An Indirect ELISA Method For The Detection Of Staphylococcus Aureus IsdB_137-361 Antibody In Bovine Serum Was Established

Bovine mastitis is an important and common dairy cow disease that harms the health of dairy co ws and affects the development of dairy industry.Staphylococcus aureus(S.aureus)is an important pa thogen that causes bovine mastitis.With the increasing number of drug-resistant strains,especially mul ti-drug resistant strains,as well as the demand for antibiotic free milk,treatment of cow mastitis indu ced by S.aureus with antibiotics is both ineffective and severely limited.Therefore,people are activel y exploring the use of vaccine to prevent bovine mastitis induced by S.ureus.In previous studies,we have constructed a vaccine candidate for bovine mastitis(named GIT)against both S.aureus and stre ptococcal infections using the immunodominant fragment,iron regulated surface determination cluster B _137-361(IsdB_137-361),of S.aureus and a streptococcal antigen component.In order to detect serum anti bodies against S.ureus IsdB_137-361antigen after immunization with this vaccine candidate,we establishe d an indirect ELISA method for the detection of antibodies in bovine serum using the recombinant Is dB_137-361expressed by pGEX-6P-1 vector as the antigen.The concentration of the coating antigen,ser um dilution ratio and reaction conditions were optimized,and cut-off value was determined by ROC analysis,and the serum cross-reactivity and sensitivity were tested.The results showed that the optim al concentration of coating antigen was 2.5?g/m L,the serum dilution was 1:1000,and the antigen w as coated at 4?for 12 h after being diluted in carbonate buffer solution with p H9.6,and sealed by PBST containing 5%skimmed milk at 37?for 30 min,and incubated at 37?for 15 min after the serum was added.The intra-batch coefficient of variation was 4.3%-9.7%,and the inter-batch coefficie nt of variation was 3.3%-9.2%,both of which were less than 10%,indicating that the method had go od stability.When the cut-off value was 0.45,the established indirect ELISA showed good specificity and high sensitivity,Eighty-four clinical samples were tested,and 25 samples were positive,with a p ositive rate of 28.74%.In conclusion,an indirect ELISA method based on IsdB_137-361expressed by pGEX-6P-1 vector as the antigen can be used for the detection of bovine serum antibody to the antigen associated with Is dB_137-361after vaccination with GIT.This study provides a support for the evaluation of humoral imm une response induced by GIT vaccine candidate against S.aureus-and streptococcal-caused mastitis in cows.