Diagnosis of Staphylococcus aureus Infection in Bovine Mastitis using its Affinity Purified Fraction
Diagnosis of Staphylococcus aureus Infection in Bovine Mastitis using its Affinity Purified Fraction
Ehab A. Fouad1*, Khaled A. Abd El-Razik2 , Eman H. Abdel-Rahman3
ABSTRACT
Affinity purified fraction from Staphylococcus aureus (S. aureus) was extracted, characterized and evaluated for its potency in diagnosis of S. aureus mastitis through indirect Enzyme Linked Immunosorbent Assay (ELISA). For purification of fraction, bacterial fresh cultures were centrifuged at 3000 rpm / 20 min and were homogenate in phosphate buffer saline and centrifuged at 15000 rpm / 20 min that known as the crude antigen. It entered to affinity column chromatography [Cyanogen Bromide-Sepharose 4B (CNBr-Sepharose 4B)] (Sigma Chemical Co.), obtain the fraction. In brief, IgG of cows S. aureus positive sera were purified by precipitation with 50% ammonium sulphate followed by purification with Protein A Sepharose gel. The separated IgG was dialyzed for 3 days against a 0.1 M NaHCO3 buffer pH 8.3 with 0.5 M sodium chloride and 0.02 percent NaN3 before being linked to CNBr-Sepharose 4B swollen beads. Bound fraction was eluted with mixture of glycine (0.1 M) and sodium chloride (0.5 M) at pH (2.3). Purified antigens and unbound were inspected of protein concentration. In the crude antigen, there were two bands with molecular weights of 73 and 64 KDa, compared to ten bands with molecular weights ranging from 209 to 24 KDa. The isolated fraction showed diagnostic properties of S. aureus mastitis using indirect ELISA with 100% sensitivity and 95 % specificity. The fraction validity lasted for more than one year of storage at -20 ºC. The current study introduces effective way for S. aureus mastitis diagnosis through the using of purified fraction instead of the classical way.
Keywords | Staphylococcus aureus, Affinity column chromatography, Antigen, SDS-PAGE, Indirect ELISA
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September 2022
Vol. 10, Iss. 9, Pages 1887-2089
