The Development Of ELISA Kit For Eosinophil Cationic Protein And Its Clinical Application

Objective 1 .Develope a sandwich enzyme-immunoassay of double antibodies kit (ELISA) for ECP with a high sensitivity and accuracy. 2.Compare different coating methods. 3.Evaculate the significance of ECP test in the diagnosis and treatment of anaphylactic disease.Methods 1.The development of sandwich enzyme-immunoassay of double antibodies kit (ELISA) for ECP:(1)Purify monoclonal antibody against ECP with staphylococcal protein A ;( 2) Label polyclonal antibody against ECP with horseradish peroxidase by NaIO₄; (3)Develop and optimize the ELISA kit;(4)Evaluate the test performance. 2.The comparison of different coating methods:(1) Biotinylation of monoclonal antibody against ECP;(2) Biotinylation of bovine serum albumin;(3) The development and optimization of the ELISA kit which was first coated with biotin;(4) The development and optimization of the ELISA kit which was first coated with avidin;(5) The comparison of three methods.3.Evaluate the importance of ECP detection in the diagnosis and treatment of anaphylactic disease: detect the ECP level and IgE level of patients with allergic skin disease and appraise the correlation; compare the ECP level of patients with urticaria before and after treatment ,and evaluate the correlation between ECP and symptom score,total IgE and specific IgE; evaluate the correlation between ECP and symptom score,total IgE,EOS count of patients with atopic dermatitis.4. Evaluate the importance of ECP detection in the diagnosis and treatment of allergic rhinitis.Result 1.The development of ELISA kit for ECP: (1)We explored various factors and conditions of ELISA and determine the optimal reaction conditions: the dilution of mAb and HRP-1abeled pAb was 1:500 and 1:8000,respectively; the best blocking buffer was 1%BSA;the optimal serum dilution was sample bufferâ…¡;the optimal reaction time and temperature was 60min and 37℃, respectively.(2) The linear range of our kit was 2.5～200 ng/ml; sensitivity was 2.75ng/ml; relative deviation,intra coefficient of variation and inter coefficient of variation was 11.5%,<10% and <15%, respectively. The comparison of three methods:(1) The linear range,sensitivity and relative deviation of biotin-avidin coating test was 2.5～500 ng/ml,2.61ng/ml and 10.9%,respectively.(2) The linear range,sensitivity and relative deviation of avidin coating test was 2.5～500 ng/ml,2.68ng/ml and 11.9%,respectively.(3)There was no significant difference among their reproducibility and precision. Avidin coating test was the stablest method and antibody coating test was the simplest method,but its antibody dosage was the highest. 3.The significance of ECP detection in allergic disease diagnosis and treatment:(1)The ECP level of 37℃group was significantly higher than room temperature group and 0℃group, and decoagulant had no impact on ECP level, while hematolysis could increase ECP notably. (2)ECP and total IgE were both higher than normal control, but there was no correlation between them. The ECP levels of patients with urticaria before and after treatment had significant difference, and had positive relationship with symptom score and specific IgE. The ECP level of patients with atopic dermatitis directly correlated with symptom score. 4.The ECP level of patients with allergic rhinitis was higher than normal control, and directly correlated with specific IgE.Conclusion 1 .We developed a sandwich enzyme-immunoassay of double antibodies kit(ELISA) for ECP with advantages such as high sensitivity,accuracy and simple operation. 2.Among three methods, the linear range and sensitivity of biotin-avidin coating test were better and it could save antibody, but its stability was worse. 3. ECP detection had significance to the diagnosis and treatment of allergic disease.