Establishment Of Three Rapid Immunology Detection Methods For MC-LR

Microcystins(MCs)are a class of toxins produced by cyanobacteria and they are widely present in freshwater.MCs have pentapeptide or heptapeptide-related structures and they have hepatotoxicity and other toxicity.MCs are enriched in fish and shellfish or other products and they enter into human body through the food chain and drinking water.MCs have a variety of isomers.microcystin-LR(MC-LR)is the most toxic and the most widely distributed of MCs.They caused a serious threat gof human health and food safety.The detection methods of MC-LR include the biological detection method,the high performance liquid phase detection method and the immunological detection method.The immunological tests are based primarily on the principle of the specific response between antigen and antibody,among them,ELSIA detection method has the characteristics include high-specificity,high-sensitivity,rapidity and it is easy to operate and suitable for mass detection.Colloidal gold immunochromatographic test strip has the characteristics of rapidity,simple operation and intuition.It is suitable for the initial detection of the site.Rapid detection of immunoassay has been paid more attention in MC-LR detection.In this study,based on the specific reaction between antigen and antibody,we prepared a detection of antigen as a solid-phase coating antigen,then the coating antigen was conjugated with the carboxy beads to prepare the magnetic bead probe and we used colloidal gold to label the MC-LR monoclonal antibody to obtain the gold standard probe.We established the indirect competitive ELSIA(ic-ELSIA)detection method,the MC-LR indirect competition ELSIA detection method based on nano-beads(IMB-ic-ELSIA)and and the colloidal gold immunochromatographic test strip detection method for the detection for MC-LR analysis.In this study,small-molecule MC-LR was conjugated with carrier protein-bovine serum albumin(BSA)to prepare solid-phase coated antigens by using the carbodiimide method.Ic-ELSIA was established by using the free target in the test and the solid-phase antigen in the microtiter plate competing for the specific monoclonal antibody.The main conditions for ic-ESLIA detection are optimized.The results showed that the concentration of the primary antibody was 2 ?g / m L and incubated at 4 ? for 12 h.The blocking agent was skim milk powder and incubated at 37 ? for 2 h.The optimal dilution ratio of monoclonal antibody was 1:4 000 and incubated at 37 ? for 1 h.TMB substrate was incubated at 37 ? for 10 min.The linear regression equation of MC-LR was y = 0.4485 x + 0.1014,the correlation coefficient R2 =0.9908.The detection range of MC-LR was 0.99 ng/m L-60.34 ng m L,IC50 = 7.74 ng/m L.The concentration of the standard was 40 ng/m L,20 ng/m L,10 ng/m L,5 ng/m L and the recoveries were 90.78%,88.90%,87.10% and 88.00%,and the average recoveries were 88.70%.We respectively collected South Lake water,the moon lake water,North Lake water and tap water for testing.MC-LR not detected;Detection of simulated samples,detection of the corresponding content of MC-LR.The solid-phase coated antigen and the carboxyl bead were used to prepare the magnetic bead probe,and the IMB-ic-ELSIA detection method was established by the antisense competition under the action of the magnetic field and the main conditions for IMB-ic-ESLIA detection are optimized.The results showed that the amount of magnetic beads was 40 ?g,the optimal dilution ratio of monoclonal antibody was 1: 16 000,and incubated at 37 ? for 10 min.The dilution ratio of enzyme-labeled secondary antibody was 1: 3 000 and incubated at 37 ?for 20 min.The TMB substrate was incubated at 37 ? for 8 min.The linear regression equation of MC-LR was y = 0.4303 x + 0.0248,the correlation coefficient R2 = 0.9904,the detection range of MC-LR was 1.50 ng/m L-107.15 ng/m L,IC50 = 12.59 ng/m L.The concentration of the standard was 100 ng/m L,60 ng/m L,20 ng/m L,5 ng/m L and the recoveries were 84.72%,91.38%,82.20% and 105.80%,and the average recoveries were 91.02%.We respectively collected South Lake water,the moon lake water,North Lake water and tap water for testing.MC-LR not detected;Detection of simulated samples,detection of the corresponding content of MC-LR.Colloidal gold solution with a diameter of about 20 nm was prepared by the method of sodium citrate reduction and then colloidal gold solution was incubated with MC-LR monoclonal antibody to prepare the labeled probe.Complete antigen as a detection line,anti-mouse secondantibody as a quality control line.The colloidal gold immunochromatographic test strip was established by using the free target in the analyte and the solid phase antigen on the NC membrane to compete with the colloidal gold probe,and the main reaction conditions were optimized.The main parameters were as follows: 100 ?L colloidal gold solution was added 0.9?L 0.2 mol/m L K2CO3 solution,the amount of monoclonal antibody was 1 ?g.The antigen concentration was 4 mg/m L,the secondary antibody concentration was 0.3 mg/m L,antigen fixative was 0.01 mol/m L PBS solution,NC film was sartorius 140.The test strip test limit was60 ng/m L.