Syphilis Division of the Department of Medicine, Johns Hopkins Medical School, Baltimore.
J Exp Med. 1930 Oct 31;52(5):747-68. doi: 10.1084/jem.52.5.747.
When cholesterinized antigen is dropped into an excess of water, the rapid flocculation of cholesterin crystals is prevented by the fact that, as tiny aggregates form, they adsorb a protective surface of hydrophilic lecithin (i.e., antigen) which endows the particles with its own stable surface properties and thus prevents further aggregation. The colloidally dispersed antigen-cholesterin particles have approximately the same isoelectric point (pH 1.9), critical potential (1 to 5 millivolts) and coagulation value (0.75 M NaCl) as pure antigen particles of the same concentration, while the corresponding values for cholesterin are pH 2.1 to 3.4 (probably due to an associated impurity), >100 millivolts, and <0.001 N NaCl, respectively. Presumably, this adsorption of antigen by the cholesterin nucleus is determined by the fact that the former has a lower surface tension against water. At any rate, many surface active substances (serum; alcoholic extract of milk, egg or any animal tissue; Na-oleate; Na-glycocholeate; Na-taurocholate) cause a similar stable dispersion of cholesterin; and conversely, many otherwise water-insoluble substances of the most diverse chemical structure can be made to form a stable colloidal suspension by adding antigen to their alcoholic solutions before dropping into water. The colloidal suspension formed by antigen alone is very finely dispersed: only a few of the particles exceed the limits of dark field visibility. Cholesterin causes a marked increase in the number of these particles, out of all proportion to its mass; thus, one part of cholesterin to five of antigen causes a ten-fold increase in such visible particles, at the expense of the submicroscopic micellae formed by antigen alone. At the same time, the suspension becomes much more turbid. The particles remain discrete until the cholesterin: antigen ratio exceeds 1:1, when slight microscopic aggregation is observed; microscopic flocculation is seen only when this ratio exceeds 5:1, when there is not sufficient antigen to act as an efficient protective colloid. Cholesterin therefore causes a coarsened dispersion of antigen by forming a relatively large nucleus upon which antigen is adsorbed. As shown in the text, the larger the antigen particle the greater is its avidity for reagin per unit surface or mass. Thus, the coarse sol formed by dropping water-into-antigen is about twice as efficient as a finely dispersed antigen-into-water sol of the same concentration. The coarsened dispersion caused by cholesterin completely explains the greater sensitivity of the cholesterinized antigen in complement fixation. The same factor obtains in the flocculation reactions. In addition, the coarsened dispersion acts as a preliminary quasi-aggregation, facilitating by just so much the subsequent formation of visible clumps (or sedimenting aggregates) upon the addition of syphilitic serum; moreover, there is less surface in a coarse sol, with more reagin per unit surface, and correspondingly more efficient flocculation. The foregoing would be of purely academic interest were it not for the following considerations. From several points of view cholesterin is unsatisfactory as a sensitizer for antigen. Its solubility in alcohol is small. Even the 0.6 per cent concentration used in the Kahn test is difficult to keep in solution. Yet, as our experiments show, its sensitizing action increases indefinitely with its concentration. If it were sufficiently soluble, even 3 per cent could be used to advantage, increasing the sensitivity of 1(1/2) per cent antigen for complement fixation some 200 to 400 per cent, instead of about 50 per cent, as does 0.2 per cent cholesterin. Since, as we have shown, the sensitizing action of cholesterin upon antigen is due solely to the coarse dispersion it causes, and since it is quite inert during the actual combination of the lipoid particles with reagin, it can be replaced by any substance with similar physical properties. The problem in hand was therefore to find a water-insoluble substance, very soluble in alcohol, with so high an interfacial tension against water that, as in the case of cholesterin, microscopic particles would adsorb antigen when the alcoholic solution of the two is dropped into water. Given such a substance, it would be possible to obtain a more sensitive antigen for both complement fixation and flocculation, but particularly for the former. These theoretical expectations have been realized in a group of substances shortly to be reported: they make possible an antigen which is from 2 to 10 times as efficient in the Wassermann test as any now available.
当胆固醇化抗原被投入过量的水中时，由于微小的聚集物形成时，它们会吸附一层保护性的亲水性卵磷脂（即抗原）表面，从而赋予颗粒自身稳定的表面特性，从而防止进一步聚集，因此可以防止胆固醇晶体的快速絮凝。胶体分散的抗原-胆固醇颗粒具有与相同浓度的纯抗原颗粒大致相同的等电点（pH1.9）、临界电位（1 至 5 毫伏）和凝固值（0.75MNaCl），而胆固醇的相应值为 pH2.1 至 3.4（可能是由于相关杂质）、>100 毫伏和<0.001NNaCl。推测，这种胆固醇核对抗原的吸附是由前者对水的表面张力较低决定的。无论如何，许多表面活性剂（血清；牛奶、鸡蛋或任何动物组织的酒精提取物；Na-油酸酯；Na-甘胆酸盐；Na-牛磺胆酸盐）都会导致胆固醇的类似稳定分散；相反，许多其他水溶性差的化学结构的物质可以通过在滴入水之前向其酒精溶液中添加抗原来形成稳定的胶体悬浮液。仅由抗原形成的胶体悬浮液非常细分散：只有少数颗粒超过暗场可见性的限制。胆固醇会导致可见颗粒的数量显著增加，不成比例地增加其质量；因此，胆固醇与抗原的比例为 1:5 会使这种可见颗粒增加十倍，而牺牲由抗原单独形成的亚微观胶束。同时，悬浮液变得更加浑浊。只要胆固醇与抗原的比例不超过 1:1，颗粒就保持离散，此时会观察到轻微的微观聚集；当该比例超过 5:1 时，会看到微观絮凝，因为没有足够的抗原作为有效的保护性胶体。因此，胆固醇通过形成相对较大的核来使抗原的分散变粗糙，在其上吸附抗原。如文本所示，抗原颗粒越大，其每单位表面或质量对反应原的亲和力就越大。因此，与相同浓度的精细分散的抗原入水溶胶相比，由入水形成的粗溶胶大约有效两倍。胆固醇引起的粗分散完全解释了胆固醇化抗原在补体固定中的更高敏感性。在絮凝反应中也是如此。此外，粗分散作为初步的准聚集，通过这种方式稍微促进了随后在添加梅毒血清时形成可见团块（或沉降的聚集体）的形成；此外，在粗溶胶中，单位表面的表面积更小，具有更多的反应原，相应地更有效地絮凝。如果不是因为以下考虑，这将纯粹是学术上的兴趣。从几个角度来看，胆固醇作为抗原的敏化剂并不令人满意。它在酒精中的溶解度很小。即使在 Kahn 测试中使用的 0.6%浓度也难以保持溶解状态。然而，正如我们的实验所示，其敏化作用会随着浓度的增加而无限增加。如果它足够溶解，即使使用 3%也可以带来好处，使 1(1/2)%抗原的补体固定敏感性提高约 200%至 400%，而不是像 0.2%胆固醇那样提高约 50%。由于正如我们已经表明的，胆固醇对抗原的敏化作用仅归因于它引起的粗分散，并且由于在脂质颗粒与反应原实际结合期间它是完全惰性的，因此可以用具有类似物理性质的任何物质代替。手头的问题是找到一种不溶于水但非常溶于酒精的物质，其与水的界面张力非常高，以至于当两种物质的酒精溶液滴入水时，微小颗粒会吸附抗原。有了这样的物质，就有可能获得一种更敏感的抗原，既用于补体固定，也用于絮凝，但特别是用于前者。这些理论预期已经在即将报告的一组物质中得到实现：它们使能够获得比任何现有物质在 Wassermann 测试中效率高 2 至 10 倍的抗原。
