Study On Matrix Effects Of Five Kinds Of Meat On Colloidal Gold Immunochromatographic Assay And Pretreatment Methods Of Chicken For Detection Of Sulfadimidine

Sulfadimidine?SM₂?,which is a kind of sulfonamides,is widely used in fisheries and animal husbandry.It can affect the synthesis of folic acid in bacteria by preventing bacteria from using para-aminobenzoic acid,leading to the inability to synthesize the nucleoprotein of bacterial and achieved the purpose of bacteriostasis.However,SM₂ and its metabolites can be accumulated in muscle tissues,organs,or other edible parts of animals and enter the human body through the food chain,thus causing great harm to human health.At present,there are instrument detection methods and immunochromatographic assay for the detection of SM₂ in foods.Compared with traditional instrument detection methods,Colloidal gold immunochromatographic assay?CG-ICA?based on antigen-antibody specific binding has many advantages of convenience,rapidity,and sensitivity.However,the CG-ICA method may be influenced by the different samples,causing large errors in test results.In this paper,a method for the quantitative detection of SM₂ with CG-ICA was developed.Some important parameters of the method were optimized.The signal intensities of test line and control line were recorded by a portable colloidal gold strip reader and recorded as A_T and A_C,respectively.The values of A_T/A_C of negative and positive samples were defined as B₀ and B,respectively.The standard curve was established by plotting the B/B₀ against the concentration of SM₂.The results showed that when the value of pH of colloidal gold solution was 7.0,the labeling amount of anti-SM₂ mAb was 3.2?g in 1 mL colloidal gold solution,the concentration of complete antigen?SM₂-BSA?was 0.8 mg/mL,and the detection time was 20 min,the established detection method had a linear range of 0.1 ng/m L to 96 ng/m L with good linear correlation?R²=0.9938?.The values of LOD and IC₅₀ were 0.104 ng/m L and0.308 ng/mL,respectively.The recoveries of the method in chicken,pork,shrimp,beef,and fish were ranging from 93.7%to 102.9%,85.6%to 86.5%,80.6%to 89.6%,76.7%to 81.6%,and 70.1%to 73.4%,respectively.The matrix effects of five meat matrices?chicken,pork,shrimp,fish,and beef?on detection of SM₂ with CG-ICA were systematically explored.The results showed that the regression equations of the standard curves based on chicken,pork,shrimp,beef,and fish extracts were y=-0.4795 x-0.6206,y=-0.4436 x-0.6989,y=-0.4539 x-0.5567,y=-0.4237 x-0.3771,and y=-0.381 x-0.6351 with a reliable correlation coefficiencies?R²=0.9945,0.9819,0.9949,0.9824,and 0.9743?,respectively.The values of LOD of detecting SM₂ with CG-ICA in chicken,pork,shrimp,beef,and fish were 0.063,0.075,0.030,0.028,and 0.165 ng/mL,respectively.The linear ranges of standard curves based on chicken,pork,and shrimp were from0.1 ng/m L to 48 ng/m L.The linear ranges of standard curves based on fish and beef were both from 0.04 ng/m L to 6 ng/m L.By comparing the standard curves based on the five meat extracts with the standard curve based on PBS,the result showed that the standard curve based on the chicken extract was closest to that based on PBS,indicating that the matrix effect of the chicken sample was the weakest.The standard curves based on pork and shrimp extracts deviated from that based on PBS,indicating that the matrix effect of pork and shrimp was strong,and the standard curves of fish and beef samples had a greater deviation from that based on PBS.It showed that the matrix effect of fish and beef was stronger,especially the beef.In this paper,three pretreatment methods for extracting SM₂ in chicken were studied:phosphate buffer saline method,immunomagnetic beads adsorption method,and GB29694-2013 method.The parameters of immunomagnetic beads adsorption method were optimized.The 0.01 M pH 5.0 PBS was chosen as the buffer solution of coupling 80?g anti-SM₂ mAb to 1 mg magnetic beads.When the dosage of immunomagnetic beads was 350?g and the time to capture sulfadimidine was 45minutes,the recovery and the coefficient of variation?CV?of the immunomagnetic beads adsorption method were 98.8%to 102.9%and 4.7%to 6.2%,respectively.The recovery and CV of the phosphate buffer saline method were 40.5%to 78.5%and8.7%to 10.4%,respectively while the recovery and CV of the GB29694-2013method were 90%to 107.1%and 8.5%to 9.8%,respectively.The phosphate buffer saline method had low recovery and high CV although it was rapid and convenient.Both GB29694-2013 method and immunomagnetic beads adsorption method had high and stable recovery.And the immunomagnetic beads adsorption method have advantage of time-saving and have remarkable potential for practical application.