Novel Immunoassays For Staphylococcus Aureus Based On IgY As Recognition Element

BackgroundInfectious diseases account for six of the ten major health threats of the world declared by the WHO in 2019,and 25%of deaths worldwide were caused by infection.The effective control of infectious diseases has received wide attention.Staphylococcus aureus(S.aureus),as a common pathogenic bacterium,can lead to suppurative infection of superficial skin and critical pneumonia,bacteremia,organ abscess and endocarditis.In our country,the multiple incidences of bromatoxism were closely related to the food contaminated by S.aureus.Hence,the timely quantitative analysis of S.aureus has actual implications for the diagnosis of clinical diseases and the control of food safety.ObjectiveConventional immunoassay techniques often use mammalian Ig G for S.aureus detection.Since protein G-producing streptococcus can non-specifically bind to the Fc segment of Ig G antibody,it will cause false positive results in the detection of S.aureus.To deal with this scientific problem,we used chicken anti-protein A IgY as the specific recognition element of S.aureus,and two new immunoassays for S.aureus were built based on magnetic immunoassay and enzyme-catalyzed colorimetric reaction.Methods1.Highly sensitive and specific determination of S.aureus based on chicken anti-protein A IgYStaphylococcal protein A(SPA)could be selected as epitopes since they were specifically expressed on the cell wall surface of S.aureus.In this study,chicken anti-protein A IgY was utilized as the recognition element to bind with S.aureus via antigen-antibody interaction.Immunomagnetic beads were prepared by attaching chicken anti-protein A IgY to the beads.The immunomagnetic beads could separate S.aureus from the sample matrix to form"immunomagnetic beads-S.aureus"immune-complex.Subsequently,horseradish peroxidase(HRP)labeled chicken anti-protein A IgY was added to form"sandwich structure",which catalyzed Tetramethylbenzidine(TMB)/hydrogen peroxide(H2O2)substrate to produce color signal.2.Ultrasensitive and specific determination of S.aureus based on dual-recognition mode of SPA.One SPA molecule has five binding sites of Fc segments of mammalian Ig G.Therefore,IgY and Ig G were used as the dual-recognition mode of SPA to detect S.aureus in this study,which could enhance the sensitivity under the lower cost.Immunomagnetic beads were prepared by attaching chicken anti-protein A IgY to the beads,which were used to capture S.aureus in the samples,and then HRP-labeled rabbit anti-mouse Ig G was employed as the revealing partner to form"sandwich structure",followed by an HRP catalyzed colorimetric reaction.Results1.Highly sensitive and specific determination of S.aureus based on chicken anti-protein A IgY.The immunoassay for S.aureus could complete within 60 min with high specificity and selectivity.Some foodborne pathogens produced no interfere on the detection system.The detection limit of this developed immunoassay was 2.3×10~2 CFU/m L with a linear analysis range from 7.8×10~2 CFU/m L to 2.0×10~5 CFU/m L.This method was not affected by complex food matrices.The recoveries of S.aureus in milk and apple juice without any pretreatment ranged from 95.1%to 124.9%,and the limits of S.aureus detection in milk and apple juice were 2.3×10~2CFU/m L and 2.4×10~2 CFU/m L,which was lower than the limit set in European Union Legislation for food microbiological restrictions.The designed method was appropriate for the field of food detection.2.Ultrasensitive and specific determination of S.aureus based on dual-recognition mode of SPA.Optimized reaction conditions constructed a calibration curve for S.aureus in PBS ranging from 5×10~2CFU/m L to 5×10~4 CFU/m L with a detection limit of 110 CFU/m L,which was comparable to PCR(Polymerase Chain Reaction)method.This method could clearly distinguish S.aureus from several interfering bacteria including streptococcus with good specificity.The recoveries of S.aureus in sodium chloride injection,fruit juice and human urine were tested ranged from 89.1%to 121.1%,which indicated that this immunoassay possessed a good practical application value.ConclusionNovel immunoassays for S.aureus were proposed using chicken anti-protein A IgY as the recognition element,both of which had good sensitivity and specificity.These techniques exhibited high application potential for S.aureus monitoring in various kinds of samples,which were of significance for the diagnosis and treatment of clinical diseases and the control of food safety.