Accurate Quantitative Method Of Protein Health Markers

The aim of this study is to determine the protein health markers.The problem of protein health markers quantification is classified into three categories:the total protein quantitative,the specific target protein quantitative without considering the activity and the active target protein quantitation.The solutions are put forward respectively,and the system of accurate quantitative method for protein health markers is systematically formed.First,for the quantitative system of different protein mixtures as health markers,the determination of serum total protein was taken as an example,and an internationally recognized reference method was used for accurate quantitative analysis.IFCC provides biuret reagent as a reference method for the determination of serum total protein.It has the advantages of simple operation,strong anti-interference ability and high accuracy.But even if the experimental parameters and experimental procedures are strictly regulated,the experimental data obtained from different laboratories may still be quite different.Therefore,it is of great significance to calibrate experimental instruments and control methodological data with a strict reference method to obtain accurate and reliable data by using biuret method.During the experiment,the wavelength and absorbance of the ultraviolet visible spectrophotometer used in the experiment are calibrated one by one using the filter standard material(GBW(E)130225,GBW(E)130226)according to the requirements of the reference method,And the pure water weighing method is used to calibrate the pipette.The traceable standard-bovine serum albumin(BW3627-1)and freeze-dried human serum total protein(GBW09187)are used as the samples to test the accuracy,repeatability,reproducibility,detection limit and quantitation limit of the biuret method.After confirming the accuracy and reliability of the method,we made quantitative determination and uncertainty evaluation of RELA's international comparative serum samples A and B.The results of the international comparison feedback show that our results are in good agreement with other laboratories,indicating the accuracy of the method and the reliability of the measurement technology.Secondly,for the accurate quantitative problem of the single protein health marker without consideration of activity,the determination of H-FABP was taken as an example,and the isotope dilution mass spectrometry was used for accurate quantification.The detection of myocardial damage markers is one of the important means of early diagnosis of cardiovascular disease.H-FABP is one of the markers of myocardial injury.It is of great significance for the diagnosis and treatment of cardiovascular disease.The purity of H-FABP was measured by mass balance method and isotope dilution mass spectrometry.The results were 0.795 g/g and 0.781 g/g respectively,and the accuracy,repeatability and uncertainty of the isotope dilution mass spectrometry were evaluated.Then,the isotope dilution mass spectrometry method of H-FABP in serum was established.The results were 2.56 ng/g,and the results were in good agreement with the results of ELISA kit.The accuracy,repeatability,recovery rate,detection limit and quantitative limit of the method were evaluated.Isotopic dilution mass spectrometry method of pure H-FABP and isotopic dilution mass spectrometry method of H-FABP in serum can be traced to SI units.The two methods can be used as standard methods for the pure substance of H-FABP and the standard substance of the H-FABP in serum and have great significance for the accuracy and interoperability of H-FABP.Finally,aiming at the quantification of active target protein,taking the soybean transgenic G2 protein as an example,the PCR protein quantificative method was used for accurate quantification..Most of the current methods of protein detection require standard substances to make a standard curve for relative quantification,which can not realize absolutely quantification foe target protein.Digital PCR quantitative technology is an absolute quantitative technique that can give the number of copies of nucleic acid without relying on the standard curve.Because of its simple operation,high sensitivity and high accuracy,it has become one of the essential technologies in molecular biology.Therefore,using the specific binding of antigen antibody to capture active protein,and then using digital PCR instrument as a measuring instrument,the absolute quantitation of active protein a can be realized.Based on the above assumption,we carried out relative quantitative experiments on fluorescent PCR and digital PCR to ensure the feasibility of the absolute quantitative method.In this paper,the fluorescent PCR G2 protein quantitative method and the digital PCR G2 protein quantitative method were successfully established by using the two differents point monoclonal antibodies mbAl,mbA2,G2 protein and TaqMan protein series kits.The accuracy,repeatability,reproducibility,detection limit and quantitation limit of the two methods were evaluated by using the G2 protein reference material as the test sample.The method parameters of the two methods are compared with those of the established ELISA method.The results show that the PCR protein quantitative method has high accuracy and repeatability.At the same time,the digital PCR G2 protein quantitative method is more accurate and stable than the fluorescence PCR G2 protein quantitative method,In addition,the digital PCR can directly give the copy number of the sample without relying on the standard curve,so the quantitative analysis of the concentration of the active protein is carried out on the digital PCR G2 protein quantitative method.