Kontsek P, Martens E, Vandenbroeck K, Kontseková E, Waschütza G, Sareneva T, Billiau A
Rega Institute, Catholic University Leuven, Leuven, 3000, Belgium.
Cytokine. 1997 Aug;9(8):550-5. doi: 10.1006/cyto.1997.0200.
We examined the antigenic resemblance between human (h) and porcine (p) interferon (IFN)-gamma by binding (ELISA) and neutralization assays. The murine polyclonal antisera and sets of murine monoclonal antibodies (mAbs) raised against either IFN were tested in confrontation with recombinant IFNs of either species, and with site-specific mutants of hIFN-gamma. Several of the mAbs raised against pIFN-gamma cross-reacted in ELISA with hIFN-gamma. In contrast, none of the anti-hIFN-gamma mAbs cross-reacted. By employing site-specific mutants of recombinant hIFN-gamma as antigens in ELISA we succeeded in identifying the C-terminal portion 97-111 as the antigenic site in hIFN-gamma recognized by the cross-reactive anti-pIFn-gamma mAbs. None of the mAbs recognizing the common antigenic structure had neutralizing potency, although His111 was determined by others as the residue important for bioactivity of hIFN-gamma. Mutations in the domain 97-111 had no or little influence on homospecific reactivity of anti-hIFN-gamma mAbs, indicating that this domain, while being mouse-immunodominant in the case of pIFn-gamma was poorly immunogenic in the case of hIFN-gamma. The epitopes of three out of five anti-hIFN-gamma mAbs mapped in the N-terminal region 1-23, indicating immunodominance of this region in hIFN-gamma. Another mAb (D9D10), also directed to the N-terminus of hIFN-gamma, apparently recognized a conformational epitope. This antibody lacked ELISA-reactivity with the wild-type hIFN-gamma but strongly bound mutant protein with an engineered disulfide bridge Cys7-Cys69. Surprisingly, D9D10 showed high reactivity also with the wild type hIFN-gamma produced by baculovirus construct coding for the mature protein with signal sequence or with wild type protein possessing residues Cys-Tyr-Cys from the signal sequence.
我们通过结合（酶联免疫吸附测定法，ELISA）和中和试验研究了人（h）干扰素-γ与猪（p）干扰素-γ之间的抗原相似性。用针对任一干扰素产生的鼠多克隆抗血清和鼠单克隆抗体（mAb）组与任一物种的重组干扰素以及人干扰素-γ的位点特异性突变体进行对抗测试。几种针对猪干扰素-γ产生的单克隆抗体在ELISA中与人干扰素-γ发生交叉反应。相比之下，抗人干扰素-γ单克隆抗体均未发生交叉反应。通过在ELISA中使用重组人干扰素-γ的位点特异性突变体作为抗原，我们成功鉴定出C末端部分97 - 111是人干扰素-γ中被交叉反应性抗猪干扰素-γ单克隆抗体识别的抗原位点。尽管其他人确定His111是人干扰素-γ生物活性的重要残基，但识别共同抗原结构的单克隆抗体均无中和效力。97 - 111结构域中的突变对抗人干扰素-γ单克隆抗体的同种特异性反应性没有或几乎没有影响，这表明该结构域在猪干扰素-γ的情况下是小鼠免疫显性的，但在人干扰素-γ的情况下免疫原性较差。五个抗人干扰素-γ单克隆抗体中有三个的表位定位于N末端区域1 - 23，表明该区域在人干扰素-γ中具有免疫显性。另一种单克隆抗体（D9D10）也针对人干扰素-γ的N末端，显然识别一个构象表位。该抗体与野生型人干扰素-γ缺乏ELISA反应性，但与具有工程化二硫键Cys7 - Cys69的突变蛋白强烈结合。令人惊讶的是，D9D10对由编码带有信号序列的成熟蛋白的杆状病毒构建体产生的野生型人干扰素-γ或具有来自信号序列的Cys - Tyr - Cys残基的野生型蛋白也显示出高反应性。
