Chen S, Sawicka J, Prentice L, Sanders J F, Tanaka H, Petersen V, Betterle C, Volpato M, Roberts S, Powell M, Smith B R, Furmaniak J
FIRS Laboratories, RSR Limited, Cardiff, Wales, United Kingdom.
J Clin Endocrinol Metab. 1998 Aug;83(8):2977-86. doi: 10.1210/jcem.83.8.5010.
A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.
制备、鉴定了一组针对人重组类固醇21-羟化酶（21-OH）的五种小鼠单克隆抗体（MAb），并用于研究21-OH自身抗体（AAb）与人类21-OH上不同表位的相互作用。对孤立性自身免疫性艾迪生病、Ⅰ型和Ⅱ型自身免疫性多腺体综合征患者以及无明显艾迪生病的21-OH抗体阳性患者（共25例）的AAb进行了研究。四种MAb为IgG1亚类，一种为IgG2a，均具有κ轻链。其中四种抗体的亲和力在2.0×10⁸M⁻¹至7.0×10⁸M⁻¹范围内，另一种的亲和力为2.3×10⁷M⁻¹。21-OH MAb与17α-羟化酶（17α-OH）或P450侧链裂解酶无交叉反应。使用一系列21-OH片段进行的研究能够鉴定出参与形成MAb结合位点的短氨基酸（AA）序列。发现定义为表位区域（ER）1的AA 391 - 405对M21-OH1和M21-OH2的结合很重要，AA 406 - 411（ER2）对M21-OH3和M21-OH4的结合很重要，AA 335 - 339（ER3）对M21-OH5的结合很重要。此外，在竞争实验中使用MAb Fab或F(ab')₂片段来研究21-OH AAb表位。这些研究表明，21-OH AAb识别的表位与MAb相似，ER2和ER3是两个不同主要表位的一部分，ER1是一个次要表位的一部分。M21-OH抗体Fab或F(ab')₂片段的混合物在25份血清中的24份中几乎完全抑制（80% - 95%）AAb结合，对于其余一份血清，抑制作用明显但不完全（67%抑制）。不同形式自身免疫性肾上腺疾病患者的AAb结合特征之间没有重大差异。在免疫荧光试验中，所有五种21-OH MAb均与人肾上腺组织反应，但在这些实验中只有M21-OH1和M21-OH2与牛肾上腺组织反应。在免疫荧光试验中，没有一种MAb与人类卵巢组织反应。总体而言，这些研究表明，21-OH AAb与21-OH C末端部分的至少三个不同表位结合，其中两个表位似乎是人类21-OH特有的。
