| Macrolides (Macrolides, MALs) have outstanding antibacterial activity on most of gram-positive bacteria and mycoplasma, such as Staphylococcus aureus, Streptomyces, etc. In the veterinary clinic, they are primarily used for the prevention and treatment of respiratory and gastrointestinal diseases of livestock and poultry, but because MALs drugs can cause ototoxicity and renal toxicity and other side effects, a serious threat to human health, their residues in animal foods caused concern at home and abroad in recent years. MALs multi-residue rapid detection immunoassay was studied in this paper, in order to provide technical support for MALs residue monitoring in animal foods.In this study, an anti-tilmicosin (TIM) PAb was prepared. The IC5o values of anti-TIM was 1.1 μg/L. The PAb had 45.8% cross-reactivity (CR) with tylosin (TYL). In addition, the effect of different types of hapten heterology on TIM ELISA sensitivity was evaluated. The results showed that the degree of hapten heterology parallels the improvement of ELISA sensitivity.A simultaneous multi-residue colloidal gold immunochromatographic (IGCA) methods for MALs in milk was established. Major factors including colloidal gold-labeled antibody system, antigen-coated system, materials technology system, sample adding mode were screened and optimized. Then, the product adaptability for area sample, low temperature anti-jamming capability, acceleration and long-term stability were investigated. The cut-off value for erythromycin (ERY), spiramycin (SPI), TIM and TYL was 5,5,10, and 20 μg/L, respectively. Milk sample without pre-treatment, four drugs synchronization detection time is within 10 min,the false positive and false negative rates are both 0. The Long-term stability test results showed that:MALs colloidal gold test strip is valid up to 12 months.A simultaneous multi-residue fluorescent microspheres immunochromatographic (FMCA) methods for MALs in milk was established. The fluorescent microspheres markers were firstly screened, then, fluorescent microspheres labeled antibody system, antigen-coated system, materials technology system, sample adding mode were researched. Then, the reaction amplification system stability, low temperature anti-jamming capability, accelerated and long-term stability were assessed. Under the UV light, The cut-off value for ERY, SPI, TIM and TYL was 2.5,2.5,2.5 and 5 μg/L, respectively. Milk samples were simply diluted, four drugs synchronization detection time is within 20 min. With the fluorescence signal reader, the LOD for the four MALs were 0.13 μg/L. The recoveries of ERY. SPI and TIM at levels of 1.25-5 μg/L ranged from 91.8-109.2%,89.6-114.4% and 84.8-111.6%, respectively, with the CV ranged from 5.4-11.3%,7.9-15.7% and 6.2-13.7%, respectively. The recoveries of TYL at levels of 2.5-10 μg/L ranged from 85.8-115.2%, with the CV ranged from 8.5-14.9%. The long-term stability test results show that:the strip is valid up to 12 months.ELISA method for four MALs in milk residue detection was established. Various parameters which impact on the performance of ELISA method were optimized, including the antigens or antibody concentration, reaction time, temperature, pH value, ion concentration, organic solvent and sample pretreatment.The LOD of the ELISA for ERY, SPI, TIM and TYL was 0.272,0.609,0.358 and 0.962 ug/L, respectively. The recoveries of ERY, SPI, TIM and TYL at levels of 1.0-4.0 μg/L ranged from 72.8-103.7%,75.3-106.8%,84.9-113.6% and 75.2-105.7%, with the CV ranged from 5.4-11.8%, 6.6-9.4%,3.7-10.3% and 6.6-12.3%. Stability test results showed that:ELISA kits stored at 4℃, the validity is up to 12 months.Finally,20 blind milk samples were detected by the IGCA, FMCA and ELISA, with HPLC-MS/MS method results for parallel. The results showed that:The correlation coefficient (R2) of the test results for the IGCA, FMCA, ELISA.and HPLC-MS/MS was> 0.99, a good correlation between the four methods. This showed that the high accuracy and good reliability for the established immunological method. |
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