Novel Labels And Heterologous Competitive Systems For Improving Sensitivity Of Immunoassay

Immunoassay has attracted much attention in the field of rapid detection because of its unique advantages.Lateral flow immunochromatography(LFI)and enzyme-linked immunosorbent assay(ELISA),as two conventional screening platforms,are widely used in clinical diagnosis,environmental monitoring,food safety test,and other fields to determine target molecules because of their advantages of simplicity,short detection time,powerful specificity,and low cost.However,the wide application of these two methods is limited by their low detection sensitivity,which makes it difficult to carry out more accurate qualitative and quantitative analysis for the target compounds with low concentration in specific samples.Therefore,improving the sensitivity of the two immuonoassays is an urgent task for the development of LFI and ELISA.Previous studies indicated that novel labels and optimized antigen-antibody(Ag-Ab)system were two effective strategies to improve the sensitivity of conventional LFI and ELISA.In this systematical study,we simultaneously explored the influence of three novel labels and two heterogeneous competition systems on the detection sensitivity of LFI and ELISA,respectively,which could provide new insight to improve the detection sensitivity of LFI and ELISA or even other immunoassays.The first chapter reviewed the development of LFI and ELISA,including their priciples,original establishment model,and practical application in different fields.Meanwhile,quinolones(FQs)and sulfanilamides(SAs)drugs involved in the study were shown in this chapter separately,including their hazards,limited standards,and existing detection methods.In chapter two,a novel LFI by introducing time-resolved fluorescent microspheres(Eu(Ⅲ)-time-resolved fluorescence microspheres doped polystyrene nanoparticles,EuNP)and the new designed heterologous detective antigen was established to detect sulfamethazine(SMZ)residue in raw milk.The dynamic light scattering results showed that EuNP-mAb complex system had good dispersion coefficient,and the biolayer interference results indicated that the affinity between heterologous competitive Ag and Ab(3.214×10-5 M)was much lower than that between Ag and Ab(3.875×10-9 M),which has a positive impact on improving sensitivity of LFI.Under the optimal working conditions,the limit of detection(LOD)and linear range of the proposed method were 0.0045 and 0.05-10 ng/mL,respectively.The recovery of LFI for the detection of SMZ in raw milk was 96.1%-108.2%.The proposed LFI provided a rapid and convenient strategy for fast and ultra-sensitive screening of SMZ in raw milk.EuNP-LFI may be a remarkable method for detection of other targets at low concentrations to ensure food safety.In chapter three,another LFI was developed by introducing magnetic beads with colloidal gold nanoparticles(AuMB)to LFI test strips for simultanously qualitative and quantitative detection of SMZ in raw milk.In the proposed assay,colloidal gold is used as label for qualitative detection on the basis of naked-eye and fluorescence signal is used for quantitative detection of SMZ in positive samples.Results indicated that the threshold concentration for determining whether a test sample is positive or negative is 5 ng/mL.The LOD and linear range of the proposed AuMB-LFI for quantitative detection were 0.39 and 0.5200 ng/mL,respectively,and its recovery for detecting SMZ in raw milk was 97.4%-110.2%.The developed AuMB-LFI provides novel insights into efforts to maintain balance between efficient screening of targets in large samples and saving time and money.In chapter four,an aggregation-induced emission fluorescent microbeads(AIEFM)based-LFI was established to detect norfloxacin(NOR)residues in nine types of animal-derived food samples.The fluorescence spectrum characterization results showed that AIEFM fluorescence intensity and Stoke’s shift were much bigger than that of commercial FITC fluorescence microspheres.Dynamic light scattering characterization results indicated that AIEFM-mAb complex system has good dispersion coefficient.Under the optimized working conditions,the LODs of AIEFM-LFI for detection of NOR in honey,eggs,chicken,milk,beef,lamb,fish,swine liver,and pork samples were 0.03,0.03,0.02,0.04,0,14,0.30,0.15,0.04 and 0.22 ng/mL,respectively,the corresponding linear ranges were 0.05-20,0.05-10,0.05-10,0.05-20,0.5-100,1-200,0.5-100,0.05-10 and 0.5-200 ng/mL,respectively.The detection results of AIE-LFI were compatible with HPLC-MS/MS in detecting NOR in 135 real samples from above nine types of animal-derived food.The developed AIEFM-LFI was sensitive and reliable for NOR detection in those real samples.In chapter five,molecular descriptors of haptens and machine learning methods were used to successfully screen the heterologous competitive antigen(HCA)for improving the ELISA sensitivity.In this chapter,the anti-enrofloxacin(ENR)monoclonal antibody(mAb)was first prepared with the ENR-bovine serum albumin(BSA)as immunogen.The molecular descriptors of quinolones were then utilized to screen heterologous coating antigens for the detection of ENR based on ensemble learning method to improve the ELISA sensitivity.Results indicated that six of the seven heterologous competitive antigens we selected could enhance the sensitivity of ELISA.The ELISA sensitivity for the detection of ENR with sarafloxacin-BSA as heterologous coating antigen was improved 10-fold(in PBS)and 6-fold(in milk sample),respectively,compared with that with ENR-BSA as homologous antigen.The strategy can effectively screen the suitable heterologous competitive antigens to improve the sensitivity of ELISA,followed by the preparation of mAb with no additional modification to corresponding immunogen.In chapter six,heterologous competitive antigens(HCAs)suitable for improving the sensitivity of ELISA were successfully screened based on their cross-reactivities(CRs)with 19 quinolone analogues;each containing the norfloxacin amino derivative(NOR0)coupled with bovine serum albumin(BSA)as a coating antigen.HCAs prepared with hapten analogues(CRs of 0.77%-49.92%)remarkably enhanced the sensitivity of the subsequent ELISA.ELISA sensitivity for NOR detection improved 26-fold when moxifloxacin-BSA was used as a heterologous coating antigen relative to when NOR0-BSA was used as a homologous coating antigen.This work,therefore,represents a detailed screening method to select suitable heterologous competitive antigens that improve ELISA sensitivity.Secondly,we present new theoretical tools to estimate hapten structures for use in the method,which may also be applied to improve the sensitivity of other immunoassays.Chapter seven drew a conclusion of the full text.We also proposed the direction of improving the sensitivity of immunoassay,which may provide some new insights to develop immunoassays.