Leder L, Wendt H, Schwab C, Jelesarov I, Bornhauser S, Ackermann F, Bosshard H R
Biochemisches Institut, Universität, Zürich, Switzerland.
Eur J Biochem. 1994 Jan 15;219(1-2):73-81. doi: 10.1111/j.1432-1033.1994.tb19916.x.
Antiserum to a native protein may cross-react with the corresponding denatured protein or with peptides. The cross-reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation-sensitive immunoassays must be employed to distinguish a genuine from an apparent cross-reaction. In the present study, we have analyzed critically the cross-reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross-reaction with the help of the protein A antibody-capture ELISA, a new conformation-sensitive ELISA format. Three systems were analyzed: cross-reaction of antisera to native yeast and horse cytochrome c with unfolded apo-cytochrome c; cross-reaction of antisera to a coiled-coil leucine-zipper peptide with a homologous random-coil peptide obtained by introducing two proline residues into the leucine-zipper sequence; cross-reaction of antisera to two peptides that correspond to the N-terminal and an internal sequence of ferredoxin: NADP+ reductase (FNR), with the native enzyme. The reaction of the anti-(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross-reaction. Furthermore, the apparently cross-reactive antibodies to horse cytochrome c did not discriminate against sequence-related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine-zipper peptide did cross-react in a genuine way with the homologous random-coil peptide, that is, the cross-reactive antibodies do not seem to have been produced against the unfolded form of the leucine-zipper peptide. Of the two anti-peptide sera the one against the unstructured and highly accessible N-terminal segment reacted strongly with the native protein. The second serum against a solvent-accessible turn-like sequence of FNR showed apparent cross-reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide. The generation of cross-reactive antibodies depends on the conformational stability and integrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier-bound. The results clarify the different nature of cross-reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation-specific probes.
针对天然蛋白质的抗血清可能会与相应的变性蛋白质或肽发生交叉反应。这种交叉反应要么是抗体的固有特性，要么是由针对某些污染用于免疫的天然蛋白质的未折叠蛋白质产生的抗体所引起。必须采用合适的构象敏感免疫测定法来区分真正的交叉反应和表面上的交叉反应。在本研究中，我们严格分析了兔抗血清对蛋白质和肽的交叉反应。我们借助蛋白A抗体捕获ELISA（一种新的构象敏感ELISA形式）区分了真正的交叉反应和表面上的交叉反应。分析了三个系统：抗天然酵母和马细胞色素c的抗血清与未折叠的脱辅基细胞色素c的交叉反应；抗卷曲螺旋亮氨酸拉链肽的抗血清与通过在亮氨酸拉链序列中引入两个脯氨酸残基获得的同源无规卷曲肽的交叉反应；抗两种与铁氧化还原蛋白：NADP+还原酶（FNR）的N端和内部序列相对应的肽的抗血清与天然酶的交叉反应。抗（细胞色素c）血清的反应显然是由于针对未折叠蛋白质产生的抗体，这是一种表面上的而非真正的交叉反应。此外，针对马细胞色素c的表面上交叉反应性抗体不能区分来自狗、牛、兔和鸽的序列相关蛋白质。相比之下，针对亮氨酸拉链肽的抗体确实与同源无规卷曲肽发生了真正的交叉反应，也就是说，交叉反应性抗体似乎不是针对亮氨酸拉链肽的未折叠形式产生的。两种抗肽血清中，一种针对无结构且高度易接近的N端片段的血清与天然蛋白质强烈反应。第二种针对FNR的溶剂可及的类似转角序列的血清表现出表面交叉反应性：识别天然蛋白质的抗体针对游离肽的一种次要构象异构体，并且不与游离肽的主要形式反应。交叉反应性抗体的产生取决于免疫原的构象稳定性和完整性及其应用的分子形式，即游离的、聚合的或与载体结合的。这些结果阐明了抗血清对蛋白质和肽的交叉反应性的不同性质。如果要将抗血清用作构象特异性探针，这一知识至关重要。
