人生长激素的生物素-亲和素放大酶联免疫法的建立

Establishment of biotin avdin magnify enzyme linked immunosorbent assay for human growth hormone in serum

为建立有生物素和亲和素放大系统的人血清生长激素 (hGH)酶联免疫分析法 (BA ELISA) ,以微量的GH抗原免疫BALB/C小鼠 ,利用杂交瘤技术制备出 2 9株分泌抗hGH单克隆抗体 (MCA)的杂交瘤株。选择最佳配对MCA ,利用亲和素与生物素高亲和力的特性 ,建立了血清hGH双位点的BA ELISA法。我们的MCA滴度为 (0 5～ 5 0 0 )× 10 4,亲和常数Ka =(0 13～ 1 40 )× 10 10 L/mol。血清hGH的ELISA的灵敏度为 0 0 4± 0 0 1μg/L ;在血清中分别加入 1,2 5 ,5 μg/L的hGH ,其回收率为 90 7%～ 10 7 6 % (平均为 10 1 2 0 %± 0 0 3 % ) ;hGH含量为 2 8、10 0、2 9 8μg/L的血清批内、批间变异系数分别为 4 9%、3 8%、7 9%和 6 2 %、3 5 %、9 5 % ;用本法测定了正常儿童、生长激素缺乏症 (GHD)患儿和活动性肢端肥大症患者空腹血清hGH水平 ,分别为 1 87± 3 0、0 30± 0 42和 8 71± 6 95 μg/L。上述结果表明 ,本ELISA方法灵敏、特异、稳定、准确 ,能确切反映hGH分泌功能 ,在诸多方面均优于RIA。

To establish the biotin avidin enzyme linked immunosorbent assay for measuring human Growth hormone Method: After immunizing BALB/C mice with micro dosage hGH,we syncretized spleen cell of BALB/C mice immunized and murine myeloma cell ,and got 29 strain hybridized cell lines which could secrete anti hGH monoclonal antibodies(MCA) We selected the best matched two kind of MCA, one as solid phase antibody after particular appraising the characteristic of MCA ,The other one as biotin labeled antibody, to establish BA ELISA of serum hGH Results: The titer and affinity of the MCA were (0 5～50 0)×10 4 and(0 13～1 40)×10 10 L/mol, respectively With a detection limit of the method was 0 04μg/L, the hGH level in the range of 0 02～100 μg/L can be measured by this method Specificity is high: the cross reaction rate with hPRL was just 0 29% Accurate: After adding 1,2 5 and 5μg/L hGH into serum, the recovery rate was 90 7%～107 6 %, by the average of 101 2 % Precision: When hGH level were 2 8 ,10 0 and 29 81μg/L, the coefficients of intra assay and inter assay variations were 4 9%, 3 8%,7 9% and 6 2%,3 5%, 9 5%, respectively Validity: After gradient dilution of high concentration of hGH, the values measured by this method were significantly close and correlated with expected value(r= 0 9960, P <0 001), the curve of dilution was paralleled to the curve of hGH standard The values of 61 samples that maesured simultaneously by this method and radioimmunoassay (RIA) , showed a good correlation (r=0 9681, P <0 01) Serum level of hGH of 10 normal children , 8 GH deficiency children, and 11 active acromegaly patients were 1 87±3 0、0 30±0 42和8 71±6 95μg/L, respectively Conclusion: Our results indicate that the BA ELISA assay is sensitive, specific, stable, valid, and is better than RIA in kinds of aspects