Research On Applications Of Immunoassay In Detection Of Antibiotics And Proteins

In recent years,the development of animal husbandry is rapid along with the massive products and wide use of veterinary drugs.However,the veterinary drugs residues in animal-derived foods are potentially hazardous to human health,which became the vital issue to be solved urgently.Proteins acting as direct performers of life activities are biomarkers for various diseases.The selective separation and detection of protein biomarkers from complex biological samples has become a challenge in proteomics,diagnostics,and genetics.There are several difficulties involved in the detection of veterinary drugs residues and proteins in real samples,especially the low concentration of target analytes and the complexity of sample matrix.Therefore,the development of rapid and effective method for detection of veterinary drugs residues and proteins is required.Immunoassay based on highly specific antigen-antibody interactions has been widely applied in food inspection,drug analysis,and diagnosis owing to its super properties,including high sensitivity,specificity,and throughput.In this study,we report the development of an indirect competitive biotin-streptavidin-amplified-based BA-ELISA for the determination of kanamycin and a magnetic immunoaffinity extraction-fluorescence spectroscopy assay(MIAE-FSA)for the separation and detection of bovine serum albumin(BSA).The major research works are listed in the following two aspects:(1)We report the preparation of anti-kanamycin polyclonal antibodies and the development of BA-ELISA based on the biotin-streptavidin amplification system for the specific detection of kanamycin residues in milk and honey.Several significant parameters were investigated and optimized in detail.Under optimum conditions,the linear range of kanamycin was 0.3-30 ng mL-1 and the 50%inhibitory concentration was 6.48 ng mL-1.The limit of detection and limit of quantitation for kanamycin were 0.07 ng mL-1 and 0.22 ng mL-1,respectively.Cross-reactivity values of the primary antibody with tobramycin,gentamicin,amikacin,and apramycin were all less than 1%.This novel BA-ELISA method was applied for analysis of kanamycin in milk and honey samples with satisfactory recoveries of 91.0%-103.3%.The developed protocol was also validated against liquid chromatography-mass spectrometry,returning a significant correlation(R2>0.99).These results indicate that this BA-ELISA is highly sensitive,specific,and precise,providing a viable option for monitoring kanamycin residues in milk and honey samples.(2)We report the preparation of anti-BSA polyclonal antibodies and the development of MIAE-FSA for the selective separation and detection of BSA from human serum matrix.The immunomagnetic microspheres(IMMs)with a unique core-shell structure was synthesized using hydrothermal,sol-gel,and glutaraldehyde conjugation techniques.The as-prepared IMMs were used as adsorbent for the rapid capture of target proteins,and the target proteins were then detected by fluorescence spectroscopy.The IMMs showed good dispersibility,favorable stability,and strong magnetic responsiveness.The maximum binding amount of polyclonal antibodies on the magnetic microspheres reached 32.2 mg g-1 after optimizing the antibody immobilizaiton conditions.Under optimal extraction conditions,the maximum adsorption capacity of target proteins was 107.7 ?g g-1.After the IMMs being recycled for ten cycles,the extraction efficiency remained about 80%.The proposed method was applied for the capture of BSA from spiked human serum with satisfactory recoveries more than 89.3%.The proteins separated by IMMs were of high purity,which was confirmed by MALDI-TOF-MS.These above results demonstrate that this MIAE-FSA is highly specific,selective,rapid,and reusable,providing an effective method for selective purification and detection target proteins form spiked human serum samples.