Rapid Detection Of Mycotoxin And IL-6 By Time-resolved Immunoassay Strip

Objectives:An immunochromatographic strip based on europium nanoparticles?EuNPs?fluorescent microspheres was constructed for rapid quantitative detection of mycotoxin and IL-6 in human serum.These two indicators may serve as potential markers of fungal infection.To provide new ideas and technical methods for clinical diagnosis of fungal infection.Methods:1.After crushing,grinding and sieving,80%methanol solution was added to extract mycotoxins from the samples of Chinese traditional medicine pills by solvent extraction.The time-resolved immunoassay strip and ELISA were used to detect the mycotoxins in 5samples with the same dilution concentration.The time-resolved immunoassay strip was used to detect the mycotoxins in 22 kinds of Chinese traditional medicine pills sold in the market.They were divided into four groups:clearing away heat,regulating qi and eliminating food,activating blood circulation and removing blood stasis,and tonifying deficiency.2.AuNPs-chicken IgY and AuNPs-MIS02 complexes,EuNPs-chicken IgY and EuNPs-MIS02 complexes were obtained by coupling 70-nm colloidal gold with chicken IgY and IL-6 labeled antibody?MIS02?,respectively.The complexes were evenly mixed and sprayed onto the bonding pad to dry.Diluted sheep anti-chicken IgY antibody and IL-6coated antibody?MIS01?were used to mark quality control line?C line?and detection line?T line?on the fixed position of NC membrane,respectively,and dried at 37?.According to the order of sample pad,bonding pad,NC film and absorbent paper,they are assembled on PVC hard board and packed into test strip clamp.The sample was directly added into the sample adding hole.After reaction for a certain time,it was detected by gold standard paper reader and fluorescence analyzer.The T/C ratio was obtained,and the results were obtained by standard curve.Recombinant IL-6 antigen solution diluted by Tris-HCl buffer was used to evaluate the linearity,sensitivity,tureness,accuracy and specificity of two kinds of test strips.214 clinical serum samples were divided into two groups.Colloidal gold test strip and time-resolved test strip were used to detect the clinical serum samples,and the results were lined with chemiluminescence method by linear regression fitting analysis.Results:1.Linear regression fitting of time-resolved immunoassay strips and ELISA results is highly correlated.AFB₁:y=0.2718x+7.0017,n=5,R2=0.9925,?p<0.01?;DON:y=0.3824x+26.447,n=5,R2=0.9955?p<0.01?;ZEN:y=3.9444x-82.296,n=5,R2=0.9455?p<0.01?.The 22 kinds of Chinese traditional medicine pills sold in the market have different degrees of AFB1,DON and ZEN pollution.The content of AFB₁ was1.0²9.94?g/kg,DON was 100⁶13.49?g/kg and ZEN was 57.08⁸398.43?g/kg.2.IL-6 colloidal gold strip has good linearity and good specificity.The linear range is2-200pg/mL.The sensitivity is 1.26pg/mL.The recoveries ranged from 101%to102%.In-batch precision CV and inter-batch precision CV were 8.31-8.95%and 9.15-14.71%respectively.The results are in good agreement with those of Siemens automatic chemiluminescence immunoassay:y=0.9357x+7.2941?n=206,R2=0.8306,p<0.01?.IL-6 time-resolved strip has good linearity and good specificity.The linear range is2-500pg/mL.The sensitivity is 0.37pg/mL.The recoveries ranged from 102%to106%.In-batch precision CV and inter-batch precision CV were 5.92-8.87%and 7.59-9.04%respectively.The results are in good agreement with those of Siemens automatic chemiluminescence immunoassay:y=0.9502 x+6.1397?n=214,R2=0.9520,p<0.01?.3.Statistical methods:The comparison test and linear evaluation were fitted and analyzed by the linear regression method,and the regression equation and fitting degree were obtained;the sensitivity calculation was based on the linear equation,which was obtained by the formula 2*3 SD₀/??X₂-?X₀?;the precision was evaluated by the coefficient of variation within and between batches;the accuracy was evaluated by the recovery rate;and the specificity was evaluated by the coefficient of variation between batches.The results of PCT,CRP and IL-8 high-value samples were evaluated.Conclusions:1.The time-resolved strips can be used for rapid detection of AFB₁,DON and ZEN in traditional Chinese medicine pills.The toxins of these three fungi may become potential markers of fungal infections and serve as one of the indicators for the diagnosis of fungal infections.2.In this study,an immunochromatographic assay for IL-6 based on colloidal gold and Eu nanospheres was established.Both methods are simple,fast and low-cost.By comparison,time-resolved immunofluorescence based on europium nanospheres has higher sensitivity and precision than colloidal gold method and wider linear range and the linear fitting effect is better in clinical serum sample comparison test.Indicating that the performance of time-resolved immunofluorescence test strip based on europium nanoparticles is better than that of colloidal gold test strip.3.In this study,a time-resolved test strip was established to detect mycotoxin and IL-6.It provides a new way of thinking and technology for clinical diagnosis of fungal infections.