A Preliminary Exploration On Separating Microcystins From Water By Immunoaffinity Chromatography

Objective:Microcystins(MCs) are a group of natural toxins known to be produced by certain species of cyanobacteria. Microcystin-LR(MC-LR) is known to be of the acute hepatic toxicity and many other hazards to human. Many analytical methods are used for MCs detection, and some of the more popular methods include phosphatase assay, ELISA immunoassay, liquid chromatography(LC), LC with mass spectrometric (MS) detection. However, there are many interfering substances which largely influence the test results. Reversed C18 has been widely used for concentrating MCs and reducing unrelated substances before MCs analysis, but some studies demonstrated that some trace interfering substances still remained even after C18 clean-up, which indicating that a more effective clean-up method is required. Immunoaffinity chromatography (IAC) with its high specificity and high efficiency has attracted many attentions. This article explores the main influencing factors in Immunoaffinity chromatography preparation, including the antibody(ligand) purification, the vector selection, the optium concertration of coupling antibody, the coupling time of ligand with vector and the blocking time of vector's residual active groups. We try to establish a method of immunoaffinity chromatography with high specificity, high adsorption and complete elution characteristics to separate MCs from water, which is conducive to following accurately quantitative detection of MCs in the environment.Methods:In the first segment of overall research, anti-microcystin polyclonal antibodies are precipitated and purified by saturated ammonium sulfate method, and protein concertration is detected by ultraviolet spectrophotometry (UVS). The purified antibodies'titer is measured by indirect-ELISA. The purified antibody and the Sepharose CL-4B act respectively as a ligand and a vector to form an immunoaffinity column. The preparation process is as follows:the vector is activatedby N,N-Carbonyldiimidazole (CDI) and then coupled with ligand in boric acid buffer solution. The conditions of IAC preparation are improved in three aspects involving the concentration of antibody-coupling, the coupling time and the blocking time. Coupling rate and the amount of ligand binding are detected by UVS. In the process of IAC adsorbing algal toxins, methanol is used as eluant and the optium volume of methanol is selected. The efficacy of IAC is evaluated by column capacity, the antigenic specificity and the differences between columns. In the second segment, the method of IAC combiad with indirect competitive ELISA (idc-ELISA) is applied to detect the amount of MCs in environmental water. The comparison between IAC column and reverse C18 column are made for evaluation of their clean-up effects. The recovery rate of IAC comined with idc-ELISA system is measured to test its accuracy through adding standard MC-LR to the sample.Results:The concentration of purified antibody is 85.78mg/ml, and its titer ranges from 1:16000 to 1:32000. Antibody affinity constant ranges from 2.798×107 to 5.596×107. The conditions of IAC preparation are as follows:the concentration of antibody-coupling is 8mg/ml, the coupling time is 24h at 4℃, and the blocking time of the actived groups by ethanolamine is 4h at 4℃. In the process of IAC adsorbing MC-LR, the eluent is methanol and its volume is 10ml. The IAC is specific for MC-LR, and its capacities are between 233.1ng and 284.5ng. The degree of the difference among five IACs is small, the standard deviation is 97.27%±2.86%, and the coefficient of variation (CV) is 0.029. The recovery rate of the same IAC adsorbing the standard MC-LR is around 95%. The effect of the IAC adsorbing MCs in environment water is the same as C18, and the recoveries after adding standard MC-LR to the samples detected by IAC combined with idc-ELISA are 75.97%±2.7% and 72.49%±2.1%.Conclusion:An immunoaffinity chromatography column for MC-LR has been developed. The purified antibody affinity constant ranges from 2.798×107 to 5.596×107, which satisfys the requirement of antibody affinity constant (between 107 and 1012). The IAC is specific for MC-LR, and the degree of the difference among five IAC columns is small. The effect of the IAC adsorbing MCs in environmental water is the same as C18, which demonstrates that IAC is a good column for adsorbing MC-LR.