1. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences 2. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences 3. Immunology Research Center, Mashhad University of Medical Sciences 4. Faculty of Medicine, Mashhad University of Medical Sciences 5. Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences
Abstract
Introduction
Developing new tb diagnostics is a research priority because traditional methods suffer from low sensitivity or being time-consuming. to introduce an applicable diagnostic reagent for tb, this study was designed for production, purification and validation of anti-tb igy.
Methods
Chicken egg yolk immunoglobulins (igy) have added advantages of being economical and ethical compared to their mammals analogues. white leghorn laying chickens were immunized intramuscularly with formalin-inactivated cells of mycobacterium tuberculosis (mtb). eggs were collected before immunization and at different time intervals after immunization. igy antibody was purified from egg yolks using isopropyl alcohol-ammonium sulfate-caprylic acid method. the purity of igy was assessed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis in each step. igy concentration was estimated by bradford protein assay.
Results
An average purity of about 87% was determined by densitometric scanning of sds-page analysis. the maximum production of 2.91 mg/ml igy (58.2 mg/each egg yolk) was obtained in this study. a minimum of 104 cfu/ml mtb cells could be specifically detected by capture enzyme-linked immunosorbent assay (elisa) using the polyclonal anti-tb igy and hrp-conjugated rabbit anti-mtb igg. moreover, the specific reactivity of the polyclonal anti-tb igy against mtb and eight non-mycobacterium clinical isolates was confirmed by indirect elisa and dot-blot assay using hrp-conjugated rabbit anti-chicken igy (igg).
Conclusion
The resulting anti-tb igy can be used for a wide range of applications including immunomagnetic separation of mtb from specimens, especially in paucibacillary tb cases such as extrapulmonary or hiv co-infected tb pateints.
