Study On The Preparation And Immunological Detection Of The Erythromycin Molecularly Imprinted Polymer

In the paper, the erythromycin molecularly imprinted polymer(EM-MIP) prepared by bulk polymerization is used as artificial antibody to replaced erythromycin(EM) biological antibody. And then a method detecting erythromycin residues, CLIA, is built. The results are as follows: The preparing of EM-MIP 1、It is prepared by EM as template molecules, MAA as functional monomer, EGDMA as cross-linker, acetonitrile as solvent and AIBN as imitator, and by the thermostatic thermal polymerization method. 2、 Condition optimizing:(1) The usage amount of template molecule, functional monomer, crosslinker: when the proportion is 1:4:20 the adsorption is best;(2) The choice of solvent(Tetrahydrofuran, methanol, acetonitrile): acetonitrile is the best;(3) The usage amount of Initiator(0.01g、0.05g、0.1g、0.2g). The optimal aggregation effect of template molecule and functional monomer occurs when the usage is 0.1g;(4) The different bath temperature(20℃、40℃、60℃、80℃) of polymerization is 60℃;(5) The optimal times of polymerization is 24h;(6) The choice of elution conditions: when the Volume ratio between Methanol and acetic acid is 3:1, eluting in the soxhlet extractor, the eluting time is shorter and the affect is better. 3、The research of performance of Erythromycin molecularly imprinted polymer(1) The research of adsorption properties: The adsorption capacity of molecularly imprinted polymer is better than the non- molecularly imprinted polymer. And when the concentration of the solution of erythromycin is 170mg/m L the adsorption, 130.8mg/mg, reaches the maximum, which is more than 34.6mg/mg of the non- molecularly imprinted polymer. It can be clear that the molecularly imprinted polymer shows predominant absorption to Erythromycin.(2) The research of Adsorption kinetics: The Adsorption kinetics Curve don’t change along with initial substrate concentration. When the concentration of Erythromycin reaches 140mg/m L, the curve sharply raises. Along with the time going the hole of MIP hunts more and more Erythromycin molecules, and then the absorption condition achieves balance at the 4h.(3) The adsorption quantity of Erythromycin molecularly imprinted polymer in ervthromycin,luo ervthromycin and chloramphenicol solution for 4 hours,concentration of 140mg/m L was respectively 90.4mg/mg、30.2mg/mg、18.6mg/mg. It can be concluded that the absorption capacity of molecules imprinted polymer is far greater than the absorption capacity of its structural analogs of chloramphenicol and roxithromycin, namely the MIP takes the specific adsorption capacity with erythromycin.(4) The research of Thermal stability:Overall the absorption property goes down gently with the rising temperature,The amount of adsorbed polymer reached 108.4mg/mg, and the highest absorption capacity is still 83% when the polymer is dealed with 100%. So it can be concluded that the molecule imprinted polymer is not affected by temperature.(5) The research of resistance to acid and alkali corrosive: The polymer’s shape remained unchanged after soaking acid ﹠ alkaline solution and its absorption capacity goes 92.9%-96.8% of the highest capacity. Specially the absorption by weak acid﹠alkaline is slightly higher than the strong. It indicates that the MIP is almost not affected by chemistry.(6) The research of repeatability: The MIP’s absorption goes down, one after the previous time. The fifth adsorption quantity is 112.3mg/mg. The absorption of the fifth adsorption-desorption experiments is approximately 87.46% than the first. Thus, the MIP reused does not affect the absorption markedly. 4、Combine the Direct competitive ELISA, built by the ME-MIP replacing the biological antibody, and CLIA to detect the EM residues in the water. And then the standard curve is established that abscissa is EM’s concentration and the ordinate is luminescence value. The linear equations is y=-3154.5x+27563 and the correlation coefficient is 0.988, the limit of detection of CLIA is 0.87μg/L and the Linear range 0.9-8.1μg/L, and the results of CLIA is that the recovery is 87.7%-93.2%, the intra-assay RSD is less than 8.5% and the inter-assay RSD is less than 13.2%. 5、The established EM-CLIA method and EM ELISA kit were used to detect EM in real samples. The results showed that when the spiked level in the sample was 1, 3 and 9μg·kg-1, results of EM residues by the EM-ELISA kit and EM-CLIA were 0.926±0.02, 2.786±0.05, 8.674±0.04mg/kg and 0, 2.568±0.17, 7.963±0.13mg/kg, highly significant difference between the two methods.