Sawada M, Inagawa T, Frölich J C
Prostaglandins. 1985 Jun;29(6):1039-48. doi: 10.1016/0090-6980(85)90227-8.
A highly sensitive and reproducible enzyme-immunoassay for the measurement of thromboxane B2 was developed. Thromboxane B2 (TxB2) was coupled with beta-D-galactosidase by mixed anhydride reaction. Thromboxane B2-antiserum was generated in rabbits and used at a final dilution of 1:480,000. The separation of immunocomplex from the free form of TxB2 was accomplished by the double antibody method. The second antibody was sheep anti rabbit IgG. The precipitated enzyme activity was measured fluorometrically with 4-methyl-umbelliferyl-beta-D-galactoside as substrate. This method allowed to measure TxB2 in the range of 0.002-5 picomole per tube. The cross-reactivity of the anti-thromboxane B2-antiserum with 2,3-dinor thromboxane B2 was about 20%, but it was less than 0.2% for the other prostanoids tested. TxB2 extracted from human urine was measured by enzyme-immunoassay (y) and radioimmunoassay (x) which has been found closely correlated to values obtained by gas chromatography-mass spectrometry. Regression analysis of the data comparing enzyme-immunoassay and radioimmunoassay gave the equation y = 0.996 x + 0.470, correlation coefficient r = 0.9947. Inter-assay coefficient of variation was 3.1%. The assay was further simplified by coating the second antibody on glass beads. The regression equation between this solid-phase enzyme immunoassay (y) and radioimmunoassay (x) was y = 0.9860 X 1.927, r = 0.9895, and enzyme immunoassay (y) was y = 0.9749 X -0.94808, r = 0.9887. Thus, the enzyme-immunoassay shows specificity and sensitivity comparable to radioimmunoassay making use of radioactive tracer unnecessary.
已开发出一种用于测量血栓素B2的高灵敏度且可重复的酶免疫测定法。血栓素B2（TxB2）通过混合酸酐反应与β-D-半乳糖苷酶偶联。在兔体内产生了血栓素B2抗血清，并以1:480,000的最终稀释度使用。通过双抗体法实现免疫复合物与游离形式的TxB2的分离。第二抗体是羊抗兔IgG。以4-甲基伞形酮基-β-D-半乳糖苷为底物，通过荧光法测量沉淀的酶活性。该方法能够测量每管0.002 - 5皮摩尔范围内的TxB2。抗血栓素B2抗血清与2,3-二去甲血栓素B2的交叉反应性约为20%，但对于所测试的其他前列腺素，交叉反应性小于0.2%。通过酶免疫测定法（y）和放射免疫测定法（x）测量从人尿中提取的TxB2，发现其与气相色谱-质谱法获得的值密切相关。比较酶免疫测定法和放射免疫测定法的数据的回归分析得出方程y = 0.996x + 0.470，相关系数r = 0.9947。批间变异系数为3.1%。通过将第二抗体包被在玻璃珠上，该测定法进一步简化。这种固相酶免疫测定法（y）与放射免疫测定法（x）之间的回归方程为y = 0.9860x + 1.927，r = 0.9895，而酶免疫测定法（y）为y = 0.9749x - 0.94808，r = 0.9887。因此，酶免疫测定法显示出与放射免疫测定法相当的特异性和灵敏度，无需使用放射性示踪剂。
