| Atrazine is a trazine herbicide,which is one of the conventional and low-cost herbicides in corn fields around the world.As atrazine is cheap and can effectively control annual grass and broad-leaved weeds,it has gradually become one of the most widely used herbicides in the world.With the massive and widespread use of atrazine,the impact of atrazine residues in the environment on the health of non-target organisms such as humans and the safety of environmental ecosystems has attracted increasing attention.Atrazine’s long residual effect can cause serious phytotoxicity to sensitive crops such as wheat and soybean.In addition to causing serious phytotoxicity to subsequent sensitive crops,atrazine also causes serious pollution to groundwater and surface water.Therefore,the development of efficient and sensitive atrazine detection technology is crucial for real-time monitoring of trace pollutants in the environment.Nanobody-based ELISA technology has the advantages of high efficiency,rapidity,sensitivity,and real-time,and to a certain extent makes up for the lack of accurate and efficient large-scale instrument methods that cannot be used for rapid on-site detection.Rapid testing has developed rapidly.However,various atrazine detection methods based on enzyme-linked immunosorbent technology currently rely on polyclonal antibodies or monoclonal antibodies.Based on the above problems,this study works on atrazine as the target.The main research contents are as follows.1.By immunizing llamas to obtain polyclonal antibodies,a polyclonal antibody-based enzyme-linked immunoassay for atrazine was established.Llamas with strong immune responses and without background were selected for immunization through pre-select,and the antiserum of llamas was collected.The optimal coating antigen concentration was 1μg/mL and the antiserum dilution factor was 50 000 folds,the optimal concentration of NaCl was determined to be 0.2 mol/L by the checkerboard method.The concentration of methanol was 5%and the optimal buffer pH was 7.4,the IC50 was 12.13 ng/mL,the linear detection range was 5.982～32.48 ng/mL,and the minimum detection limit was 0.0516 ng/mL;the specificity test was also carried out,tested the cross-reaction test of polyclonal antibodies against other three triazine herbicides and common environmental metabolites,the results showed that the cross-reaction rate of terbuthylazine was the highest at 95.8%,and simazine was 53.1%.,desisopropylatrazine was 40.5%,and the cross-reaction rate of other compounds tested was low;the addition and recovery test of actual samples was carried out with 3 river water from the environment,and the test results were consistent with LC-MS,the recovery rate of addition is between 90.1%and 114.2%,indicating that the developed atrazine ic-ELISA method can be used to detect actual samples of the environment.2.Nanobodies against atrazine were obtained by phage display,and the yield was up to 16 mg/L through optimization.Based on the preferred llama,the prepared immunization antigen is used to immunize the high-quality llama species.The whole blood after the 5th immunization was collected to extract lymphocytes,and RNA was extracted from it,reverse transcribed into cDNA,and two rounds of PCR were performed using the cDNA as a template,and the first round of amplification of all heavy chain antibodies(VHs and VHHs).The variable domains of IgG2,IgG3b,and IgG3a were amplified in the second round using 3 primer pairs to increase the diversity of the phage display library.On this basis,solid-phase panning was performed using both homologous and heterologous coating antigen at the same time.The concentration of the coating antigen was increased round by round,the washing solution and the number of plate washings were increased,and the concentration of atrazine used for elution was decreased.Nanobodies were successfully obtained after panning.The IPTG induction concentration,induction time and induction temperature during the prokaryotic expression and purification of nanobodies were optimized using the self-developed nanobody concentration test method.The optimal IPTG concentration was 1.0 mM,and the optimal induction time was 16 h.The optimal induction temperature was 30℃,and the B-Per lysis method was compared with the ultrasonic lysis method.The results showed that the B-Per lysis efficiency was higher than that of the ultrasonic lysis method.The effects of two different buffers,PBS and PB,on purification were compared.It showed that PB buffer was more suitable for purifying nanobodies,and the final yield of nanobody is 16 mg/L.3.An atrazine enzyme-linked immunosorbent assay method based on nanobodies was constructed and the key amino acid sites of nanobodies binding to small molecular compounds were predicted by molecular docking.An atrazine enzyme-linked immunosorbent assay method based on nanobodies was constructed,and the optimal concentration of coating antigen was 1.0 μg/mL,the optimal concentration of nanobody was 1.2 μg/mL,and the optimal NaCl concentration is 0.4 mol/L,the optimal methanol concentration in the system is 5%and the optimal buffer pH value is 7.4,the IC50 is 7.732 ng/mL,the linear detection range is 5.460～10.70 ng/mL,and the minimum detection limit is 0.0213 ng/mL;at the same time,the specificity test was carried out to test the cross-reaction test of nanobody to other three triazine herbicides and common environmental metabolites.The cross-reaction rate of simazine was 26.19%,the cross-reaction rate to desisopropylatrazine was 27.25%,and the cross-reaction rate of other compounds tested was low;the actual samples were taken from 3 river waters from the environment.Compared with LC-MS,the recovery rate of addition was between 97.2%and 114%,indicating that the developed atrazine ic-ELISA method can be used to detect actual samples of the environment.To initially explore the research mechanism,the nanobody protein sequence homology modeling was used,and high-quality models were obtained after ERRAT scoring,model Procheck evaluation and model Verify3D evaluation.Molecular docking,predicting the key amino acids for the binding of nanobodies to small molecular compounds.4.Developed a double-antibody sandwich enzyme-linked immunosorbent assay method for nanobody concentration,using two detection antibodies,when HRP-labeled anti-VHH mouse monoclonal antibody was used as the detection antibody.The optimal capture antibody concentration was 2.50 mg/L,the optimal NaCl concentration was 0.400 mol/L,the optimal methanol concentration was 5%,and the optimal buffer pH was 7.4.The EC50 value was 18.71 ng/mL.The range is 2.0～31.25 ng/mL;when HRP-labeled anti-HA secondary antibody is used as the detection antibody,the optimal capture antibody concentration is 1.00 mg/L,the optimal NaCl concentration is 0.400 mol/L,and the optimal methanol concentration is 5%,the optimal buffer pH is 7.0,the EC50 value is 2.145 ng/mL,and the detection range is 0.5～5 ng/mL;at the same time,this method was used to test 3-PBA nanobody and mEH nanobody,indicating that the developed nanobody Antibody concentration analysis method can be used for concentration testing of other Nanobodies.In summary,this study established an atrazine enzyme-linked immunosorbent assay method based on polyclonal antibodies through llama immunization,which provided ideas and experience for the subsequent application of camel polyclonal antibodies;through phage display technology,8 strains of nanobodies were successfully obtained by panning;high-quality models were obtained by using the homology modeling of nanobody protein sequences,and the nanobody models were molecularly docked with small molecule compounds with high cross-reactivity rates,and predicted nanobodies.Key amino acids for the binding of antibodies and small molecule compounds;through commercialized mouse monoclonal antibodies against nanobodies,an enzyme-linked immunoassay method for detecting the concentration of nanobodies was developed,and the quantitative analysis of nanobodies was realized. |
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