Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders
Adsorption-induced antigenic changes and their significance in ELISA and immunological disorders
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DOI：
10.3109/08820139709048914
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发表时间：
1997-01-01
影响因子：
2.8
通讯作者：
Sun, J
中科院分区：
文献类型：
--
作者：
Butler, JE;Navarro, P;Sun, J
比较了吸附在聚苯乙烯和硅胶上的I-125标记抗体和抗原与非吸附方法固定的抗体和抗原的功能特性。不到20%的多克隆(Pab)和1-2%的单抗捕获抗体等效物在吸附后作为单层保留了功能。当相同的抗体分别通过链霉亲和素桥固定或使用第一阶段多克隆抗球蛋白捕获抗体固定时，存活率大约增加一倍或完全挽救。同样，当免疫球蛋白通过链霉亲和素桥固定或二次吸附在白蛋白单层上时，牛免疫球蛋白对Pab和mAb抗IgS的抗原性最高。这些构型的免疫球蛋白明显比直接吸附在聚苯乙烯或硅橡胶上时具有更强的抗原性。在聚苯乙烯或硅胶上吸附后，也观察到类似的活性。有趣的是，这些免疫球蛋白在变性后在6M盐酸胍中的吸附与在PBS中的吸附没有预先变性时具有同样的抗原性。虽然上述关于抗体和抗原的许多发现可以解释为当分子被某种类型的底层分子固定时，抗原表位或抗体结合部位更容易获得，但我们也表明，当IgG2a被吸附时，某些mAb和PAb优先识别IgG2a上的同种异体。此外，当IgG2a被吸附在较低的亚单层浓度时，这种抗原性最高。最后，我们发现抗原性的差异不需要与固定化方法有关，但也可能是表位微环境的差异。这是用一种针对荧光素(Flu)的丝状噬菌体克隆来证明的。根据所使用的载体蛋白和连接方法的不同，该克隆识别荧光素半抗原的方式也不同。本报告提供的数据表明，吸附在疏水聚合物上的抗体和抗原发生了功能性质的变化。数据表明，最有可能发生的是抗原表位或抗体结合部位的构象和可及性的变化。尤其是在后者的情况下，功能浓度可能比抗体蛋白浓度低1-2个数量级。这些观察结果对免疫诊断学有影响，并强调需要在使用抗体的分析中确定抗体的特异性，并且不能根据固相抗原和抗体在溶液中的行为来假设它们的行为。我们的研究也与硅胶医疗假体的使用有关。以多层(第二层)形式吸附在硅胶表面的免疫球蛋白的抗原性远高于直接吸附的。由于这样的表面会暴露在体内非常高的蛋白质浓度下，因此可以预期的是多层而不是单层。因此，从这些研究来看，吸附在硅胶上的宿主蛋白似乎会在多层膜的表面表达给免疫系统。在这种情况下，宿主蛋白在体内的吸附似乎不太可能产生新的表位，宿主的免疫系统可以对这些表位做出反应，从而引发自身免疫综合症。
The functional properties of I-125-labeled antibodies and antigens adsorbed on polystyrene and silicone were compared to their counterparts immobilized by non-adsorptive methods. Less than 20% of polyclonal (pAb) and 1-2% of monoclonal (mAb) capture antibody equivalents remained functional after adsorption as a monolayer. Survivability circa doubled or was totally rescued, when the same antibodies were immobilized via a streptavidin bridge or by using a first stage polyclonal antiglobulin capture antibody, respectively. Similarly, the antigenicity of bovine IgGs for pAb and mAb anti-IgGs was highest when the IgGs were immobilized via a streptavidin bridge or when secondarily adsorbed to an albumin monolayer. IgGs in these configurations were significantly more antigenic than when directly adsorbed on polystyrene or a silicone elastomer. Similar activity was seen after adsorption on polystyrene or silicone. Interestingly, these IgGs were equally antigenic when denatured and subsequently adsorbed in 6M guanidine-HCl Versus adsorption in PBS without prior denaturation. Although many of the above finding on antibodies and antigens could be explained by the greater accessibility of antigenic epitopes or antibody binding sites when molecules are immobilized by some type of underlying molecular layer, we also show that certain mAb and pAbs preferentially recognized allotopes on IgG2a when IgG2a was adsorbed. Furthermore, such antigenicity was highest when IgG2a was adsorbed at low, sub-monolayer concentrations.Finally, we show that differences in antigenicity need not be related to the method of immobilization, but can also result from differences in the microenvironment of the epitope. This was demonstrated using a filamentous phage clone specific for fluorescein (FLU). This clone recognizes the fluorescein hapten differently depending on the carrier protein used and the method of conjugation.Data presented in this report indicate that antibodies and antigens adsorbed on hydrophobic polymers undergo changes in their functional properties. Data suggest that both changes in conformation and the accessibility of antigen epitopes or antibody binding sites, most likely occur. Especially in the case of the latter, the functional concentration may be 1-2 orders of magnitude lower than the antibody protein concentration. These observations have implications for immunodiagnostics and emphasize the need to determine the specificity of an antibody in the assay in which it is employed and to make no assumptions about the behavior of solid-phase antigens and antibodies from their behavior in solution.Our studies are also relevant to the use of silicone medical prostheses. The antigenicity of IgGs adsorbed on silicone as a multilayer (secondary layer) is much higher than when directly adsorbed. Since such surfaces would be exposed to very high protein concentrations in vivo, multilayers not a monolayer, would be expected. Thus it would seem from these studies that host protein adsorbed on silicone would be expressed to the immune system at the surface of multilayers. This being the case, it seems unlikely that the adsorption of host protein in vivo would generate new epitopes against which the host's immune system could respond and subsequently initiate an autoimmune syndrome.