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Standardization Of Indirect Sandwich Enzyme Linked Immunosorbent Assay For Detection Of Foot And Mouth Disease

By: Muhammad Mujahid Amjed | Prof. Dr. Khushi Muhammad.
Contributor(s): Professor Dr. Masood Rabbani | Faculty of Veterinary Sciences.
Material type: materialTypeLabelBookPublisher: 2005Subject(s): Department of MicrobiologyDDC classification: 0883,T Dissertation note: Foot and Mouth Disease (FMD) is one of the most troublesome and infectious diseases of livestock, caused by the FMD virus. In this study Indirect Sandwich Enzyme Linked Immunosorbent Assay (IS-ELISA) was standardized to characterize the FMD serotype "0" virus. Oil based FMD serotype "0" vaccine was prepared and injected at the neck region of guinea pigs and rabbits. The vaccine induced anti-FMD serotype "0" virus antibodies in the vaccinated animals after 21 days post boosting. The serum thus separated was purified through ammonium sulfate salt (NH4)2S04 and ion exchange column chromatography. Total protein content in the guinea pig serum (whole serum), Ammonium Sulfate Precipitated Guinea Pig Serum (ASPGPS) protein and Ion Exchange based purified Guinea Pig Serum (IEGPS) protein when analyzed through spectrophotometer at 280 nm and 260 nm was found to be 52 ug/mi, 24 ug/ml and 10 ug/mi respectively. Virus Neutralization (VN) test was performed to monitor the neutralizing antibody titer. The whole serum of guinea pigs and rabbits showed the 1:32 and 1:64 anti-FMD serotype "0" virus neutralizing antibody titers. While anti-FMD serotype "0" virus neutralizing antibody titer was 1:128 in the IEGPS proteins. IEGPS protein with 1:128 neutralizing antibody titer were used as capture/trapping antibody in the standardization of the assay. The IEGPS protein 1:1000 diluted with 10 ug/ml of protein content was found to be optimum as capture/trapping antibody. To cover residual blank spaces, different available blocking buffers were evaluated and Skimmed Milk Solution 5 % in Phosphate Buffered Saline (PBSSKIVI-5%) proved best amongst blocking buffers. Coating of 1:1000 diluted IEGPS at 37 °C for 1 hour followed by storage at 4 °C for overnight was best incubation time in the study. FMD serotype "0" virus 1:100 diluted was optimum in IS-ELISA. Similarly rabbit anti-FMD serotype "0" virus specific immune serum 1:10,000 diluted and goat anti-rabbit IgG horseradish peroxidase conjugate 1:4000 diluted were found to be optimum during the standardization of the assay. Lastly ELISA plates were proved to be best amongst the available plates for assay. In each experiment, plateau region, test background and plate background were recorded. Results of the study helped for establishment of an economical, sensitive, reliable, robust IS-ELISA technique in research and diagnostic laboratories in the country.
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Foot and Mouth Disease (FMD) is one of the most troublesome and infectious diseases of livestock, caused by the FMD virus. In this study Indirect Sandwich Enzyme Linked Immunosorbent Assay (IS-ELISA) was standardized to characterize the FMD serotype "0" virus. Oil based FMD serotype "0" vaccine was prepared and injected at the neck region of guinea pigs and rabbits. The vaccine induced anti-FMD serotype "0" virus antibodies in the vaccinated animals after 21 days post boosting. The serum thus separated was purified through ammonium sulfate salt (NH4)2S04 and ion exchange column chromatography. Total protein content in the guinea pig serum (whole serum), Ammonium Sulfate Precipitated Guinea Pig Serum (ASPGPS) protein and Ion Exchange based purified Guinea Pig Serum (IEGPS) protein when analyzed through spectrophotometer at 280 nm and 260 nm was found to be 52 ug/mi, 24 ug/ml and 10 ug/mi respectively. Virus Neutralization (VN) test was performed to monitor the neutralizing antibody titer. The whole serum of guinea pigs and rabbits showed the 1:32 and 1:64 anti-FMD serotype "0" virus neutralizing antibody titers. While anti-FMD serotype "0" virus neutralizing antibody titer was 1:128 in the IEGPS proteins. IEGPS protein with 1:128 neutralizing antibody titer were used as capture/trapping antibody in the standardization of the assay. The IEGPS protein 1:1000 diluted with 10 ug/ml of protein content was found to be optimum as capture/trapping antibody. To cover residual blank spaces, different available blocking buffers were evaluated and Skimmed Milk Solution 5 % in Phosphate Buffered Saline (PBSSKIVI-5%) proved best amongst blocking buffers. Coating of 1:1000 diluted IEGPS at 37 °C for 1 hour followed by storage at 4 °C for overnight was best incubation time in the study. FMD serotype "0" virus 1:100 diluted was optimum in IS-ELISA. Similarly rabbit anti-FMD serotype "0" virus specific immune serum 1:10,000 diluted and goat anti-rabbit IgG horseradish peroxidase conjugate 1:4000 diluted were found to be optimum during the standardization of the assay. Lastly ELISA plates were proved to be best amongst the available plates for assay. In each experiment, plateau region, test background and plate background were recorded. Results of the study helped for establishment of an economical, sensitive, reliable, robust IS-ELISA technique in research and diagnostic laboratories in the country.

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