【摘要】：現(xiàn)已證實,Nodal參與了腫瘤惡性生物學(xué)過程,但對其高敏感檢測法尚未建立。采用基因工程表達(dá)人源Nodal作為抗原免疫BALB/c小鼠,通過經(jīng)典PEG誘導(dǎo)的細(xì)胞融合技術(shù)篩選出針對Nodal的特異性單克隆抗體7株。夾心ELISA確證7株抗體組成了15種可配對的抗體對。經(jīng)篩選后選取抗體對AF12-DG5建立標(biāo)準(zhǔn)化夾心ELISA法,結(jié)合生物素-親和素檢測系統(tǒng),DG5抗體標(biāo)記生物素,采用鏈親和素與辣根過氧化物酶標(biāo)記的生物素(HRP-Biotin)按質(zhì)量比4∶1預(yù)先混合孵育的ABC混合物進(jìn)行檢測,以提高ELISA法的靈敏度。棋盤滴定確定抗體工作最佳濃度為:捕獲抗體(AF12)2μg/ml,檢測抗體(生物素化DG5)2μg/ml。此條件下的夾心ELISA法線性范圍為0~3 000pg/ml,檢測限為68pg/ml,平均回收率為99.6%,精密度準(zhǔn)確度良好。以正常人血清作為陰性對照,使用該夾心ELISA法測定結(jié)直腸癌、鼻咽癌和膽囊癌患者血清,發(fā)現(xiàn)三種腫瘤患者血清與正常人血清中的Nodal濃度均存在明顯的統(tǒng)計學(xué)差異,可作為臨床使用參考。
[Abstract]:It has been proved that Nodal is involved in malignant biological process of tumor, but its high sensitivity detection has not been established. BALB/c mice were immunized with genetically engineered human Nodal as antigen. Seven specific monoclonal antibodies against Nodal were screened by classical PEG-induced cell fusion technique. Sandwich ELISA confirmed that 15 pairing antibody pairs were made up of 7 antibodies. After screening, the antibody was selected to set up a standardized sandwich ELISA method for AF12-DG5, combined with biotin-avidin detection system, and DG5 antibody was used to label biotin. In order to improve the sensitivity of ELISA method, the mixture of HRP-Biotin and HRP labeled biotin (ABC) was detected by pre-incubation with ABC mixture at a mass ratio of 4:1 in order to improve the sensitivity of the method. The optimal concentration of antibody determined by chessboard titration was as follows: capture antibody (AF12) 2 渭 g / ml, to detect antibody (biotin DG5) 2 渭 g / ml. Under these conditions, the linear range of sandwich ELISA method is 0 ~ 3 000 PG / ml, the detection limit is 68 PG / ml, the average recovery is 99.6%, and the accuracy of precision is good. The serum levels of Nodal in patients with colorectal cancer, nasopharyngeal carcinoma and gallbladder carcinoma were determined by sandwich ELISA method with normal human serum as negative control. The results showed that there were significant differences in serum Nodal concentrations between the three kinds of tumor patients and normal controls. It can be used as a reference for clinical use.
【作者單位】： 中山大學(xué)藥學(xué)院微生物與生化藥學(xué)實驗室;
【基金】：國家自然科學(xué)基金資助項目(81672943、81472643)
【分類號】：R730.4
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本文編號：2440016