A single-chain-Fv-based immunofluorometric assay specific for the CEA variant NCA-2.

摘要

Carcinoembryonic antigen (CEA) is a member of the immunoglobulin gene superfamily. In meconium, a C-terminal truncated form of CEA, denominated NCA-2, is predominant. This molecular variant is present also in sera from adult cancer patients, but no NCA-2-specific assay is yet available, and its possible clinical significance is largely unknown. We have used phage display technology to produce a single-chain antibody (scFv) suitable for an NCA-2-specific assay and compared the NCA-2 results with CEA values in patient sera. A phagemid library with a diversity of 10(7) was constructed from splenic mRNA obtained from a mouse immunized with NCA-2 purified from human meconium. Following phage rescue and three rounds of panning on solid phases coated with NCA-2, several clones were isolated, which displayed high specificity for NCA-2. These were sub-cloned into an Escherichia coli expression vector for high-level expression of soluble scFv. From BIAcore studies a single scFv with low k(off) and a K(D) of 10(-10) mol/l was selected for europium labelling and an immunofluorometric assay with a sensitivity of 0.2 microg/l was established. The assay detects a protein in serum with a molecular weight significantly lower than CEA. For comparison, a routine assay for CEA, which measures both CEA and NCA-2 equivalently, and a CEA-specific assay based on the T84.66 antibody were employed. The difference between serum values measured with the two CEA assays corresponded to the values determined with the NCA-2-specific assay.