Establishment Of A Quantitative Method For Homodimer Neutrophil Gelatinase-associated Lipocalin (NGAL) And Its Use As A Biomarker Of Bacterial Infection

The prevention and treatment of pneumonia are serious problems that governments,medical researchers and clinical medical staff,of all countries,need to address.The lack of quick and easy methods for identifying the pathogen causing pneumonia and the lack of means of monitoring the efficacy of antibiotics are key factors affecting the prevention and treatment of pneumonia.In the absence of evidence of the exact cause,it is common to use antibiotics to treat pneumonia based on experience and a few clinical test indicators.This has contributed to the negative effects of antibiotic abuse;adverse drug effects and the emergence of resistant bacteria.In recent years,although a few studies have shown that neutrophil gelatinase-associated lipocalin(NGAL)has the ability to distinguish bacterial infections from viral infections.However,the use of NGAL as a bacterial marker needs further verification and in-depth research.Acute bacterial infection can cause neutrophils to activate and release the homodimer NGAL pre-existing in the second granule.Therefore,this paper uses Community-acquired pneumonia(CAP)in children as the research field to study the possibility of homodimerNGAL as a biomarker of acute bacterial infection.In order to specifically quantify the homodimer NGAL,this paper first established a sandwich ELISA in which the capture and detection antibodies are the same monoclonal antibody.Subsequently,by analyzing the concentration of homodimer NGAL in bacterial CAP,viral CAP,and healthy children’s serum samples,as well as the correlation between NGAL and clinical bacterial infection indicators such as PCT,CRP,and neutrophil count,the homology was evaluated.The source dimer NGAL is used to distinguish the ability to diagnose bacterial CAP and viral CAP.The main research contents and results are as follows:1.Established a quantitative method for homodimer NGAL.Compared with monomers or heterodimers,homodimer NGAL molecules have the same epitope.Based on this feature,this thesis uses the same monoclonal antibody obtained in the previous screening as the capture antibody and detection antibody to establish a sandwich ELISA that specifically detects homodimer NGAL.The checkerboard method was used to determine the concentration of the coating antibody(1 μg/mL)and the concentration of the detection antibody(1:5000),and the ELISA methodology was carried out.Finally,a specific immune-quantitative method for homodimer NGAL was established.The specific methodological parameters are as follows:the linear range is 0.1～6.4 μg/L(R2>0.99,the range is 0.99～1.0),the minimum detection limit is 0.045 μg/L(0.032～0.054 μg/L),average intra-assay and inter-assay coefficient of variation(CV)was 7.63%(1.42%～18.10%)and 12.6%(7.37%～23.23%),respectively.The average recovery rate for homodimer was 99.25%(81.3 5%～119.11%).The average recovery rates of heterodimer and heterodimer were 6.79%(5.40%～8.64%)and 5.68%(4.20%～9.62%),respectively.The results of the gradient dilution study showed no significant difference.The above data show that the NGAL homodimer quantitative method established in this paper has good specificity and reproducibility,and can be used for subsequent research work.2.Homodimer NGAL has the ability to be used as a diagnostic marker for bacterial pneumonia in children.This paper selects children with CAP as the research,and analyzes the concentration of homodimer NGAL in the serum of CAP(133 cases),viral CAP(65 cases)and healthy control group(47 cases)and its relationship with other bacteria.Correlation of infection markers(PCT,CRP and neutrophil count),the ability of homodimer NGAL to distinguish acute bacterial infections from viral infections was evaluated in more detail.The results showed:(1)The homodimer NGAL level(arithmetic mean±SD,73.25±52.68 μg/L)of the bacterial pneumonia group was significantly higher than that of the viral pneumonia group(22.63± 14.47 μg/L)and the healthy group(15.54±9.11 μg/L),the P values are all less than 0.0001.(2)In the bacterial CAP group,NGAL was significantly correlated with PCT,CRP,and the number of neutrophils,and the R values were 0.699,0.566,and 0.590,respectively;while in the viral CAP group and healthy control group,the above correlation between the markers is significantly reduced.(3)The area under the receiver operating characteristic curve(AU-ROC)value of NGAL used to distinguish bacterial pneumonia and normal control group is 0.94(95%CI=0.89～0.97);used to distinguish bacterial CAP from viral AU-ROC value of CAP is 0.86(0.80～0.91);the AU-ROC value used to distinguish between bacterial CAP and non-bacterial infection volunteers is 0.90(0.85～0.93).In addition,the AU-ROC values of the above-mentioned NGAL differential diagnosis were significantly higher than the AU-ROC values corresponding to the other three markers.The above data indicate that the homodimer NGAL can distinguish between bacterial and viral CAP and is a good marker of bacterial infection.In summary,this paper establishes a specific quantitative method for homodimer NGAL;It is preliminarily confirmed that homodimer NGAL has the ability to distinguish between bacterial pneumonia and viral pneumonia.The thesis research can provide the theoretical basis and technical means for the differentiated diagnosis of the cause of acute infectious diseases based on NGAL in the future and to guide doctors in the scientific use of antibiotics.