Establishment Of A Dual Fluorescence-Linked Immunosorbent Assay For Detecting IL-1Ra/IL-1β And Its Primary Application In Distinguishing Brucellosis Infection And Immunity

Interleukin-1(IL-1)is an inflammatory cytokine,including interleukin 1α(IL-1α)and interleukin 1β(IL-1β),which has various physiological functions and pathological implications.It is of great significance in linking innate immunity to a wide range of diseases other than inflammatory diseases.IL-1RN encoded interleukin-1 receptor antagonists(IL-1Ra)and IL-1β are both members of the IL-1family.It has been reported that the balance between IL-1Ra and IL-1β is related to many diseases,such as arthritis,diabetes,leukemia,brucellosis and so on.In another study,the prognosis of leukemia patients was judged by the changes in the levels of IL-1Ra and IL-1β in the patients’ serum.Therefore,the analysis of IL-1Ra and IL-1βlevels and their ratios in serum are indicative of disease to some extent.Brucellosis is a disease caused by the gram negative intracellular bacterium that is endemic between humans and animals and belongs to zoonotic diseases.Brucellosis seriously endangers human health and causes serious economic losses to the animal husbandry industry.At present,the prevention and control of brucellosis is carried out in the world by immunizing susceptible animals with vaccines and culling infected animals.However,brucellosis still cannot be eradicated,one of the reasons is that the infected and vaccine immunized animals cannot be differentiated,which leads to the accidental killing of healthy immunized animals.Infected animals with failed or missed immunizations are not culled in time,resulting in economic losses and causing difficulties in brucellosis control and decontamination.Therefore,based on the previous results of our research group,the experiment established a dual fluorescence-linked immunosorbent assay method for IL-1Ra/IL-1β on the basis of monoclonal antibodies,and applied this method to detect clinical serum samples of cattle and sheep to further explore the diagnostic significance of IL-1Ra and IL-1β ratio in distinguishing brucellosis infection and immunity.In the early stage of our research,we selected the conserved homologous sequences of IL-1Ra and IL-1β from cattle and sheep respectively,and named the differential protein sequences of IL-1Ra and IL-1β as IL-1Ra-1 and IL-1β-1,and some similar sequences were named as IL-1β-1Ra-2.By optimizing protein expression and purification conditions,high-purity IL-1Ra and IL-1β standards were prepared,and IL-1Ra,IL-1β and IL-1β-1Ra-2 immunogen proteins were obtained.We prepared IL-1Ra,IL-1β,IL-1β-1Ra-2,IL-1Ra-1 and IL-1β-1 monoclonal antibodies,selected Ab IL-1β-1Ra-2 monoclonal antibody with similar affinity to IL-1Ra and IL-1β as the capture antibody;selected Ab IL-1Ra-1 and Ab IL-1β-1specific monoclonal antibodies,conjugated with red and orange fluorescent microspheres respectively as detection antibodies.Through optimization of the conditions,a dual FLISA detection method for IL-1Ra and IL-1β was established,and the standard curve of IL-1Ra was finally obtained as y=0.0906x-0.0654,R2 was0.9904;the standard curve equation of IL-1β was y=0.1327x-0.1068,and R2 was0.9965.The lowest detection limit of IL-1Ra was 4.129 pg/m L,and the lowest detection limit of IL-1β was 3.197 pg/m L.The average recovery rate of IL-1Ra standard product in bovine serum was 112.18%,and the average recovery rate in goat serum was 108.9%;the average recovery rate of IL-1β standard product in bovine serum was 112.13%,and the average recovery rate in goat serum was126.56%,and the recovery rate and specificity were good.The established method was applied to clinical serum samples from cattle and sheep to further explore the diagnostic value of IL-1Ra to IL-1β ratio in distinguishing the infection and immunity of brucellosis.A total of 458 sheep serum samples and 163 bovine serum samples from the negative group,132 sheep serum samples and 58 bovine serum samples from brucellosis vaccine immunization group,180 sheep serum samples and 100 cattle serum samples from brucellosis infection group contained were detected.The results showed that the IL-1Ra/IL-1β value of the bovine brucellosis immune group was significantly different from that of the infected group;the IL-1Ra/IL-1β value of the sheep brucellosis immune group was significantly different from that of the infected group.In addition,the ROC curve analysis of cattle and sheep showed that the area under the curve of the immune group and the infection group was 1,and both were greater than 0.5,which has differential diagnosis significance.Therefore,the method in this study,with the help of the Rose Bengal Test,can be preliminarily applied to distinguish brucellosis infection and immunity in cattle and sheep.In summary,this study successfully established a dual-fluorescence immunosorbent assay method for the IL-1Ra/IL-1β,providing a new reference method for the differential diagnosis of brucellosis natural infection and vaccine immunity as well as the prevention and purification of brucellosis.