Studies On Chemiluminescence Immunoassay And Time-Resolved Fluorescence Immunochromatography Based On Olaquindox

Olaquindox,as known as quinamide alcohol,had a broad-spectrum antibacterial effect and could improve feed protein conversion rate.It was often used in feed additives.After research,it had been found to have toxicities such as accumulation toxicity and teratogenicity.2018 The Ministry of Agriculture of China issued Announcement No.2638,deciding to stop the use of three veterinary drugs such as quinethanol in animal foods,so it was particularly important to develop fast,efficient and accurate detection methods.Based on the antigen and monoclonal antibodies,the chemiluminescence system was studied in this paper.Lumilo chemiluminescence enzyme-linked immunoassay and time-resolved immunochromatographic techniques were developed,which were performed with conventional immunoadsorption tests and instrumental analysis comparison and verification,the main research contents were as follows:1.Monoclonal antibody production and identification of olaquindoxThree kinds of monoclonal hybridoma cells were resuscitated by rapid melting method,ascites was prepared by in vivo induction,and ascites was purified by caprylic acid-ammonium sulfate method.Identified the type and subtype of the monoclonal antibody as IgG1,its IC₅₀ was 22.013 ng/mL,and the cross-reaction rate with other commonly antibiotics was less than 0.01%.In this section,highly sensitive and highly specific quinethanol monoclonal antibodies were obtained.It laid the foundation for the subsequent immunoassay methods.2.Chemiluminescence immunoassayThis part the luminol-HRP-H₂O₂-Enhance chemiluminescence system was established.Through research on the dissolution characteristics and enhancement effects of four enhancers,the concentration of 4-bromophenol dissolved in 0.02 mol/ml Tris HCl（0.02 mol/L,pH8.5）with 4%DMSO was determined as the enhancer.The concentration of luminol was 1 mmol/L.the mixture of the two equal volumes was called liquid A,the dilution of 30%H₂O₂ by 500times was called liquid B.The detection time was 5min.With the RLU_max/IC₅₀ as the optimization standard,it was determined by the checkerboard method to use a white light-emitting board and indirect competition.The working concentration of the coated antigen and antibody was 0.94μg/mL and 1:4800.With the optimized conditions,the competition reaction time was 45min,the HRP enzyme-labeled sheep anti-mouse antibody was diluted to 6000 times,and the inhibition rate and the spiked concentration were finally fitted with a curve to obtain an"S"curve with IC₁₀=1.446 ng/mL and IC₅₀=14.6 ng/mL.Detection recovery rate was b etween 90%and 105%,and the CV was lower than 5%by precision analysis.Through the test of spiked samples,the results of CLEIA analysis were consistent with those of traditional instruments.3.Time-resolved fluorescence immunochromatographyIn this part,a time-resolved fluorescence immunochromatographic test strip was developed,which was traced by Eu³⁺labeled monoclonal antibody,and used indirect competitive immunoassay.The fluorescent microspheres were coupled with 60μg/ml antibody of equal volume in 0.05 mol/ml pH8.0 HEPES solution by EDC/NHS two-step method for 3 h.The activation time of carboxyl group in 0.02 mol/ml pH5.5 MES was 40 min.OlA-HS-BSA and Goat anti mouse antibody were scribed on NC membrane with 1μL/cm as T-line and C-line respectively.T/C value was used as optimization standard,Taking the T/C value as the optimization standard,it was determined that the coating concentrationt of T-line and C-line was 0.4 mg/ml and 0.6 mg/ml,coupling microsphere amount of 5μL,immunochromatography detection time and microplate mixing time of 15min and 6min respectively.Based on the optimal process conditions,the detection limit of naked eye is4ng/ml,the detection limit of instrument was 2.5ng/ml,the quantitative detection range was2.5 ng/ml²0ng/ml,the recovery rate was between 90%and 110%,and the CV was less than10%.The results of fluorescence immunochromatography were consistent with those of traditional instrument,and the quality of it was stable,It can be preserved for at least one year.In conclusion,this paper established the chemiluminescent immunoassay and time-resolved immunochromatography,which have the characteristics of high sensitivity,high accuracy and high stability,and have great research and application value.