MEASUREMENT OF AFFINITY IN SERUM SAMPLES OF ANTIGEN-FREE, GERM-FREE AND CONVENTIONAL MICE AFTER HYPERIMMUNIZATION WITH 2,4-DINITROPHENYL KEYHOLE LIMPET HEMOCYANIN, USING SURFACE-PLASMON RESONANCE
MEASUREMENT OF AFFINITY IN SERUM SAMPLES OF ANTIGEN-FREE, GERM-FREE AND CONVENTIONAL MICE AFTER HYPERIMMUNIZATION WITH 2,4-DINITROPHENYL KEYHOLE LIMPET HEMOCYANIN, USING SURFACE-PLASMON RESONANCE
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DOI：
10.1002/eji.1830250630
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发表时间：
1995-06-01
影响因子：
5.4
通讯作者：
BOS, NA
中科院分区：
文献类型：
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作者：
BAKKER, R;LASONDER, E;BOS, NA
我们先前研究了在无抗原（AF）、无菌（GF）和常规（CV）小鼠中对T细胞依赖性抗原2，4-二硝基苯基钥孔血蓝蛋白（DNP-KLH）的初级和次级应答以及超免疫。AF小鼠脾脏中DNP特异性IgG分泌细胞的绝对和相对数量均显著高于GF和CV小鼠，特别是在超免疫后。在本研究中，我们测量了总的和DNP特异性IgG浓度在这些超免疫小鼠的血清中使用敏感的夹心酶联免疫吸附试验。关于超免疫接种前后的总IgG浓度，AF小鼠在用抗原加强后显示出几乎13倍的增加; GF小鼠显示出大约8倍的增加。在CV小鼠中观察到轻微但不显著的增加。AF免疫小鼠中的总的以及DNP特异性IgG水平分别是GF和CV小鼠的2倍和5倍。使用表面等离子体共振仪器（BIAcore（TM），Pharmacia，Uppsala，Sweden），我们获得了三组超免疫小鼠的多克隆样品的平均结合亲和力（K-A）。伊加和IgM样品显示对DNP-赖氨酸的低亲和力。在IgG抗体中，AF小鼠显示出最高的K-A值，其次是GF小鼠，而CV小鼠显示出比AF小鼠低3倍的K-A。这些差异主要由解离速率常数（k（diss））决定，因为在缔合速率常数（k（ass））中未观察到显著变化。此外，与GF和AF小鼠相比，CV小鼠的血清具有较低百分比的高亲和力抗体。这些结果表明，除了在AF小鼠中观察到的较高的总体结合亲和力以及在GF小鼠中较小程度的结合亲和力之外，与CV小鼠相比，AF小鼠中高亲和力IgG的相对贡献更大。
We previously investigated the primary and secondary responses and hyperimmunization to the T cell-dependent antigen 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH) in antigen-free (AF), germ-free (GF) and conventional (CV) mice. Both the absolute and relative numbers of DNP-specific IgG-secreting cells in the spleen of AF mice were considerably higher compared to GF and CV mice, especially after hyperimmunization. In the present study we measured the total and DNP-specific IgG concentration in the sera of these hyperimmunized mice using a sensitive sandwich enzyme-linked immunosorbent assay. With respect to the total IgG concentration before and after hyperimmunization, the AF mice showed an almost 13-fold increase after boosting with the antigen; the GF mice showed an approximately 8-fold increase. A slight but non-significant increase was observed in the CV mice. The total as well as the DNP-specific IgG levels in the AF-immunized mice were 2-fold and 5-fold higher compared to GF and CV mice, respectively. With the use of Surface Plasmon Resonance instrumentation (BIAcore(TM), Pharmacia, Uppsala, Sweden) we obtained mean binding affinities (K-A) of the polyclonal samples of the three groups of hyperimmunized mice. IgA and IgM samples displayed low affinity for DNP-lysine. The AF mice displayed the highest K-A value among IgG antibodies, followed by GF mice, while CV mice showed a 3-fold lower K-A compared to AF mice. These differences were mainly determined by the dissociation rate constant (k(diss)), since no significant changes were observed in the association rate constant (k(ass)). Furthermore, the sera of the CV mice have a lower percentage of high-affinity antibodies compared to GF and AF mice. These results suggest that besides a higher overall binding affinity seen in AF mice, and to a lesser extent in GF mice, the relative contribution of high-affinity IgG is greater in AF mice compared to CV mice.