Study On The Immunoassay Methods For Determination Of Phenylethanolamine A In Animal Food

Phenylethanolamine A is a phenethanolamine member of the β-adrenergicagonists family. The illegally use of it in animal production can bring a lot of adverseeffects to animal and human. So, the Ministry of Agriculture of China prohibitedPEAA from being used in feed and animal drinking water.In this study, two Immunoassay methods were developed for determination ofPEAA residue in animal food, including enzyme-linked immunosorbent assay (ELISA)and colloidal gold immunochromatography assay (CGIA).The results are shown asfollows:1. A PEAA derivative, PEAA-NH2was synthesized by hydrogenation usingRaney nickel as catalyst, before it was coupled to carrier proteins to generatePEAA-BSA, PEAA-OVA by diazotization. Using the conventional immunizationprotocol, three Balb/C mice were immunized with PEAA-BSA as the immunogen.The No.2mouse whose antiserum exhibited the strongest inhibition was selected forfurther fusion. The splenocytes from the selected mouse were fused with Sp2/0. Afterthe subcloning, three hybridoma clones, namely2H8,3D11and5C6, were shown toproduce antibodies stably. Based on titer and inhibition ratio, the antibody of2H8wasbetter than the other two clones and therefore2H8antibody was selected for furtheruse.2. The PEAA-HRP conjugate was synthesized by the sodium periodate method.Subsequently, a dcELISA method was established using the monoclonal antibody(clone2H8) and PEAA-HRP. With the PEAA concentration range of0.025-2.025ng/mL, the IC50value was0.204ng/mL and the LOD (IC10) was0.022ng/ml. Thecross-reactivity experiment demonstrated that the monoclonal antibody was highlyspecific for PEAA and its derivative (PEAA-NH2), with minor cross-reactivity (CR)with ractopamine (CR=8.3%) and negligible cross-reactivity with other15β-agonistscompounds (CR<0.1%). For sample testing, the LOD (limit of detection) values ofblank samples (pork, swine liver, swine urine and feed) were shown to be0.431,0.455,0.225and8.693ng/mL, respectively; the corresponding LOQ (limit ofquantification) values were0.578,0.647,0.360and14.477ng/mL, respectively. Therecovery rates ranged from84%to114%for intra-assay and82%to120%for inter-assay. The coefficients of variation were blow13.33%for inter-assay and blow10.53%for intra-assay. Furthermore, the dcELSIA was validated by liquidchromatography tandem mass spectrometry (LC-MS/MS) method and the resultshowed a high correlation coeffcient between the two methods. Overall the resultssuggested the dcELISA method established in this study could be used to detectPEAA residues in real samples reliably.3. The colloidal gold was prepared by citrate reduction method and the averagediameter of the colloidal gold was40nm estimated with the Spectrophotometer.Subsequently, a colloidal gold test strip was prepared using the monoclonal antibody(clone2H8). The optimum concentration of the monoclonal antibody was60μg/mL.The IC50value with strip reader was0.52±0.11ng/mL and the LOD (limit of detection)value with naked eye was2.7ng/mL. The detection result could be obtained within10min. The cross-reactivity experiment demonstrated that the monoclonal antibody washighly specific for PEAA and its derivative (PEAA-NH2), with minor cross-reactivity(CR) with ractopamine (CR=0.52%) and negligible cross-reactivity with other10β-agonists compounds (CR<0.1%). For sample testing, the LOD (limit of detection)value of blank swine urine samples was shown to be0.188ng/mL; the correspondingLOQ (limit of quantification) value was0.263ng/mL. The recovery rates ranged from92.00%to96.00%for intra-assay and95.00%to102.00%for inter-assay. Thecoefficients of variation were blow17.65%for inter-assay and blow10.75%forintra-assay. Furthermore, the test strip was validated by liquid chromatographytandem mass spectrometry (LC-MS/MS) method and the result showed a highcorrelation coeffcient between the two methods. Overall the results suggested the teststrip prepared in this study could be used to detect PEAA residues in swine urinesamples reliably.In this study, the anti-PEAA monoclonal antibody was prepared by hybridomatechnique. With the monoclonal antibody, two Immunoassay methods (including adcELISA method and a colloidal gold test strip) were established, which can detectPEAA residue in real samples reliably.