Establishment and characterization of antibodies against Ciz1b for detection of early-stage lungcancer

Abstract

In Sweden lung cancer is the fifth most common cancer form. One problem with lung cancer is that it often is diagnosed late when the patient already suffers from symptoms, leading to low survival compared to other cancer forms. New and improved screening methods for risk groups could lead to earlier detection of lung cancer and increased survival. Higgins et al. 2012 presented in the article “Variant Ciz1 is a circulating biomarker for early-stage lung cancer” Ciz1b as a new marker for lung cancer. Ciz1b is an alternative splice variant of the normally occurring Ciz1 protein and has been shown to be a promising biomarker for early-stage lung cancer detection being able to distinguish lung cancer patients from healthy individuals. Higgins et al.2012 has done this study using Western blot which is a method not suitable for diagnostic purposes. Therefore they suggest that a method generating simplified quantitative data, such as an ELISA, should be established. The problem to be solved is that there are no Ciz1b specific reagents to be used in ELISA format (Higgins et al., 2012) and therefore this master thesis project was done. The aim of this project was to establish, characterize and evaluate antibodies specific for detection of Ciz1b to be used in ELISA format. Ciz1b is an alternative splicing of Ciz1 where eight amino acids have been spliced. To be able to generate antibodies directed toward this small splice site site-specific immunization was used with phage particles. Filamentous phages were used to express Ciz1/Ciz1b specific peptides on the phage surface by phage display to generate an immune response against specific regions of the Ciz1/Ciz1b proteins, such as the Ciz1b-specific splice site. Phage particles used for immunization were analyzed to verify that the recombinant peptides were located in desired reading frame and that the recombinant pVIII molecules were expressed on the phage surface. The phage particles were immunized in Balb/c female mice several times and blood samples were collected from tail vein. An ELISA was established to analyze anti-Ciz1/Ciz1b titer in blood samples to verify which mice raised an immune response against Ciz1/Ciz1b. Mice with anti-Ciz1/Ciz1b titer were used for hybridoma technology. Five mice had anti-Ciz1/Ciz1b titer; two were used for hybridoma technology. From the hybridoma technology 15 hybridomas show selectivity towards Ciz1b. Those hybridomas produce antibodies with promising specificity against Ciz1b but further analysis has to be done to verify the specificity.