建立雌酮硫酸钠胶体金免疫层析快速限量检测方法

目的：建立一种快速检测孕马尿中雌酮硫酸钠的胶体金免疫层析试纸方法。方法：采用化学合成法将雌酮制备为ESS-17-肟（半抗原），应用混合酸酐法将ESS-17-肟与牛血清白蛋白（BSA）偶联制备为ESS-17-肟-BSA（免疫原），对所得偶联物溶液进行紫外扫描鉴定并计算偶联率。ESS-17-肟-BSA免疫BALB/C小鼠，取血清高滴度小鼠脾脏淋巴细胞与SP2/0细胞融合制备杂交瘤细胞，用ELISA法进行阳性筛选。获得阳性克隆细胞株后于BALB/C小鼠体内诱生腹水制备单克隆抗体，抗体纯化后用胶体金标记，通过正交设计确定最佳实验参数，研制出雌酮硫酸钠胶体金免疫层析试纸条。结果：获得经化学结构修饰后的目标抗原ESS-17-肟-BSA，融合克隆后得到4株稳定分泌抗体的杂交瘤细胞株2D1、7G4、5F3、2A7，其中2D1抗体纯化后特异强，效价达1.0×10⁶。该抗体制备的胶体金层析试纸条的检测限为1 μg·L^-1，与结构类似物均无交叉反应，此法检测样品结果与高效液相色谱法测定结果比对阳性符合率为85.45%（45/52），阴性符合率为89.58%（43/48），总符合率为88.00%（88/100），结果一致性较好。结论：本研究制备了雌酮硫酸钠免疫原，融合制得抗雌酮硫酸钠特异性单克隆抗体，并研制出以雌酮硫酸钠单克隆抗体为基础的胶体金免疫层析试纸条，能够快速、灵敏地检测孕马尿中的雌酮硫酸钠。

Objective: To establish a method for rapid detection of sodium estrone sulfate in pregnant mare's urine by colloidal gold immunochromatographic test strip. Methods: Use chemical synthesis method to prepare haptene from oestrone. Apply mixed anhydride method to prepare ESS-17-oxime-BSA from ESS-17-oxime and BSA. Use UV scanning to authenticate and calculate coupling rate. BALB/C mice were immunized with ESS-17-oxime-BSA. Get high serum titer mice splenic lymphocytes and SP2/0 cells to mix them to prepare hybridoma cells. It positive clones were screened by ELISA method. After obtaining positive clone cell lines, induce ascites in mice's body to prepare monoclonal antibody and labeled with colloidal gold. Confirm the best experimental parameter, preparing the sodium estrone sulfate colloidal gold immunochromatographic test strip. Results: The target antigen ESS-17-oxime-BSA modified chemical structurally was obtained. After mixing and clone, obtaining 4 lines stable antibody secreting hybridoma cell lines 2D1,7G4, 5F3, 2A7. The purified 2D1 antibody's specificity is high, titer was 1×10⁶. The detection limit of colloidal gold chromatography test paper prepared by 2D1 antibody was 1 μg·L^-1. There was no cross reaction with structural analogues. The results were compared with that determined by high performance liquid chromatography. The positive coincidence rate was 85.45%(45/52), the negative coincidence rate was 89.58%(43/48) and the total coincidence rate was 88.00%(88/100). The consistency of the result is good. Conclusion: Estrone sulfate sodium's immunogen was synthesized, the anti-estrone sodium sulfate specificity monoclonal antibody was prepared and developed. The colloidal gold immunochromatographic test strip can be used to detect sodium sulfate rapidly and sensitive in pregnant mare' surineestrone.

[1] RIŽNER TL. The Important roles of steroid sulfatase and sulfotransferases in gynecological diseases[J]. Front Pharmacol, 2016, 7(2):30
[2] LABRIE F,MARTEL C. A low dose (6.5 mg) of intravaginal DHEA permits a strictly local action while maintaining all serum estrogens or androgens as well as their metabolites within normal values[J]. Horm Mol Biol Clin Investig,2017, 29(2):39
[3] REZVANPOU A, DON-WAUCHOPE AC. Clinical implications of estrone sulfate measurement in laboratory medicine[J]. Crit Rev Clin Lab Sci,2017, 54(2):73
[4] GEYER J, BAKHAUS K, BERNHARDT R, et al. The role of sulfated steroid hormones in reproductive processes[J]. J Steroid Biochem Mol Biol,2017, 172(9):207
[5] HIGUCHI T, ENDO M, HANAMURA T, et al. Contribution of estrone sulfate to cell proliferation in aromatase inhibitor(AI) -resistant,hormone receptor-positive breast cancer[J]. PLoS One, 2016, 11(5):26
[6] KOYAMA AK,TWOROGER SS,ELIASSEN AH,et al. Endogenous sex hormones and cognitive function in older women[J]. Alzheimers Dement,2016, 12(7):758
[7] HOSOGI J, TANAKA H, FUJITA K, et al. LC-MS/MS coupled with immunoaffinity extraction for determination of estrone,17 betaestradiol and estrone 3-sulfate in human plasma[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2010, 878(2):222
[8] SPEARS KJ, ROSS J, STENHOUSE A, et al. Directional transepithelial transport of organic anions in porcine LLC-PK1 cells that co-express human OATP1B1(OATP-C) and MRP2[J]. Biochem Pharmacol,2005, 69(3):415
[9] O'CONNOR KA,BRINDLE E,HOLMAN DJ,et al. Urinaryestrone conjugate and pregnanediol 3-glucuronide enzyme immunoassays for population research[J]. Clin Chem,2003, 49(7):1139
[10] 秦立得,南文龙,陈义平.胶体金免疫层析试纸条在禽病检测方面的应用与展望[J].中国动物检疫,2016, 33(8):78. QIN LD, NAN WL, CHEN YP. The application and expectation of using immunochromatographic strip in detection of avian diseases[J]. China Anim Quarant,2016, 33(8):78
[11] 施海燕,盛恩泽,马明,等.氯噻啉胶体金增强免疫层析分析方法的建立[J].分析化学,2017, 45(3):403. SHI HY, SHENG EZ, MA M, et al. Development of an enhanced colloidal gold immunochromatographic assay for detection of imidaclothiz[J]. Chin J Anal Chem,2017, 45(3):403
[12] 曹德康,苏建忠,张瑛,等.胶体金免疫层析技术在食品安全检测中的应用研究进展[J].解放军预防医学杂志, 2017(10):1322 CAO DK, SU JZ, ZHANG Y, et al. Research progress in colloidal gold immune chromatographic techniques in food safety detection[J]. J Prev Med Chin PLA,2017(10):1322
[13] HATZIDAKIS G, KATRAKILI K, KRAMBOVITIS E. Development of a direct and specific enzymeimmunoassay for the measurement of oestrone sulfate in bovine milk[J]. J Reprod Fertil,1993,98(1):235
[14] MORTAUD S, DONSEZ-DARCEL E, ROUBERTOUX PL, et al. Murine steroid sulfatase(mSTS):purification, characterization and measurement by ELISA[J]. J Steroid Biochem Mol Biol, 1995, 52(1):91
[15] HIRAKO M, TAKAHASHI H, DOMEKI I. Establishment of a reliable extraction method of estrone sulfate from bovine plasma for detection of the peripheral level during the regular estrous cycle by radioimmunoassay[J]. Domest Anim Endocrinol, 1996, 13(3):187