Study On Broad-spectrum And Rapid Determination Of α-zearalanol Compounds And β-lactam Antibiotics

In recent years,the problem of veterinary drugs residues in animal-derived food has caused wide public concern.Long-term exposure to veterinary drug residues in food could adversely affect human health,and even increase the levels of antimicrobial resistance.To strengthen the supervision of veterinary drug residues,it’s necessary to establish fast,low-cost,high-sensitive and high-throughput detection methods for the initial screenings of a large number of samples.α-Zearalanol（α-ZAL）andβ-lactams antibiotics（BLAs）are the most commonly used veterinary drugs that can promote animal growth,prevent and treat animal disease.The metabolic profile ofα-ZAL could result in more than oneα-ZAL class residues in food;and in animal husbandry,multiple BLAs are used simultaneously and residue in food.The study focused on broad-spectrum recognition materials,a broad-spectrum monoclonal antibody（m Ab）against all six ofα-ZAL class was prepared,then an ultrasensitive indirect competitive enzyme linked immunosorbent assay（ic-ELISA）and a colloidal gold immunochromatography assay（GICA）were established for the rapid multi-residue detection ofα-ZAL class;penicillin-binding protein that can be bound with most ofβ-lactams antibiotics was expressed in vitro,and a receptor-based assay was established for the rapid multi-residue detection ofβ-lactams antibiotics.（1）Based on the structural characteristics ofα-ZAL class,the position-7 of ZAN was modified and then coupled with the carrier proteins BSA and OVA.anti-α-ZALs m Ab were obtained and identified,the subclass type was Ig G1,the titer was up to 10⁵,the affinity constant was 1.20×10⁸L/mol,and the cross-reaction rates of common mycotoxin and growth promotion were less than 0.1%.（2）Based on anti-α-ZALs m Ab,an indirect competitive ELISA for simultaneous detection ofα-ZAL class was developed.The IC₅₀ values ofα-ZAL and its analogues ranged from0.080～0.194 ng/m L,and the detection range was 0.020～0.835 ng/m L.After analyzing and processing the matrix effect of the real samples,the ic-ELISA can be used in the detection of real sample such as muscle,liver,kidney,milk and milk powder,and the recovery rates ranged from 79.2%～104.2%,the coefficients of variation（CVs）were less than 11.4%.（3）Based on anti-α-ZALs m Ab,a colloidal gold immunochromatography assay（GICA）was developed to specifically determine residues ofα-ZAL class.The cut-off concentrations ofα-ZAL class tested by GICA were all confirmed to be 5 ng/m L by unaided visual assessment.Quantitative detection was performed with a test strip reader,the sensitivity(IC₅₀)values ranged from 1.225～1.800 ng/m L,and the limit of detection(IC₁₀)values ranged from0.105～0.227 ng/m L.After analyzing and processing the matrix effect of the real samples,the GICA can be used in the detection of milk sample,and the recovery rates of milk sample were85.6%～93.9%,the CV values were lower than 12.4%.（4）Based on the mechanism ofβ-lactam antibiotics,penicillin-binding proteins（PBPs）can be bound with most ofβ-lactams antibiotics,thus PBPs were selected as the broad-spectrum recognition material forβ-lactam antibiotics.Select the DNA fragment pbp3 from S.pneumoniae R6 as the template,via gene recombination,in vitro expression and affinity purification,the purified PBP3*at a concentration of 0.5 mg/m L was obtained,and it was confirmed to have biological activity.（5）By using the specific binding betweenβ-lactam antibiotics and receptor protein PBP3*,the purified receptor protein was directly immobilized on the high adsorption 96-well polyethylene reaction plate.Based on color development reaction and enzymatic chemiluminescence,a receptor-based microplate assay was developed for simultaneous detection of fifteenβ-lactam antibiotics in milk,the limits of detection ranged from 0.28～100.30ng/m L,which were lower than the EU MRLs.Based on the receptor-based microplate,a receptor-based chemiluminescence assay was developed,and IC₅₀values was reduced by 8～10times.Through the treatment of matrix effects,the two methods can be used in the detection of milk sample,and the recovery rates of milk sample ranged from 72.9%～89.3%,the CV values were lower than 12.6%.