Conjugation of Anti-Antibodies with Peroxidase for Detection of Haemorrhagic Septicaemia through ELISA
The demand for highly sensitive, precise and accurate immunoassays has led to the development of numerous labeled reagent tests. The sensitivity imparted by the label, such as an isotope or fluorescent dye is combined with the specificity provided by an antibody/antigen reaction. Enzyme linked immunosorbent assay (ELISA) that involved enzyme labeled antibody, is a highly sensitive and specific method of choice to detect a variety of antigens or antibodies in a wide range of microorganisms. In the present research work, ELISA was standardized for the detection of haemorrhagic septicemia (HS). HS has a wide geographic distribution in the world. The disease primarily affects cattle and buffaloes although a sporadic outbreak of the disease has been reported in other domesticated animals. In several Asian countries, HS ranked as a major fatal disease in the buffalo, a species of great importance in rural economy of the region. There is a need to evaluate and improve the diagnostic methods for such diseases. This paper includes the development of such diagnostic ELISA kit through indigenously prepared reagents and conjugates by periodate oxidation method. Methods: Rabbit anti-buffalo antibodies horseradish peroxidase conjugates were prepared and subjected to standardize indirect ELISA for detection of HS-antibodies. Isolated HS-antibodies of buffalo calves were inoculated into rabbits and sera was collected after 14 days, purified by ammonium sulfate precipitation technique. Qualitative analysis of anti-antibodies was carried out by applying agar gel precipitation test. An indirect ELISA was performed on the conjugates that were diluted as 1:100, 1:200, 1:400 and 1:800 in PBS, to seek the best one dilution. Results: It was observed that precipitation lines of group A were relatively better than that of group B. Protein contents of controlled rabbits were less than immunized rabbits, where as protein contents of group A were maximum of all. An amount of 10 mg horseradish peroxidase was conjugated with rabbit-antibuffalo antibodies by periodate oxidation method. Pasteurella multocida was prepared and coated to flat-bottomed microtitre plates. The dilutions (1:100, 1:200, 1:400 and 1:800) of anti-antibodies peroxidase conjugates were used for the performance of ELISA. Conclusion: It was concluded that periodate oxidation coupling is an efficient method and 1:400 dilution of conjugate is best for ELISA. New work to be presented: The method used in this research work is not widely used for the diagnosis of various infectious diseases. The utilization of such method i.e. periodate method of conjugation will open new principles and methodologies to prepare the diagnostic kits as it also reflects high sensitivity.
Indirect ELISA periodate conjugation antispecies antibodies haemorrhagic septicaemia.
M. Anjum Zia Fozia Anjum M. Khalid Saeed Khalil-ur-Rehman Azeem I. Khan
Department of Chemistry (Biochemistry), University of Agriculture, Faisalabad, Pakistan National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Food and Biotechnology Research Center, PCSIR Laboratories Complex, Lahore, Pakistan Centre for Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad, Pakis
国际会议
北京
英文
1987-1991
2007-05-23（万方平台首次上网日期，不代表论文的发表时间）