Development and evaluation of an immunoassay for Keratin 5/19 complex in lung cancer

Abstract

14 antibody combinations were evaluated as candidates for a keratin 5/19 enzymeimmunoassay. By enzyme immune assay and western blot, 1A12+Ks19.1 was determined as the superior combination. The most successful test assay was the two-step enzyme immunoassay with 2 hours incubation time for the biotinylated antibody and 1 hour incubation for the HRPconjugated antibody. The experiments used to evaluate antibody combinations also indicates a structural difference between keratin 5/19 in pleura liquid compared to serum and that K5/19 complex containing the N-terminal is likely to be present in serum in higher extent than complex with intact C-terminal. The western blot also revealed cross-reaction of G-2 antibody with keratin 8, although G-2 is stated specific for keratin 5 by its manufacturer. [1] A randomized lung cancer study including 110 lung cancer plasma samples, 52 serum samples of benign lung disease and 52 plasma samples from healthy blood donors was performed. The study was carried out by analyzing the samples in the constructed 1A12+Ks19.1 test and CYFRA21-1. The constructed test was not able to discriminate between histological classes of lung cancer in the lung cancer study. The ability of the test to detect early stage lung cancer varied among the different histological classes. Adenocarcinoma and large cell carcinoma samples detected by the 1A12+Ks19.1 test were progressed cancer, stage III or stage IV. For small cell lung cancer 3 of the 13 early stage lung cancer were detected, indicating the test is thus not suitable for detecting early stages of these histological classes. For Squamous cell carcinoma in lung early stages are detected in larger extent than stage III and IV for K5/19 (1A12+Ks19.1) as a separate test, but primarily in combination with CYFRA21-1 where 9 of 13 early stage samples were detected. From this study one can recommend to perform further studies on early stage SCC in lung to determine the relevance of CYFRA21-1 in combination with 1A12 as diagnostic tool.