Research On Separation Techology Of Prostate Specific Antigen

Prostate-specific antigen protein(PSA)in human serum is a tumor marker for prostate cancer,and detection kits made from it are in great demand.Since the content of PSA in human serum is extremely low,it is necessary to develop a separation and concentration process.Starting from the establishment of an anti-interference PSA detection method,the paper designs and studies the separation process of PSA from serum,obtains the optimized process conditions for PSA separation and concentration,and detects the concentrated PSA by the signature peptide-liquid mass method.The main research contents of the thesis are as follows.Firstly,signature peptide-liquid mass spectrometry method for measuring PSA concentration is established.The accurate detection of PSA concentration is the basis of its separation process.The method is based on the principle that the signature peptides of PSA has a specific response on liquid mass spectrometry,and the concentration of PSA is detected.When establishing the method,firstly,by simulating the enzymolysis peptides and combining the enzymolysis experiment data,it is concluded that HR,FR and LK are the signature peptides of PSA,and the labeled peptides is synthesized.Then trypsin was used as the enzymatic hydrolyzing agent to hydrolyze PSA as signature peptides,and the single factor test method was used to investigate the influence of the enzymatic hydrolyzing agent dosage and enzymatic hydrolysis time on the enzymatic hydrolysis effect.When the mass ratio of enzymolysis agent to PSA is 1:70,the enzymatic hydrolysis effect is the best;further investigation of the enzymatic hydrolysis time factor shows that the optimal enzymatic hydrolysis time is 10 hours.Under the optimized conditions,six groups of PSA solutions with a concentration of 75.31 μg/g were detected by the characteristic peptide-liquid mass spectrometry method.The determined concentration was74.74 μg/g and the RSD value was 0.96%,indicating that the method established in this study It is reliable and accurate,and since the method is a detection method based on characteristic peptides,it has the characteristics of strong anti-interference and wide applicability.Then,based on the principle of affinity between PSA and the prepared antibody-magnetic beads,a process for separating and concentrating PSA was designed.The components of this process are(1)an affinity unit that uses antibody-magnetic beads to enrich and adsorb serum PSA;(2)an enrichment and separation unit that separates PSA-antibody-magnetic beads using the principle of magnetic attraction;(3)the elution unit of the PSA concentrate is obtained by elution of the eluent and the separation of the magnetic beads;(4)a detection unit for detecting the concentration of the PSA concentrate by the signature peptide-liquid mass method.In this study,human serum supplemented with PSA was used as raw material.First,antibodies with good affinity for PSA were selected in the affinity unit by surface plasmon resonance technology to prepare antibody-magnetic bead solution,it was found that when the volume ratio of PSA to serum was 1:10,the enrichment efficiency of PSA reached 95.53%,and the separation was basically complete.On this basis,the single factor test method was used to systematically study the influence of factors such as the type,dosage and elution time of the eluent in the elution unit on the separation and concentration of PSA.The experimental results showed that the 5 vol% acetic acid solution with a volume ratio of 1:5 to serum was used as the eluent,and the elution effect was the best when the elution time was2 min.Under the optimized conditions,the serum PSA was separated according to the designed process.In the experiment,the separation efficiency was61.52%,which met the separation requirements.The good separation effect is mainly due to the high-efficiency affinity of PSA antibodies to PSA,and the affinity unit is the key unit for enriching PSA from serum.