Robertson M W, Liu F T
Division of Molecular Biology, Medical Biology Institute, La Jolla, CA 92037.
Mol Immunol. 1988 Feb;25(2):103-13. doi: 10.1016/0161-5890(88)90057-0.
Six peptides, representing contiguous amino acid sequences within the C epsilon 2, C epsilon 3 and C epsilon 4 domains of murine IgE, were selected for synthesis on the basis of overall hydropathy, degree of homology to both human and rat IgE and, where possible, inclusion of a native cysteine residue. Antibodies were produced against each peptide by immunizing rabbits with peptide-KLH conjugates. Each anti-peptide antiserum exhibited good reactivity with the corresponding immunizing peptide (titer: 10(-4) to 10(-5] and four of the six antisera exhibited a distinct preferential murine IgE reactivity compared to four other murine immunoglobulin classes (IgG1, IgG2b, IgM and IgA). In addition, one antiserum (anti-epsilon peptide 5), raised against a peptide with 80% homology to human IgE, reacted comparably with both human and murine IgE. Each IgE-reactive antiserum was screened for the ability to stimulate mediator release from IgE-sensitized rat basophilic leukemia (RBL) cells. Two of the four IgE-reactive antisera strongly stimulated 3H-serotonin release (anti-epsilon peptides 4 and 5), one antiserum showed weak activity (anti-epsilon peptide 3) and the remaining anti-peptide serum (anti-epsilon peptide 6), which exhibited the highest anti-IgE reactivity, exhibited no detectable stimulatory activity. Individual anti-peptide antibodies were subsequently tested for the potential to bind to receptor-bound IgE. Anti-epsilon peptide 3 was shown to exhibit the least binding, anti-epsilon peptide 6 showed the highest magnitude of binding while anti-epsilon peptides 4 and 5 exhibited intermediate values. We conclude from this study that sequences defined by epsilon-peptides 4 and 5 are not significantly involved in the receptor binding mechanism whereas epsilon-peptide 3 is likely to be most proximal to the IgE-receptor recognition site of those sequences studied. Finally, we suggest that the epsilon-peptide 6 sequence is in such an orientation in cell-bound IgE that, while it is accessible to external antibody, effective cross-linking of the IgE-receptor complex cannot be achieved through this determinant.
基于整体亲水性、与人和大鼠IgE的同源程度，并尽可能包含天然半胱氨酸残基，选择了代表小鼠IgE的Cε2、Cε3和Cε4结构域内连续氨基酸序列的六种肽进行合成。通过用肽-KLH缀合物免疫兔子，制备了针对每种肽的抗体。每种抗肽抗血清与相应的免疫肽表现出良好的反应性（效价：10^(-4)至10^(-5)），六种抗血清中的四种与其他四种小鼠免疫球蛋白类别（IgG1、IgG2b、IgM和IgA）相比，表现出明显的对小鼠IgE的优先反应性。此外，一种针对与人IgE具有80%同源性的肽产生的抗血清（抗ε肽5），与人IgE和小鼠IgE的反应相当。对每种与IgE反应的抗血清进行筛选，以检测其刺激IgE致敏的大鼠嗜碱性白血病（RBL）细胞释放介质的能力。四种与IgE反应的抗血清中有两种强烈刺激3H-血清素释放（抗ε肽4和5），一种抗血清显示出弱活性（抗ε肽3），而表现出最高抗IgE反应性的其余抗肽血清（抗ε肽6）未表现出可检测到的刺激活性。随后测试了各个抗肽抗体与受体结合的IgE结合的潜力。结果显示，抗ε肽3的结合最少，抗ε肽6的结合量最高，而抗ε肽4和5表现出中间值。我们从这项研究得出结论，由ε肽4和5定义的序列在受体结合机制中没有显著参与，而ε肽3可能是在所研究的那些序列中最接近IgE受体识别位点的。最后，我们认为ε肽6序列在细胞结合的IgE中的取向是，虽然它可被外部抗体识别，但通过该决定簇无法实现IgE-受体复合物的有效交联。
