Establishment Of AlphaLISA Method For Detection Of SED And SARS-CoV-2

Amplified Luminescent Proximity Homogeneous Linked Immunosorbent assay(AlphaLISA)is a homogeneous immunoassay technique that relies on photo-induced chemiluminescence.Two types of donor microspheres and acceptor microspheres with a diameter of 200 nm interact with the antigen and antibody bound to them,and finally emit light at 520-620 nm,which have been widely used in the field of molecular biology.Staphylococcus aureus(S.aureus)belongs to the genus Staphylococcus and is the most common food-borne pathogenic microorganism in the world.This strain can produce one or several major Staphylococcal Enterotoxins(Staphylococcal Enterotoxins).,SEs),including SEA,SEB,SEC,SED and SEE.There are approximately 240,000 cases of staphylococcal food poisoning each year.Since Staphylococcus is widely present in the air,sewage,and skin,food is easily contaminated by SEs.Foods containing protein,starch,and high water content are more suitable for the growth of Staphylococcus aureus,such as various poultry,Meat,aquatic products,etc.Milk and dairy products are important sources of staphylococcal enterotoxin,which can produce a large amount of SED,so the rapid detection of SED is particularly important.We used the AlphaLISA method to detect SED and compared it with the traditional enzyme-linked immunosorbent assay(ELISA)method.First,the AlphaLISA detection SED system was established,and the optimal dilution ratios of AlphaLISA luminescent microspheres and biotin-labeled antibody were determined to be 1:50 and 1:1000,respectively;and the optimal concentration of ELISA coating antibody was 3 μg/m L.The optimal ratios of the labeled antibody and streptavidin-conjugated HRP are 1:1000 and 1:2000;the detection limit of AlphaLISA for buffer and milk simulation sample SED is 100 pg/m L,Coefficient of Variance(CV)It was 0.3%～8.9%;the detection limit of ELISA for buffer and milk simulation samples SED was 781.29 pg/m L,and the CV was 1.0%～19.8%.Neither AlphaLISA nor ELISA cross-reacts with other enterotoxin antigens,but compared with ELISA,AlphaLISA has higher sensitivity and better accuracy in detecting SED.The new type of coronavirus(SARS-CoV-2)can cause new type of coronavirus pneumonia(COVID-19)by infecting the lower respiratory tract,referred to as "new coronary pneumonia".There are approximately 10,000 confirmed cases of SARS-CoV and MERS-CoV nationwide,and a total of approximately 24.67 million confirmed cases have been reported globally.Therefore,it is urgent to detect SARS-CoV-2 quickly and accurately.With the deepening of research topics,we have established an AlphaLISA detection system for SARS-CoV-2,and determined that the monoclonal antibody 1# and the polyclonal antibody group are the best antibody pairs for the new coronavirus AlphaLISA detection,and AlphaLISA is against the new coronavirus N protein.The detection limit of antigen is 0.39 ng/m L,the intra-assay difference is between 1.90% and 8.93%,and the inter-assay difference is between 1.75% and 9.04%.It shows good reproducibility and good tolerance to the detection of different clinical samples.Compared with commercial ELISA kits,the detection sensitivity of inactivated viruses is higher.The AlphaLISA we have established can efficiently and quickly detect the new coronavirus.Since the microspheres required to construct the AlphaLISA detection method system were purchased from foreign companies,the price was relatively expensive,which limited the further application of the method.Polystyrene microspheres are polymer microspheres formed by polymerizing polystyrene monomers.According to the different preparation methods,the particle size of polystyrene microspheres can reach several nanometers to hundreds of microns.The preparation methods of fluorescent microspheres include adsorption method,bonding method,surface coating method,embedding method and swelling method.Among them,the swelling method is relatively simple in operation,stable in performance,and can be dyed accurately and quantitatively,so it is widely used.In this subject,the swelling method was used to add europium and ethylenic bond-containing compounds to polystyrene microspheres to prepare AlphaLISA receptor microspheres.The stability and repeatability of the synthetic method of acceptor microspheres were evaluated,in order to replace commercial microspheres to realize the localization of AlphaLISA acceptor microspheres.Using the same method to synthesize 3 batches of microspheres,and couple them with antibodies for antigen detection,the results show that all 3 batches of microspheres can obtain better detection results.It shows that the synthesis method has good repeatability and can be used for the large-scale preparation and synthesis of microspheres.The stability of the same batch of synthetic microspheres was also tested.Three time points were selected for testing within 2weeks,and good testing results were also obtained.Afterwards,the optimal conditions for the detection of synthetic microspheres were explored,and the AlphaLISA system constructed by 1:100 dilution of receptor microspheres and 1:1000dilution of biotin-labeled antibody was selected as the optimal conditions.Its detection range is 50 ng/m L-25pg/m L,with a wide detection range and extremely high sensitivity.The synthesis method of the microspheres described in this study has good repeatability and stability,and it also has good sensitivity for AlphaLISA detection,which can be used to replace commercial microspheres and realize the localization of AlphaLISA detection materials.