Establishment Of Immunochromatographic Test Strips For Detecting IL-1Ra/IL-1β And Its Preliminary Application And Discussion On Diagnosing Infection And Immunity Of Brucellosis

Interleukin(IL),as a class of cytokines generated by leukocytes,is proved to be relatively hefty and hefty.The interleukin-1 family(IL-1F)is involved in the immune process and plays an important role in inflammatory response.Interleukin1β(IL-1β)and its receptor antagonist(IL-1Ra)are the most important members of IL-1F.During the inflammatory reaction,the expression of IL-1βincreases,promoting the inflammatory process.IL-1Ra is often antagonistic to IL-1 biological function.The ratio(IL-1Ra/IL-1β)caused by the interaction between IL-1Ra and IL-1βis an important indicator in the diagnosis and treatment of many diseases,including arthritis and brucellosis.Brucellosis cis a serious zoonotic epidemic.Because there is no reliable method to distinguish the infection of brucellosis from the differential diagnosis of immunity at present,a large number of animals immunized with brucellosis are culled as the animals with brucellosis due to the positive antibody reaction in the detection of serum,which brings huge economic losses.Our research group previously found that the expression levels of IL-1βand IL-1Ra in brucellosis presented significant changes,and further studies showed that the IL-1Ra/IL-1βratio was significantly different between the naturally infected animals and the artificially immunized animals in brucellosis.Therefore,it was proposed that IL-1Ra/IL-1βmight be an important indicator for the differential diagnosis of brucellosis infection and immunity.Based on the previous research results,this project aims to establish the detection method of double-t lines colloidal gold immunochromatographic strips for IL-1Ra/IL-1β,and initially apply the detection method to the differential diagnosis of brucellosis to explore the diagnostic value of IL-1Ra/IL-1β.In the experiment,the full-length recombinant proteins of high-purity IL-1βand IL-1Ra were used as the standard,and the multi-species conserved region recombinant proteins of IL-1βand IL-1Ra were used as the immunogen,and the anti-IL-1β-1,the anti-IL-1Ra-1,and the anti-IL-1β-1Ra-2 were successfully prepared.The colloidal gold probe was successfully conjugated with IL-1β-1Ra-2 rabbit polyclonal antibody.IL-1Ra/IL-1βdouble-T lines colloidal gold immunochromatographic strips were successfully assembled using sartorius 140 NC membrane.The working concentration of line T1(anti IL-1β-1 antibody)was 2 mg/mL,and that of line T2(anti-IL-1Ra-1 antibody)was 1 mg/mL.The working concentration of line C(sheep anti-rabbit antibody)was0.8 mg/mL.The working procedure of the strip was as follows:after the serum sample was premix and fully combined with the colloidal gold probe,centrifuge at 14000RPM for 20 min,discard the supernatant and use 100μL loading buffer to resuspend the precipitate,and then the resuspended droplets were added to the strip sample pad.After the strip dried out,the results were taken pictures and analyzed with ImageJ software.Finally,the standard curve equation for determining IL-1βwas y=24701x+9679.9,R~2 was 0.9726,and the detection limit was 0.7 ng/mL.The standard curve equation of IL-1Ra was y=35117x+3247.9,R~2 was 0.9753,and the detection limit was 1.0 ng/mL.The specificity and repeatability of the IL1Ra/IL-1βtest method were good,and the coefficient of variation was 9.63%.The double-T lines colloidal gold immunochromatographic strips detection method established in this study was preliminarily applied to the detection of IL-1Ra/IL-1βin brucellosis sheep serum samples.The results showed that there was a significant difference in IL-1Ra/IL-1βbetween the immunized sheep serum samples and the sheep serum samples infected with brucellosis.ROC curve analysis confirmed that IL-1Ra/IL-1βhas diagnostic reference value in distinguishing artificial immune sheep from naturally infected sheep.Sample to a larger extent discharge infection group,choose IL-1Ra/IL-1βof the immunized group twice beta median and standard deviation 73.69 as a differential reference line,the difference of the actual tests,get the positive samples using tiger red plate agglutination test screening sheep sheep(infection and immunity),further testing positive samples IL-1Ra/IL-1βbeta,ratio greater than 73.69 for immune group health sheep,ratio is less than 73.69 for the infection of sheep,as culling of target.In addition,we will further detect the ratio changes of IL-1Ra/IL-1βin other diseases to explore the broader diagnostic significance of IL-1Ra/IL-1β.