人绒毛膜促性腺激素游离β亚单位磁分离酶联免疫方法的建立

目的建立一种新的测定人绒毛膜促性腺激素游离β亚单位(hCGβ)磁分离酶联免疫分析法(MEIA)。方法用识别不同表位的两株单克隆抗体(mAb),一株mAb用FITC标记,另一株用碱性磷酸酶(AP)标记;以偶联羊抗FITC抗体的磁珠作为固相,以酚酞单磷酸酯溶液为底物,建立测定hCGβ磁分离酶免疫测定法。结果本MEIA法的灵敏度为0.1IU/L,批内CV小于8.5%,批间CV小于14%,平均稀释回收率为92.5%;与促黄体生成激素(leuteinizinghormone,LH)、促甲状腺激素(thyroidstimulatinghormone,TSH)和促卵泡成熟激素(folliclestimulatinghormone,FSH)均无交叉反应,与完整的hCG(1000IU/L)的交叉反应为1.2%,有效期不低于14个月。结论本方法的性能优于现有的放射免疫(RIA)和ELISA试剂盒,接近进口同类试剂的水平,有助于为市场提供一种高质量而价廉的hCGβ测定试剂盒。

Methodological study on magnetic enzymic immunoassay for detecting free hCGβ subunit

AIM: To establish a novel magnetic enzyme immunoassay (MEIA) for detecting human choriogonadotropin free beta subunit (hCGbeta). METHODS: Two monoclonal antibodies (mAb) were used for conjugating with FITC and with alkaline phosphatase(AP) respectively, which incorporated magnetic solid phase separation. Magnetic beads were coupled with sheep anti-FITC antibody as solid phase, and phenolphthalein monophosphate was used as substrate to set up MEIA for detecting hCGbeta. RESULTS: The sensitivity of hCGbeta MEIA kit reached 0.1 IU/L. The intraassay variation and inter-assay variation was 8.5% and 14% respectively, with the average recovery rate of dilution of 92.5%. The kit showed no cross reactivity to LH, TSH and FSH, while a cross reactivity of 1.2% to intact hCG at concentration of 1000 IU/L. The time of efficacy of hCGbeta MEIA kit was longer than 14 months. CONCLUSION: hCGbeta MEIA kit is better than hCGbeta radio immunoassay and ELISA kit, which can provide a high-qualitative and cheap hCGbeta kit for market.