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采用革克隆抗体ＣＣ－１（ＩｇＭ）免疫组化阳性的新鲜大肠癌组织制备粗抗原，经ＳＤＳ－ＰＡＧＥ分离，Ｗｅｓｔｅｒｎ－ｂｌｏｔ后进行免疫染色，检测刮ＣＡ－ＣＣ－１带。用附载ＣＡ－ＣＣ－１的硝酸纤维膜（ＮＣ）进行二次小鼠脾内包埋免疫和一次腹腔粗抗原加强免疫，成功地制备了两株ＣＣ－１的第二代单抗Ｗ１３５（ＩｇＭ）与Ａ１２－２（ＩｇＧｌ）。亲和力均比原代高；抗原决定族为糖链结构；Ｗｅｓｔｅｒｎ－ｂｌｏｔ免疫染色的比较发现：Ｗ１３５与Ａ１２－２除能识别ＣＡ－ＣＣ－１带外，尚能分别识别分子量为９４ＫＤ左右和３３ＫＤ的抗原带。免疫组化比较表明：对于大肠癌阳性反应率，Ａ１２－２基本与ＣＣ－１相同，而Ｗ１３５明显优于ＣＣ－１。以上研究表明：脾内包埋免疫法为一行之有效的免疫方式；第二代单抗可能较原代ＣＣ－１具有更大的临床应用价值。
Crude antigen was prepared by using fresh colon cancer tissues positive for CCL-1 (IgM) immunohistochemistry. SDS-PAGE, Western blot and immunofluorescence staining were used to detect CA-CC-1 band. The second generation of monoclonal antibodies to CC-1, W135 (IgM), was successfully prepared by secondary splenic inoculation and primary intraperitoneal immunization with nitrocellulose membrane (NC) loaded with CA-CC-1 ) And A12-2 (IgGl). The affinity group was higher than that of the original group. The antigenic determinants were sugar chain structure. The comparison of Western-blot immunostaining showed that W135 and A12-2 were able to recognize the CA-CC-1 bands except for the bands of about 94KD and 33KD Of the antigen band. Immunohistochemical comparison showed that for the positive rate of colorectal cancer, A12-2 was basically the same as CC-1, while W135 was significantly better than CC-1. The above studies show that the spleen embedded immunoassay is an effective immunization method; the second generation of monoclonal antibody may have more clinical value than the primary CC-1.