Purification of polyclonal antibody against pork extracts antigens using protein A column as material for developing halal food detection kit

Abstract

The main objective of this study was to purify polyclonal antibodies against pork extract as raw material for developing diagnostic kits for halal food made from animal products. Antibodies were obtained from rabbit serum which was immunized with raw pork extract, and which had been heated at 70° C, 80° C and 100° C. The purification process was carried out in two stages namely using 33% ammonium sulfate and continued with Protein A column affinity. The antibody purification results were analyzed using SDS-PAGE, while the antibody activity was tested using the western blot method on raw meat extracts and on those heated at 70° C, 80° C, and 100° C. The results of this study showed that antibodies purified with a combination of ammonium sulfate and Protein A column affinity produced relative purity of antibodies up to 97.6 per cent compared to those purified using ammonium sulfate alone. Western blot analysis showed that the purified antibodies were able to detect raw as well as heated pork extracts up to 70° C at a total protein content of 20 micro grams of meat extract. Antibodies obtained from immunizations using meat extracts that have been heated at 70° C gave the same potential as antibodies from rabbits immunized using raw meat extract, i.e. can only detect antigens of raw meat and antigens of pork heated at 70° C. From the results of this study it can be concluded that purification of antibodies using a combination of ammonium sulfate and Protein A column is effective in producing polyclonal antibodies with relatively high purity levels. In the future, further research needs to be carried out on the development of immunodiagnostics using polyclonal antibodies purified from animals immunized with pork extract that has been heated at a temperature of 70° C or more.The main objective of this study was to purify polyclonal antibodies against pork extract as raw material for developing diagnostic kits for halal food made from animal products. Antibodies were obtained from rabbit serum which was immunized with raw pork extract, and which had been heated at 70° C, 80° C and 100° C. The purification process was carried out in two stages namely using 33% ammonium sulfate and continued with Protein A column affinity. The antibody purification results were analyzed using SDS-PAGE, while the antibody activity was tested using the western blot method on raw meat extracts and on those heated at 70° C, 80° C, and 100° C. The results of this study showed that antibodies purified with a combination of ammonium sulfate and Protein A column affinity produced relative purity of antibodies up to 97.6 per cent compared to those purified using ammonium sulfate alone. Western blot analysis showed that the purified antibodies were able to detect raw as well as heated pork extracts up to ...