Study Of Generation And Function Of Monoclonal Antibodies Of FAT-1

Objective : n-3 polyunsaturated fatty acids(n-3 PUFAs) are essential fatty acids required for normal cellular function and have been shown to exert many preventive and therapeutic effects on many diseases. The fat-1 gene from the roundworm Caenorhabditis elegans can convert n-6 PUFAs to n-3 PUFAs and several fat-1 transgenic mammals(mouse, pig and cow) have been produced. However, because there is no commercially available FAT-1 antibody, FAT-1 expression could not be directly detected in those animals in all the reported studies. This study aimed to produce FAT-1 monoclonal antibodies.Methods: We used Escherichia(E.) coli expression system to produce recombinant FAT-1(r FAT-1) protein.The recombinant FAT-1 protein was purified by His-Trap FF affinity chromatography column and analyzed by SDS-PAGE. We used tandem mass spectrographic analysis to identify the r FAT-1 protein peptides. r FAT-1 was used to immunize mice to produce FAT-1 m Ab with a standard monoclonal antibody production procedure. The subclasses and titre values of FAT-1 m Ab were examined by ELISA. FAT-1 protein in fat-1 transgenic mice tissue samples, including liver, lung, kidney, heart, brain and spleen were detected by western blotting and immunohistochemical stains.Results: The purified recombinant FAT-1 protein which was analyzed by SDS-PAGE and Coomassie brilliant blue staining showed a protein band of approximately 46 k Da in size. The identity of the FAT-1 recombinant protein was confirmed by searching sequence databases using mass spectrometry data.Recombinant FAT-1 was used to generate FAT-1 monoclonal antibodies and after three rounds of subcloning, Coomassie brilliant blue staining revealed the presence of two protein bands 55 k Da and 25 k Da in size, respectively. ELISA showed that the FAT-1 monoclonal antibodies(3A11) between 0.05 and 100 μg/m L exhibited a linear increase in OD450. Further analysis revealed that the antibodies were Ig G2 a. Immunoblotting assays and Immunohistochemical staining using FAT-1 monoclonal antibodies(3A11) both demonstrated that FAT-1 protein was present in many tissues of the transgenic mice, but was not found in the wildtype control C57BL/6 tissues.Conclusions: The FAT-1 m Ab is a useful tool for detection of FAT-1 protein in fat-1 transgenic animal tissues in future studies.