The AlphaLISA Detection Of Staphylococcal Enterotoxin B And Preparation Of Fluorescent Functionalized Microspheres

Staphylococcal Enterotoxin B is a superantigen produced by staphylococcus aureus and has great toxicity.It can activate large numbers of CD4+ T cells at low concentrations.The production of large amounts of cytokines can trigger a "cytokine storm," leading to acute toxic effects.As an important enterotoxin,SEB is not only the main cause of food poisoning,but also a potential biological threat,which can be made for an aerosol biological weapon.Also,because of its toxicity,stability,and availability,the U.S.national center for disease control lists SEB as an important pathogenic toxin on the list of biological and chemical weapons.Therefore,rapid and accurate detection of SEB is very important and urgent.AlphaLISA is a technology based on two microspheres.When these two kinds of microspheres are close to each other,light with a wavelength of 680 nm is used to stimulate the donor microspheres to generate energy,which transforms the oxygen molecules in the surrounding reaction system into an excited state and transfers the energy to the recipient microspheres.Finally,fluorescence at 520~620nm is produced.The technology is easy to operate,less reaction time,and has high sensitivity.This experiment established the method of detecting SEB by AlphaLISA.The results were analyzed,and the standard curve of AlphaLISA was quantified using the nonlinear regression method.The linear range of SEB detection in the buffer was 25 pg/ml to 25 ng/ml,and the limit of detection was 25 pg/ml,with high sensitivity.In the simulated sample,the limit of detection can reach 50 pg/ml,and the method has good tolerance,less influence from the sample matrix and high stability.Inter-batch CV and in-batch CV in buffer and simulated samples were less than 10%,with good repeatability.The method has high specificity and does not cross-react with other classical enterotoxins,botulinum toxin,ricin and acacia toxin.This is important to identify food poisoning caused by SEB contamination.When AlphaLISA was applied to the detection of SEB in supernatant,it was found that the method was almost free from SPA interference.SPA is the main interfering factor of S.aureus immunoassay,which can produce high affinity binding with the FC segment of mammalian antibodies such as mice and rabbits.In this experiment,100ug/ml SPA was used to dilute SEB,and after detection it was found that SPA had little influence on the signal value of AlphaLISA.Therefore,the establishment of AlphaLISA detection method is applicable to the detection of SEB in food samples.when the SEB content in food samples contaminated by S.aureus is low,it also can be used to detect SEB in supernatant by isolating and cultivating S.aureus.In AlphaLISA technology,functional polymer microspheres are important materials.Polymer microspheres are polymer particles with spherical shape and particle size ranging from tens of nanometers to hundreds of microns.The main preparation materials are natural polymers and synthetic polymers,such as polystyrene.The research on the functionalization of microspheres has been the focus of related fields.The functionalization of microspheres now includes quantum dot,fluorescence,cavitation,magnetization and other methods.The commercial AlphaLISA technology microsphere was prepared by foreign companies and the specific synthesis method and the contained materials could not be obtained.However,with the development of fluorescent materials and the emergence of new photosensitizers,there are more and more room for improvement.In order to improve the performance of microspheres,how to load the functionalized materials stably is particularly important.In this experiment,the PS microspheres were loaded with fluorescent dye compositions by swelling method and the overall steps of synthesis were optimized.The microspheres synthesized under the optimized steps had good repeatability,and CV values were all less than 10%.The temperature and time of swelling were also explored.The temperature and time of swelling under 80?for 15 min microspheres signal strength value of the best.By transmission electron microscope to see external morphology of the non-swelling microspheres and the swelling microspheres under different temperature,of the swelling microspheres under 80? compared with before the swelling,dispersion was nearly unchanged,and swelling under 90?,is in a certain degree of polymerization and overlap.The results of transmission electron microscopy,may reveals a possible reason of a better swelling effect under 80?.The amount of EDC and the time of antibody conjugation were explored in the process of optimizing the steps.According to the results,in order to obtain the optimal activation effect and coupling efficiency,0.1mg EDC was used to activate the carboxyl functional groups on the surface of every 5mg microsphere,and the optimal antibody coupling time was 2h.The method of loading fluorescent dye on microspheres by swelling method has been basically completed,and tne method is stable.It lays a good foundation for the further improvement and application of fluorescent dyes.