Equine tumor necrosis factor alpha: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and development of a sensitive enzyme-linked immunosorbent assay.

The researchers have managed to clone and express equine tumor necrosis factor alpha, a significant molecule involved in the immune response, in E. coli bacteria. They also developed a sensitive enzyme-linked immunosorbent assay (ELISA) to detect the presence of these molecules.

Cloning and Expression of Equine Tumor Necrosis Factor

• The researchers decoded the DNA sequences that form the mature equine tumor necrosis factor alpha (ETNF alpha).
• Using the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis, they were able to reconstruct these sequences. The sequences were then inserted into an E. coli vector (a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell) called pFLAG-1 for expression.
• The recombinant ETNF alpha (rETNF alpha) was then extracted by anti-FLAG immunoaffinity chromatography, a lab method that isolates specific molecules in a mixture.

Generation of Monoclonal and Polyclonal Antibodies

• The extracted rETNF alpha was used as Immmunogen (a substance that is able to produce an immune response) to produce murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha.
• Three monoclonal antibodies (6H4, 9B10, and 12F6) were produced from a single fusion process. Testing confirmed these antibodies could recognize rETNF alpha in western blots, a common laboratory technique used to detect proteins.
• Two antibodies, 6H4 and 9B10, were found to neutralize both rETNF alpha and the naturally-occurring ETNF alpha in a cell cytotoxicity assay. Cell cytotoxicity assays are tests that measure the compatibility of a substance with living cells – in this case, showing the antibodies’ ability to neutralize ETNF alpha.

Development of a Sensitive ELISA

• An ELISA (enzyme-linked immunosorbent assay) method was developed using the 6H4 monoclonal antibody and biotin-labelled rabbit polyclonal antibodies. ELISA is a common lab test that measures the concentration of an antigen in a sample.
• This ELISA was capable of detecting ETNF alpha levels as low as 100 picograms per milliliter. It was also successfully used to show that levels of ETNF alpha increase in horses suffering from experimental endotoxemia.

Conclusion

• The cloned ETNF alpha, the antibodies, and the ELISA established in this study should be useful for future research into diseases in horses that involve tumor necrosis factor.