VARIATION IN AFFINITIES OF ANTIBODIES DURING IMMUNE RESPONSE
VARIATION IN AFFINITIES OF ANTIBODIES DURING IMMUNE RESPONSE
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DOI：
10.1021/bi00895a027
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发表时间：
1964-01-01
期刊：
影响因子：
2.9
通讯作者：
SISKIND, GW
中科院分区：
文献类型：
--
作者：
EISEN, HN;SISKIND, GW
用荧光猝灭法测定了抗2，4-二硝基苯（DNP）抗体对多种2，4-二硝基苯的亲和力，并在代表性实例中用平衡透析法验证了所得值。所有人口的抗DNP分子检查，无论是从合并的血清或从单个bleedles的个别兔分离，是异质性方面的亲和力二硝基苯。几乎每种情况下的异质性都可以通过Sips分布函数在广泛的结合数据范围内进行描述，因此，每次滴定均通过平均内在缔合常数（Ko）和异质性指数（a）进行表征。通过加入有限量的抗原，从血清中分级沉淀抗体，从单个兔的血清中产生抗DNP群体，其Ko差异高达10，000倍。在单次注射DNP-牛-γ-球蛋白后，通过在不同时间从血清中分离抗体，跟踪[α]-DNP-赖氨酸亲和力的变化。Ko在免疫后随时间进行性增加，当最初注射少量DNP-牛-γ-球蛋白时，增加的速率最明显。当注射大剂量抗原时，Ko的上升明显延迟。免疫后早期分离的抗体群体对[N]-DNP-L-赖氨酸和2，4-二硝基苯胺具有几乎相同的低亲和力，并且推断它们的特异性结合位点对二硝基苯胺基的适应性差，并且对[N]-DNP-L-赖氨酸的正亮氨酸部分不敏感。相比之下，在免疫后较晚分离的抗体强烈结合[N]-DNP-L-赖氨酸，并且从它们与各种二硝基苯的相互作用推断，它们的结合位点平均而言刚好大到足以容纳[N]-DNP-L-赖氨酸。复合物形成是焓驱动的：对于不同的抗体-配体对，[DELTA]Ho值的范围为[长划线]8至[长划线]20 kcal mol-1，[DELTA]So值的范围为[长划线]5至[长划线]30熵单位mol-1。当代表性的2，4-二硝基苯与抗DNP分子结合时，观察到红移和减色光谱位移;对于抗体结合的2，4-二硝基甲苯，在300 m[mu]处出现新的吸收峰。提出了电荷转移有助于抗体-二硝基苯基复合物稳定性的可能性。
The affinities of anti-2,4-dinitrophenyl (DNP) antibodies for a variety of 2,4-dinitrobenzenes have been measured by fluorescence quenching and the values obtained have been verified in representative instances by equilibrium dialysis. All populations of anti-DNP molecules examined, whether isolated from pooled sera or from single bleedings of individual rabbits, were heterogeneous in respect to affinity for dinitrobenzenes. The heterogeneity in virtually every instance could be described by the Sips distribution function over a wide range of binding data, and each titration was thus characterized by an average intrinsic association constant (Ko), and by a heterogeneity index (a). Fractional precipitation of antibodies from serum, by addition of limiting amounts of antigen, yielded from the serum of individual rabbits anti-DNP populations that differed as much as 10,000-fold in Ko. Changes in affinity for [epsilon]-DNP-lysine were followed by isolating antibodies from sera at various times after a single injection of DNP- bovine-gamma-globulin. The Ko increased progressively with time after immunization and the rate of increase was most conspicuous when small quantities of DNP-bovine-gamma-globulin were injected initially. The rise in Ko was markedly delayed when large doses of antigen were injected. Antibody populations isolated early after immunization had nearly the same low affinity for both [epsilon]-DNP-L-lysine and for 2,4-dinitroaniline, and it is inferred that their specific binding sites are poorly adapted to the dinitroanilino group and insensitive to the norleucine moiety of [epsilon]-DNP-lysine. In contrast, antibodies isolated late after immunization bind [epsilon]-DNP-lysine strongly, and from their interactions with a variety of dinitrobenzenes it is inferred that their binding sites, on the average, are just about large enough to accommodate [epsilon]-DNP-L-lysine. Complex formation was enthalpy driven: [DELTA]Ho values ranged from [long dash]8 to [long dash]20 kcal mole-1 for different antibody-ligand pairs, and [DELTA]So values ranged from [long dash]5 to [long dash]30 entropy units mole-1. When representative 2,4-dinitrobenzenes were bound by anti-DNP molecules, bathychromic and hypochromic spectral shifts were observed; in the case of antibody-bound 2,4-dinitrotoluene a new absorption peak appeared at 300 m[mu]. The possibility is raised that charge-transfer contributes to the stability of anti-body-dinitrophenyl complexes.