Pedersen Maiken K, Sorensen Nanna S, Heegaard Peter M H, Beyer Natascha Helena, Bruun Leif
Department of Autoimmunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark.
J Immunol Methods. 2006 Apr 20;311(1-2):198-206. doi: 10.1016/j.jim.2006.02.008. Epub 2006 Mar 6.
Hapten-carrier conjugate immunization is an important tool in the generation of hapten-specific antibodies for analytical purposes and in the uncovering of basic vaccine-immunological mechanisms. The affinity of antibodies is known to play an important role for the resulting sensitivity of antibody-based assay systems and in deciding whether a vaccine-induced antibody response will be protective. With ovalbumin as a carrier protein and a peptide (7.2 NY) representing a 19 amino acid sequence from the E. coli-derived Verotoxin 2e as a model hapten we investigated whether it was possible to influence the affinity and titre of antibodies raised against the hapten using different conjugation ratios and orientations. The peptide was coupled to ovalbumin in four conjugation ratios and two molecular orientations - terminal and central - and the conjugates were verified by mass spectrometry. Mice were immunised ten times at two-weeks intervals with low doses of the eight conjugates. Blood samples collected between each immunisation were analysed by ELISA for specific antibody titres and relative affinities. With both types of conjugations, the anti-peptide antibody titres increased in response to increasing conjugation ratios, but central conjugation resulted in markedly higher titres than terminal conjugation. The overall anti-peptide antibody affinities reached approximately similar levels with both orientations, whereas a reversed proportionality was observed between conjugation ratio and antibody affinity for terminal conjugation. Thus, it appears that the molar ratio of a peptide and its carrier may affect the resulting antibody affinities, and that a conjugation ratio between a terminally conjugated peptide and its carrier approaching one will result in relatively high antibody affinities. Furthermore, the molecular orientation of the coupled peptide has a major effect on the anti-peptide antibody titres induced.
半抗原-载体偶联免疫是生成用于分析目的的半抗原特异性抗体以及揭示基本疫苗免疫机制的重要工具。已知抗体的亲和力对于基于抗体的检测系统的最终灵敏度以及决定疫苗诱导的抗体反应是否具有保护性起着重要作用。以卵清蛋白作为载体蛋白，以代表大肠杆菌衍生的维罗毒素2e的19个氨基酸序列的肽（7.2 NY）作为模型半抗原，我们研究了是否有可能使用不同的偶联比和方向来影响针对半抗原产生的抗体的亲和力和效价。将该肽以四种偶联比和两种分子方向（末端和中心）与卵清蛋白偶联，并通过质谱法验证偶联物。用低剂量的八种偶联物以两周的间隔对小鼠进行十次免疫。每次免疫之间采集的血样通过ELISA分析特异性抗体效价和相对亲和力。对于两种类型的偶联，抗肽抗体效价均随着偶联比的增加而升高，但中心偶联产生的效价比末端偶联明显更高。两种方向的总体抗肽抗体亲和力达到大致相似的水平，而对于末端偶联，在偶联比和抗体亲和力之间观察到相反的比例关系。因此，似乎肽与其载体的摩尔比可能会影响产生的抗体亲和力，并且末端偶联的肽与其载体之间接近1的偶联比将导致相对较高的抗体亲和力。此外，偶联肽的分子方向对诱导的抗肽抗体效价有主要影响。
