Inouye K, Morimoto K
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
J Immunol Methods. 1994 May 16;171(2):239-44. doi: 10.1016/0022-1759(94)90043-4.
F(ab')2 fragments, herein designated as F(ab')2 mu, were prepared from rat IgM monoclonal antibodies (mAbs). The IgM was digested at a pepsin-to-IgM ratio of 1:200 (w/w) in 100 mM citrate buffer (pH 4.5) at 37 degrees C for 2 h. During digestion, the light (L) chain (27 kDa) of IgM remained undegraded, whereas the heavy (H) chain disappeared and two new bands of 44 and 48 kDa appeared. The digests were fractionated by means of hydrophobic interaction HPLC with TSKgel Phenyl-5PW. The fraction containing F(ab')2 mu was homogeneous and the recovery of antigen-binding activity was 41-52%. The molecular mass of F(ab')2 mu was estimated to be 147-153 kDa, and we concluded that the fragment was composed of two truncated H chains and two intact L chains. F(ab')2 mu was used in an enzyme immunoassay of mouse interleukin-6 and the interaction of IgM with non-specific proteins was greatly reduced, when it was converted to F(ab')2 mu fragments.
F(ab')2片段，在此命名为F(ab')2μ，是从大鼠IgM单克隆抗体（mAb）制备而来。IgM在100 mM柠檬酸盐缓冲液（pH 4.5）中，以胃蛋白酶与IgM的比例为1:200（w/w），于37℃消化2小时。消化过程中，IgM的轻链（L链，27 kDa）未被降解，而重链（H链）消失，出现了两条新的44 kDa和48 kDa条带。消化产物通过TSKgel Phenyl - 5PW疏水相互作用高效液相色谱进行分离。含有F(ab')2μ的组分是均一的，抗原结合活性的回收率为41 - 52%。F(ab')2μ的分子量估计为147 - 153 kDa，我们得出该片段由两条截短的H链和两条完整的L链组成。F(ab')2μ用于小鼠白细胞介素 - 6的酶免疫测定，当IgM转化为F(ab')2μ片段时，其与非特异性蛋白质的相互作用大大降低。
