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

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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