Study On The Separation Process Of Procalcitonin Protein

As a highly specific biomarker of bacterial infection,procalcitonin(PCT)has been widely used in the research and development of reagents and production of kits for the diagnosis of inflammatory diseases.Therefore,the research on the separation and purification process of PCT has important application requirements and research significance.The complex serum containing PCT is used as the raw material,the principle of immunoaffinity is used to design and study the separation and purification of PCT experimentally,and a liquid-mass detection method has been established which is convenient for evaluating the separation effect of PCT.The main research contents are as follows:Firstly,a peptide enzymatic hydrolysis-liquid mass spectrometry method for the detection of PCT was established to lay the foundation for the separation.The principle is to use enzymatic hydrolysis to decompose PCT into multiple peptide segments,and use liquid mass spectrometry to detect the concentration of peptide segments,and then calculate the concentration.The single factor test method was used in the experiment to study the influence of trypsin dosage,time and other factors on the effect during the enzymolysis process.The results showed that when the ratio was 1:5 and the time was 12 h,the efficiency of PCT was the best.PCT was cleaved at a specific site,and multiple peptides with high response and stable appearance were obtained on mass spectrometry.The result is 0.551 mg/g,which is consistent with the measurement of one mature technology—Amino Acid Hydrolysis Method.The relative deviation of them is 2.5%,indicating that the method is reliable.In addition,the outstanding advantage of this method is that the detection of the characteristic peptide segment adopted enhances the anti-interference ability of the detection method and reduces the measurement error.Secondly,based on the principle of immunoaffinity,the process of separating and purifying PCT from complex serum mixture was designed.The separation process includes immunoaffinity unit,enrichment unit,elution unit and enzymolysis detection unit.In the immunoaffinity unit,surface plasmon resonance system was used to characterize the affinity between the four PCT antibodies and PCT.The affinity sequence was MB1>MB3>MB4>MB2,and MB1 was selected finally.In the enrichment unit,the effect of the molar ratio of PCT to MB1 on the enrichment efficiency was explored during the separation process.The results show that the effect was better when the ratio is1:3.In the elution unit,the single factor test method was used to investigate the influence of factors such as the type of eluent,dosage and time on the separation effect of PCT.The results show that the effect was better under 5vol% acetic acid,300 m L/mg(PCT)and 2 h.In the enzymolysis unit,the separation is carried out according to the optimized conditions and the designed PCT separation process,and then the PCT is subjected to enzymatic hydrolysis and detection.The detection method adopts the established peptide enzymolysis-liquid mass method,and the separation efficiency can reach96.09%,which meets the product requirements for separating PCT from complex matrices.The high separation effect is due to the high-efficiency affinity of MB1 antibody to PCT,which is the key to the effective enrichment in actual serum.Moreover,the experimental conditions of the enrichment and elution unit have been optimized,which makes the separation process more perfect.The research of PCT can help the development of immune kits and diagnostic reagents of it.