發(fā)布時(shí)間：2018-06-01 01:01

  本文選題：嗜酸性粒細(xì)胞陽(yáng)離子蛋白 + 酶聯(lián)免疫吸附試驗(yàn)��； 參考：《天津醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的1.制備雙抗夾心ELISA(enzyme linked immunosorbent assay,酶聯(lián)免疫吸附試驗(yàn))嗜酸性粒細(xì)胞陽(yáng)離子蛋白(eosinophilia cationic protein,ECP)檢測(cè)試劑盒。2.對(duì)不同的包被方法進(jìn)行比較。3.評(píng)估ECP水平檢測(cè)在過(guò)敏性疾病診療中的意義。 方法1.雙抗夾心ELISA ECP檢測(cè)試劑盒的制備:(1)葡萄球菌A蛋白親和層析法純化抗-ECP單克隆抗體(monoclonal antibody,mAb),(2)過(guò)碘酸鈉法HRP標(biāo)記抗-ECP多克隆抗體(polyclonal antibody,pAb),(3)ELISA試劑盒的制備與條件優(yōu)化,(4)實(shí)驗(yàn)效能評(píng)價(jià)。2.ECP檢測(cè)試劑盒的方法學(xué)比較:(1)抗-ECPmAb的生物素化,(2)BSA的生物素化,(3)先包被生物素的ELISA試劑盒的制備與條件優(yōu)化,(4)先包被親和素的ELISA試劑盒的制備與條件優(yōu)化,(5)比較三種檢測(cè)方法。3.評(píng)估ECP水平檢測(cè)對(duì)過(guò)敏性疾病診療的意義:(1)評(píng)估采血方式對(duì)ECP結(jié)果的影響,(2)評(píng)估ECP檢測(cè)對(duì)過(guò)敏性皮膚病診療的意義:檢測(cè)過(guò)敏性皮膚病患者血清ECP水平:檢測(cè)患者總IgE水平,并評(píng)估其與ECP的相關(guān)性;評(píng)估蕁麻疹患者治療前后ECP水平的變化,以及其ECP水平與癥狀評(píng)分、總IgE、特異性IgE相關(guān)性;評(píng)估異位性皮炎患者疾病癥狀評(píng)分與ECP水平、總IgE、嗜酸性粒細(xì)胞(EOS)計(jì)數(shù)的相關(guān)性。4.評(píng)估ECP水平檢測(cè)在過(guò)敏性鼻炎診療中的意義。 結(jié)果1.雙抗夾心ELISA ECP檢測(cè)試劑盒的制備:(1)通過(guò)對(duì)該方法中各種反應(yīng)條件的摸索和優(yōu)化,確定了最適工作條件:單抗最適包被濃度1:500稀釋,酶標(biāo)多抗最適濃度為1:8000稀釋,最佳封閉液為1%的BSA,最適血清稀釋液為稀釋液Ⅱ,最佳反應(yīng)時(shí)間為60min,反應(yīng)溫度為37℃。(2)本研究制備的試劑盒線性范圍為2.5～200ng/ml,靈敏度為2.75ng/ml,相對(duì)偏差為11.5%,批內(nèi)變異系數(shù)＜10%,批間變異系數(shù)＜15%,穩(wěn)定性較好,與ADL試劑盒相比較相關(guān)系數(shù)為0.97。2.三種方法的比較:(1)生物素親和素包被法線性范圍為2.5～500ng/ml,靈敏度為2.61ng/ml,相對(duì)偏差為10.9%;(2)親和素包被法線性范圍為2.5～500ng/ml,靈敏度為2.68ng/ml,相對(duì)偏差為11.9%。(3)三種方法的重復(fù)性、精確性沒(méi)有顯著性差異,穩(wěn)定性親和素包被法最為穩(wěn)定,抗體包被法次之,生物素包被法最不穩(wěn)定,抗體直接包被法操作最為簡(jiǎn)易,但抗體用量最多。3.ECP水平檢測(cè)對(duì)過(guò)敏性疾病診療的意義:(1)標(biāo)本采集后37℃放置1h的結(jié)果顯著高于室溫與0℃組,抗凝劑對(duì)ECP水平無(wú)影響,而標(biāo)本溶血使ECP水平顯著升高。(2)過(guò)敏性皮膚
[Abstract]:Objective 1. Preparation of double antibody sandwich ELISA(enzyme linked immunosorbent assay, enzyme linked immunosorbent assay) eosinophilia cationic protein detection kit. 2. Compare different envelope methods. 3. 3. To evaluate the significance of ECP level in the diagnosis and treatment of allergic diseases. Method 1. Preparation of double Antibody Sandwich ELISA ECP Assay Kit: 1) purification of Monoclonal body Monoclonal Antibody of Staphylococcus Staphylococcus by Affinity Chromatography Comparison of methods for evaluating .2. ECP Detection Kit; (1) Biotinylation of anti-ECPmAb and biotinylation of BSA; preparation and condition optimization of ELISA kit coated with biotin; preparation and condition optimization of ELISA kit coated with avidin; preparation and condition optimization of ELISA kit precoated with avidin (5) Compare three detection methods. To evaluate the significance of ECP level in the diagnosis and treatment of allergic dermatosis; to evaluate the effect of blood collection on the results of ECP; to evaluate the significance of ECP detection in the diagnosis and treatment of allergic dermatosis; to detect the serum ECP level of allergic dermatosis patients; and to detect the total IgE level in patients with allergic dermatosis. To evaluate the correlation between ECP and ECP, to evaluate the changes of ECP level in patients with urticaria before and after treatment, and to evaluate the correlation between ECP level and symptom score, total IgE and specific IgE, and to evaluate the relationship between ECP and ECP level in patients with ectopic dermatitis. Correlation of total IgE, eosinophil EOS count. 4. To evaluate the significance of ECP level in the diagnosis and treatment of allergic rhinitis. Result 1. Preparation of double Antibody Sandwich ELISA ECP Detection Kit: by exploring and optimizing various reaction conditions in this method, the optimum working conditions were determined: the optimal concentration of McAb was diluted by 1: 500, the optimal concentration of enzyme labeled polyantibody was diluted by 1: 8000. The best blocking solution is 1% BSA, and the optimal serum dilution solution is diluent 鈪，

本文編號(hào)：1962179