Efficacy Evaluation Of Desensitization Therapy: Component Resolved Diagnosis And Biomarker Research

The core value of clinical pharmacy is to promote clinical rational drug use,including evaluation and design of individual therapy to improve the therapeutic efficacy.Allergic disease,affecting 10%to 40%of the population in the world,is one of the three diseases classified as the focus of prevention and cure in the 21st century by the World Health Organization.Allergen immunotherapy(AIT),also known as desensitization therapy,is the only treatment that can cure allergic diseases.However,up to 30%of patients do not respond to it.Therefore,the evaluation of the efficacy of desensitization treatment will help patients choose rational therapy and reduce unnecessary social medical costs.The clinical evaluation system of desensitization treatment is based on the patient's subjective score system for symptom improvement.There is no objective and effective biomarker,which makes it difficult to evaluate the clinical efficacy of desensitization treatment.Evidence showed that AIT-induced gene expression profiles differed between clinical responders and nonresponders,resulting in differential expression of functional proteins and differences in efficacy.Moreover,there is a dose-efficacy relationship between desensitization treatment,meaning that the match degree of patient's molecular sensitization profiles and the vaccine components will have a great impact on the AIT efficacy.Component resolved diagnosis(CRD)has become a frontier hotspot in the study of patient's molecular sensitization profiles in recent years.It can specify the true sensitization molecules of patients,thereby standardizing the diagnosis and treatment of allergic diseases.However,current CRD techniques have many drawbacks such as time-consuming,high-cost,improper protein fixation methods and low sensitivity.Based on the above two aspects,the first chapter of this study screened the serum protein biomarkers of AIT efficacy by combining proteomics and enzyme-linked immunosorbent assay(ELISA)to find an objective evaluation criterion for AIT;the last two chapters constructed a series of novel CRD advices to overcome the deficiencies in the existing CRD techniques,providing technical platforms for AIT efficacy evaluation.Chapter 1.Leukotriene A₄ hydrolase is a candidate predictive biomarker for successful allergen immunotherapyObjective:Screening of candidate biomarkers for evaluating the efficacy of pollen desensitization therapy in serumMethods:Artemisia pollen allergic patients who received at least one-year AIT were enrolled.Clinical responses before and after one-year AIT were evaluated to determine AIT responders.Artemisia specific Ig E and Ig G₄ levels were measured by using Immuno CAP and ELISA separately.Stepwise regression analysis was performed to identify which rhinitis-relevant parameters explained the most variability in AIT results.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)-based proteomics was applied to identify the potential candidate biomarkers in the sera of responders and non-responders collected before and after one-year therapy.The diagnostic performance of the potential biomarkers was then assessed using ELISA in30 responders and 15 non-responders.Results:Artemisia specific Ig E and Ig G₄ levels were elevated only in the responders.Regression analysis of allergic rhinitis-relevant parameters provided a robust model that included two most significant variables(sneeze and nasal congestion).Thirteen candidate biomarkers were identified for predicting AIT outcomes.Based on their association with allergy and protein fold change(more than 1.1 or less than 0.9),4 proteins were identified to be potential biomarkers for predicting effective AIT.However,further ELISA revealed that only leukotriene A₄ hydrolase(LTA₄H)was consistent with the proteomics data.The LTA₄H level in responders increased significantly(P<0.001)after one-year therapy,while that of non-responders remained unchanged.Assessment of LTA₄H generated area under curve(AUC)value of 0.844(95%confidence interval:0.727 to 0.962;P<0.05)in distinguishing responders from the non-responders,suggesting that serum LTA₄H might be a potential biomarker for predicting the efficiency of AIT.Conclusion Serum LTA₄H may be a potential biomarker for early prediction of an effective AIT.Chapter 2.Construction and clinical verification of a paper-based sensor for component resolved diagnosis of allergic diseases based on His-tag immobilization methodObjective:To construct a new CRD sensor to solve the problems of high-cost,time-consuming and improper allergen fixation model of exist CRD technology in clinic,and to further provide a platform for the evaluation of desensitization treatment.Methods:We first synthesized the Ni-NTA and fixed it on the surface of paper through mild and efficient Schiff base reaction.We used His-tagged recombinant Can f 1(r Can f 1)as the model allergen.The recombinant allergen protein was immobilized on paper by the interaction of his-tag and Ni-NTA.Then,the paper-based devices were characterized.The detection conditions were optimized,and the analytical performance was analyzed.At last,the paper-based device and ELISA were used to detect r Can f 1s Ig E in 50 clinical samples independently to verify the clinical practicability of the developed paper device.Results:r Can f 1 was successfully immobilized on the paper by tag-based fixation model,which was more consistent with the real binding mode of allergen and s Ig E in vivo than the exist clinical fixation methods.The optimal detection conditions were finally confirmed as follows:4?L of Ni-NTA(2 mg/m L)solution,3?L of r Can f 1(0.06?g)solution for 5 min,3?L of 10-fold diluted serum samples for 10 min,and 3?L of 1000-fold diluted horse radish peroxidase(HRP)-labeled signal goat anti-human Ig E for 5 min sequentially.The developed paper-based device showed good selectivity and high sensitivity.The signal intensity of this device displayed a good linear relationship with the concentration of s Ig E ranging from 0 to 14.02 ng/m L.The detection limit was 0.51 ng/m L,which is below the clinical cut-off value(?0.84 ng/m L,0.35 KU_A/L).The reproducibility(with the 91.9-110.7%recovery rates)and stability of paper-based device(p H,temperature and long-time stability)were also evaluated with good performance.Comparing with ELISA,our device exhibited a little higher positive rate(56.2%vs 53.1%).Conclusion:The new paper-based advice constructed in this chapter is accurate,reliable,fast and sensitive in CRD of allergic diseases,which can promote individual diagnosis and provide technical basis for evaluation system of desensitization treatment based on molecular sensitization profiles.Chapter 3.Construction and clinical verification of an ultra-sensitive nano-sensor for component resolved diagnosis of allergic diseasesObjective:On the basis of the previous chapter,we aimed to build an ultra-sensitive CRD sensor to solve the problem of relative low sensitivity of clinical CRD technology and to provide a technical platform for evaluation system of desensitization treatment based on molecular sensitization profiles.Methods:Gold nanoparticles and magnetic nanoparticles were synthesized.Ni-NTA complex was modified on the surface of magnetic nanoparticles instead of paper in the previous chapter.We used His-tagged r Can f 1 as the model allergen.Relying on the interaction between His-tag and Ni-NTA,r Can f 1 was immobilized on magnetic nanoparticles.Meanwhile,numerous HRP-labeled anti-Ig E were modified on the surface of gold nanoparticles to detect s Ig E captured by magnetic nanoparticles and acquire signal amplification.The nano-device were characterized.The detection conditions were optimized,and the detection performance was analyzed.Clinical samples were further used to verify the clinical practicability of the device.Results:r Can f 1 was successfully immobilized on the magnetic nanoparticles by His-tag.Numerous HRP-anti-Ig E were modified onto one Au NP to detect the s Ig E probed by Fe₃O₄@Si O₂-NTA@r Canf1.The optimal detection conditions were as follows:1000-fold diluted HRP-labeled goat anti-human Ig E,7.5?g/m L r Can f 1incubated for 5 min and Au NP-anti-Ig E incubated for 20 min.The LOD of our new immunosensor(0.02 ng/m L)was 10 times lower than that of free anti-Ig E method(0.20ng/m L),showing much better sensitivity.The nano-device possess good p H and temperature stability.The recovery rates were calculated to be 97.25-114.18%.The detection results of clinical samples were in good agreement with those obtained by the clinical gold standard Immuno CAP,with 1000 times less serum consumption.Conclusion:The nano-sensor constructed in this chapter can significantly improve the sensitivity of existing CRD method,which can promote individual diagnosis and provide technical basis for evaluation system of desensitization treatment based on molecular sensitization profiles.