The Establishment of KL-6 Associated New Serum Markers Using High Sensitive Electrochemiluminescence Immunoassay.

2003年，我们进一步提高了使用M8分析仪（IGEN Co.，英国），并缩短了放映时间。同时，我们将小鼠骨髓瘤细胞与经多种肿瘤细胞免疫的BALB/c小鼠脾细胞融合，制备了分泌单克隆抗体的杂交瘤细胞株。ECLIA与反向间接夹心法（RISA）联用的灵敏度是酶联免疫吸附法（ELISA）的1,000倍，筛查时间是ELISA的1/6通过这种新的筛选方法，我们可以选择16种新的多克隆抗体，其对从肺癌或间质性肺炎患者获得的合并血清显示出强亲和力。在排除非特异性抗体后，我们试图克隆这些多克隆抗体。然而，杂交瘤本身具有较弱的增殖能力和不稳定性。因此，这些缺陷导致许多杂交瘤在克隆或冻融过程中丧失抗体分泌能力或细胞死亡。虽然最终只能产生少量单克隆抗体，但其中一些可能识别与原始KL-6抗体不同的表位。尽管建立这种新的筛选方法需要相当长的时间，但我们可以成功地建立极高灵敏度的筛选方法，只需很短的时间。这种新的方法有助于建立新的单克隆抗体，这些单克隆抗体与肺癌或间质性肺炎患者血清的反应比原来的KL-6抗体更敏感、更特异

In 2003, we further improved the sensitivity of the screening method of hybridomas using M8 analyzer (IGEN Co., UK) and enabled shorter screening hours. In parallel, we repeatedly produced new hybridomas secreting monoclonal antibodies by fusing of murine myeloma cells and splenocytes from BALB/c mice previously immunized with various cancer cell lines.M8 analyzer is the machine for electrochemiluminescence immunoassay (ECLIA) applying electrochemiluminescence of Ruthenium complexes. ECLIA combined with reversed indirect sandwich assay (RISA), which we had previously established and had reported its utility, enabled approximately 1,000 times sensitivity and one sixth screening hours compared with enzyme linked immunosorbent assay (ELISA) combined with RISA (RI-ELISA).By means of this new screening method, we could have selected sixteen new multiclonal antibodies that showed strong affinity to the pooled serum obtained from lung cancer or interstitial pneumonitis patients. We tried to clone these multiclonal antibodies after exception of non-specific antibodies. Hybridomas, however, originally have weak proliferation ability and instability. These defects, therefore, induced loss of antibody secreting ability or cell death of many hybridomas in the course of cloning or freezing/thawing. Although only few monoclonal antibodies could finally be produced, some of them probably recognize different epitopes from that of original KL-6 antibody.Despite of considerable long period to establish this new screening method, we could have successfully established extremely high sensitive screening method which requires only short hours. This new method must contribute to establish new monoclonal antibodies that more sensitively and specifically react with lung cancer or interstitial pneumonitis patients' serum than original KL-6 antibody