R²=0.9517,p<0.01）.It can meet the clinical requirements.