發(fā)布時(shí)間：2018-06-09 17:34

  本文選題：金標(biāo)免疫滲濾法 + 類風(fēng)濕性關(guān)節(jié)炎�。� 參考：《東南大學(xué)》2015年碩士論文

【摘要】：膠體金標(biāo)記技術(shù)的建立催生了兩種經(jīng)典的免疫學(xué)檢測(cè)方法,斑點(diǎn)金免疫滲濾(DIGFA)和膠體金免疫層析技術(shù)(GICA),其中DIGFA由于具有快速、結(jié)果可直接判讀、可單人份檢測(cè)、結(jié)果穩(wěn)定等優(yōu)點(diǎn)逐漸成為開(kāi)發(fā)各類快速診斷試劑的技術(shù)基礎(chǔ),一系列針對(duì)臨床、食品等多個(gè)領(lǐng)域的DIGFA被建立和應(yīng)用。類風(fēng)濕性關(guān)節(jié)炎(RA)是一種自身免疫性疾病,晚期關(guān)節(jié)多發(fā)生畸形難以治愈,早期診斷對(duì)疾病的控制尤為重要,大量研究已經(jīng)表明在臨床癥狀出現(xiàn)好幾年前患者血清中就會(huì)出現(xiàn)特異的自身抗體,對(duì)這些自身抗體的檢測(cè)對(duì)RA的早期診斷非常有利。本文擬建立檢測(cè)RA兩種自身抗體的DIGFA,旨在簡(jiǎn)化檢測(cè)操作、降低檢測(cè)成本、在樣本量少的情況下可快速開(kāi)展檢測(cè)。為此,首先通過(guò)與歐蒙試劑盒對(duì)比及檢測(cè)臨床確定的血清對(duì)待檢抗體特異的抗原進(jìn)行了篩選制備,采用鏈酶親和素-生物素結(jié)合方式將環(huán)瓜氨酸肽(CCP)與載體蛋白進(jìn)行偶聯(lián),優(yōu)化了新的含18個(gè)氨基酸的環(huán)肽序列；確定了較穩(wěn)定的類風(fēng)濕因子(RF)特異抗原的原料來(lái)源,同時(shí)優(yōu)化了特異性高的HRP標(biāo)記F(ab’)2片段作為酶標(biāo)二抗,并比較了兔IgG、人IgG、Fc片段幾種常用于RF檢測(cè)的抗原性能。接著,本文制備了抗自身抗體的膠體金探針,對(duì)各反應(yīng)參數(shù)進(jìn)行了優(yōu)化,確定了較好的偶聯(lián)pH在8.2-8.5之間(7μL K2CO3 (5%)/mL Au),最小偶聯(lián)蛋白量為22μg/mLAu,最適大分子穩(wěn)定劑是BSA(免疫級(jí))和PEG-20K,較優(yōu)洗滌純化緩沖液是Tris-HCl(pH 8.2),最后優(yōu)化了探針凍干的最佳保護(hù)劑為甘露醇。最后,將前兩章制備的抗原和探針應(yīng)用于DIGFA的建立,對(duì)影響檢測(cè)的多個(gè)因素進(jìn)行了優(yōu)化,確定了RIgG最適稀釋液是Tris-HCl (pH 8.2),樣品下滲速度是1.4-3.3虬/s,大分子封閉劑是BSA,洗滌條件是PBST(pH7.4,1%Tween-20),同時(shí)發(fā)現(xiàn)PEG和Tween對(duì)斑點(diǎn)顯色有一定增強(qiáng)作用,用該試劑檢測(cè)部分臨床血清,顯示了良好的檢測(cè)效果。
[Abstract]:The establishment of colloidal gold labeling technique gave birth to two classical immunological detection methods, DIGFA and GICA, in which DIGFA can be read directly and can be detected by single person because of its rapidity. Results Stability has gradually become the technical basis for the development of various rapid diagnostic reagents, and a series of DIGFA for clinical, food and other fields have been established and applied. Rheumatoid arthritis (RA) is a kind of autoimmune disease. A large number of studies have shown that specific autoantibodies are present in patients' serum several years before the onset of clinical symptoms. The detection of these autoantibodies is very helpful for the early diagnosis of RA. The purpose of this paper is to establish a DIGFA for detecting two kinds of autoantibodies in RA. The aim is to simplify the detection operation and reduce the detection cost, so that the detection can be carried out quickly under the condition of small sample size. For this reason, the specific antigens of antibodies were screened and prepared by comparing and detecting the serum specific antigens determined by Ohm kit. The cyclic citrullinated peptide (CCPP) was coupled with the carrier protein by the way of chain enzyme affinity and biotin binding. The new cyclic peptide sequence containing 18 amino acids was optimized, the source of the stable RFRFs specific antigen was determined, and the HRP labeled FGBA 2 fragment was optimized as the enzyme labeled second antibody. The antigenicity of rabbit IgG, human IgG Fc fragment for RF detection was compared. Then, the colloidal gold probe of anti-autoantibody was prepared, and the reaction parameters were optimized. The optimum coupling pH was between 8.2-8.5 渭 L K2CO3 and 5 渭 L / mL au, the minimum coupling protein content was 22 渭 g / mL au, the optimal macromolecular stabilizers were BSA (immune level) and PEG-20K, the optimal washing and purification buffer was Tris-HCl pH 8.2, and the best protection agent for the lyophilization of the probe was mannitol. Finally, the antigens and probes prepared in the first two chapters are applied to the establishment of DIGFA, and several factors affecting the detection are optimized. The optimal diluent of RIgG was Tris-HCl (pH 8.2), the osmotic rate of the sample was 1.4-3.3 / s, the macromolecular sealant was BSA, and the washing condition was PBST-pH7.4 ~ (-1) Tween-20. At the same time, it was found that PEG and Tween had a certain enhancement effect on spot color, and some clinical sera were detected with this reagent. Good detection effect is shown.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R593.22

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