双向免疫扩散法

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双向免疫扩散法是利用琼脂凝胶为介质的一种沉淀反应。即可溶性抗原与相应抗体在琼脂介质中相互扩散，彼此相遇，在比例适当处形成沉淀线。根据沉淀线的有无、形状和位置对抗原或抗体进行定性分析，也可用于抗体的半定量检测。

将可溶性抗原(如小牛血清)与相应抗体(如兔抗小牛血清的抗体)混合，当两者比例合适并有电解质(如氯化钠、磷酸盐等)存在时，即有抗原―抗体复合物的沉淀出现，此为沉淀反应(precipitin reaction)。如以琼脂凝胶为支持介质，则在凝胶中出现可见的沉淀线、沉淀弧或沉淀峰。根据沉淀出现与否及沉淀量的多寡，可定性、定量地检测出样品中抗原或抗体的存在及含量。免疫学的一些测定方法即基于此特性。

双向扩散法(double diffusion)，最早由Ouchterlony创立，故又称Ouchterlony法。琼脂凝胶是多孔的网状结构，大分子物质可以自由通过，这种分子的扩散作用使分别两处的抗原和相应抗体相遇，形成抗原―抗体复合物，比例合适时出现沉淀。由于凝胶透明度高，可直接观察到复合物的沉淀线(弧)。沉淀线(弧)的特征与位置取决于抗原相对分子质量的大小、分子结构、扩散系数和浓度等因素。当抗原、抗体存在多种系统时，会出现多条沉淀线(弧)。依据沉淀线(弧)可以定性抗原。

此法操作简便、灵敏度高，是最为常用的免疫学测定抗原和测定抗血清效价的方法(见图8-4)。

图8-4 免疫双向扩散法

Fig8-4 In immuno-double-diffusion, agar gels are poured onto slides and allowed to set, wells are then punched in the gel and the test solutions of antigen(Ag) and antibody(Ab) are added. The solutions diffuse out and where Ag and Ab meet they bind to each other, cross-link and precipitate leaving a line of precipitation. This technique may be used to determine the relationship between antigens and a particular test antibody. Three basic patterns appear. In reaction(a) the precipitin arcs formed between the antibody and the two test antigen fuse, indicating that the antibody is precipitating identical epitopes in each preparation(epitope 1). This does not mean that the antigens are necessarily identical, they are only identical in as far as the antibody cannot distinguish a difference. In reaction(b) the antibody preparation distinguishes the three different antigens, which form independent precipitin arcs. In reaction(c) the antigens share epitope 1 but one antigen also has epitope 2. This is the same situation as in (a), but in this case the antibody can distinguish them, by virtue of being able to react against both epitopes. A line of identity forms with anti-epitope 1, with the addition of a ‘spur’ where the anti-epitope 2 has reacted with the second epitope, thus indicating partial rather than total identity between the antigen preparations.