Hodgkinson S C, Allolio B, Landon J, Lowry P J
Biochem J. 1984 Mar 15;218(3):703-11. doi: 10.1042/bj2180703.
The development of a 'two-site' immunoradiometric assay (i.r.m.a.) for the direct estimation of human corticotropin-(1-39)-peptide in plasma is described. The assay is based on the simultaneous addition of 125I-labelled sheep anti-(N-terminal corticotropin) IgG (immunoglobulin G) antibodies and rabbit anti-(C-terminal corticotropin) antiserum to standards and unknowns (0.5 ml) followed by 18h incubation. The use of solid-phase reagents was avoided in order to minimize non-specific effects and the time required for reactants to reach equilibrium. Instead, the separation of corticotropin-bound from free labelled antibody is achieved by the addition of sheep anti-(rabbit IgG) antiserum, which precipitates bound labelled antibody by complex-formation with rabbit anti-corticotropin antibodies, which are also hormone-bound. Several 125I-labelled sheep anti-(N-terminal corticotropin) IgG preparations were assessed in the i.r.m.a. Although each was derived from antisera raised to a thyroglobulin conjugate of synthetic corticotropin-(1-24)-peptide (Synacthen), purification of immunoglobulins before iodination by selective immunoadsorption resulted in preparations with distinct specificities which demonstrated marked differences in binding to intact human corticotropin-(1-39)-peptide. These preparations are compared in combination with two rabbit anti-(C-terminal corticotropin) antisera. A 'two-site' assay based on the use of 125I-labelled sheep anti-[ corticotropin-(2-16)-peptide] IgG and rabbit anti-[corticotropin-(34-39)-peptide] antiserum was optimized, since steric inhibition of antibody binding was avoided with this combination and because the measurement of only intact human corticotropin-(1-39)-peptide and not fragments was assured by the use of terminal antibodies. This i.r.m.a. is characterized by rapid equilibration of reactants, a wide 'operating range' (the precision of dose estimates was less than 4% over the range 30-2200 pg/ml) and high sensitivity [8 pg of corticotropin/ml (95% confidence interval 3.7-12.0) (4 pg minimal detectable mass) can be detected directly in plasma].
本文描述了一种用于直接测定血浆中人促肾上腺皮质激素-（1-39）肽的“双位点”免疫放射分析（i.r.m.a.）的开发。该分析基于同时向标准品和未知样品（0.5ml）中加入125I标记的羊抗-（促肾上腺皮质激素N端）IgG（免疫球蛋白G）抗体和兔抗-（促肾上腺皮质激素C端）抗血清，然后孵育18小时。为了尽量减少非特异性效应和反应物达到平衡所需的时间，避免使用固相试剂。相反，通过加入羊抗-（兔IgG）抗血清来实现结合促肾上腺皮质激素的标记抗体与游离标记抗体的分离，该抗血清通过与也与激素结合的兔抗促肾上腺皮质激素抗体形成复合物来沉淀结合的标记抗体。在i.r.m.a.中评估了几种125I标记的羊抗-（促肾上腺皮质激素N端）IgG制剂。尽管每种制剂都来自针对合成促肾上腺皮质激素-（1-24）肽（赛诺龙）的甲状腺球蛋白偶联物产生的抗血清，但在碘化前通过选择性免疫吸附对免疫球蛋白进行纯化，得到了具有不同特异性的制剂，这些制剂在与完整的人促肾上腺皮质激素-（1-39）肽结合方面表现出明显差异。将这些制剂与两种兔抗-（促肾上腺皮质激素C端）抗血清进行了比较。基于使用125I标记的羊抗-[促肾上腺皮质激素-（2-16）肽]IgG和兔抗-[促肾上腺皮质激素-（34-39）肽]抗血清的“双位点”分析得到了优化，因为这种组合避免了抗体结合的空间位阻抑制，并且通过使用末端抗体确保了仅测量完整的人促肾上腺皮质激素-（1-39）肽而不是片段。这种i.r.m.a.的特点是反应物快速平衡、“操作范围”宽（在30-2200pg/ml范围内剂量估计的精密度小于4%）和灵敏度高[血浆中可直接检测到8pg促肾上腺皮质激素/ml（95%置信区间3.7-12.0）（最小可检测质量为4pg）]。
