CN113176133A - Method for separating protein and lipid in blood plasma or blood serum and matrix blood serum - Google Patents
Method for separating protein and lipid in blood plasma or blood serum and matrix blood serum Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention relates to the technical field of biological products, and particularly discloses a method for separating protein and lipid in blood plasma or blood serum, which comprises the following steps: s1, weighing ammonium sulfate, adding the ammonium sulfate into negative plasma or negative serum, mixing, and centrifuging to obtain a supernatant I; s2, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate A; s3, adding the supernatant I into the mixed precipitate A, uniformly mixing and centrifuging, and taking a supernatant II; s4, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate B; s5, adding the supernatant II into the mixed precipitate B, uniformly mixing and centrifuging, taking the supernatant III, and filtering to obtain the matrix serum. The method can effectively remove most of protein and lipid substances in blood plasma and blood serum, eliminate the influence of matrix effect on clinical detection results, and improve the stability of matrix blood serum.
Description
Technical Field
The invention relates to the technical field of biological products, in particular to a method for separating protein and lipid in blood plasma or blood serum and matrix blood serum.
Background
The quality control aims to detect errors in the analysis process, control each link related to analysis and ensure that the laboratory result is accurate and reliable. The comprehensive quality control comprises quality control of three main processes before, during and after analysis, and the quality of the final inspection result can be ensured only by correctly recognizing, detecting and controlling errors of each link of the three processes. The key of quality assurance lies in how to correctly understand the cognition and better select the quality control serum, so the quality of the quality control serum directly influences the reliability and the accuracy of the test result.
The existence of matrix effects has plagued laboratory testing for many years because matrix effects are present in almost all samples and are affected to varying degrees by any analyte tested for. The cause of the matrix effect is related to the composition of the sample to be measured and the composition and processing technology of the quality control material. The effect of the matrix on the clinical test cannot be neglected. At present, there are many related methods for studying matrix effect reduction, for example, chinese patent CN105675772B discloses a method for matrix effect reduction in a liquid chromatography-tandem mass spectrometry detection technology, when detecting pesticide residues in fruits and vegetables by a liquid chromatography-tandem mass spectrometry detection method, a small volume sample injection is adopted: when the sample is a weak matrix effect sample, the sample introduction volume is less than 5 mu L; when the sample is a medium-intensity matrix effect sample, the sample introduction volume is less than or equal to 2 mu L; when the sample is a sample with strong matrix effect, the sample is diluted by a certain degree of matrix and then is injected in a small volume. This method can reduce the matrix effect, but is cumbersome to handle, and it does not directly improve the quality control serum itself or the separation method, and thus, there is room for improvement.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for separating protein and lipid in blood plasma or blood serum, which can effectively remove most of protein and lipid substances in the blood plasma and the blood serum, eliminate the influence of matrix effect on clinical detection results, improve the stability of the matrix blood serum, and ensure that the prepared matrix blood serum is consistent with the matrix of a clinical sample.
The first object of the present invention is to provide a method for separating proteins and lipids from plasma or serum, which comprises the steps of:
s1, weighing ammonium sulfate, adding the ammonium sulfate into negative plasma or negative serum, mixing, and centrifuging to obtain a supernatant I;
s2, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate A;
s3, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S2, uniformly mixing and centrifuging, and taking a supernatant II;
s4, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate B;
s5, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S4, uniformly mixing and centrifuging, taking the supernatant III, and filtering to obtain the matrix serum.
The inventor of the invention discovers through a great deal of research and experiments that most of protein and lipid substances in blood plasma and blood serum can be effectively removed by adopting the method, the influence of matrix effect on clinical detection results is eliminated, the prepared matrix blood serum has stronger stability, and further, the quality control substance obtained by diluting the matrix blood serum has stronger stability, and the requirement of actual measurement can be met.
Ammonium sulfate is added into negative plasma or negative serum, and can compete with protein for water molecules in the serum or plasma solution, so that a hydration film on the surface of the protein is damaged, the solubility of the protein is reduced, and the protein is precipitated from the solution;
by adding activated carbon and diatomaceous earth to the negative plasma, the porous structure of activated carbon provides a large amount of surface area, making it very easy to achieve the effect of adsorbing impurities. The diatomite has a fine porous structure and strong adsorption capacity; and the activated carbon and the diatomite are added again, so that the adsorption effect is better than that of the activated carbon and the diatomite which are added at one time, and the adsorption effect is weakened when the adsorption and desorption are in a balanced state after the activated carbon adsorbs impurities to a certain degree.
By adding active carbon and dextran into negative serum, the dextran plays a role of a molecular sieve; and the activated carbon and the dextran are added again, so that the adsorption effect is better than that of the activated carbon and the diatomite which are added at one time, and the adsorption effect is weakened when the adsorption and desorption are in a balanced state after the activated carbon adsorbs impurities to a certain degree.
The lipids include total cholesterol, triglyceride, phospholipid, etc. The protein includes total protein, albumin, globulin, etc.
In a preferred embodiment of the method of the present invention, in the steps S2 and S4, the mass concentration of the ammonium sulfate in the negative plasma is 50% to 100%, and the mass ratio of the activated carbon to the diatomaceous earth is (5-8): 1-2.
In the technical scheme of the invention, the mass concentration of ammonium sulfate in negative plasma is 50-100%;
the specific proportion of the activated carbon and the diatomite is set, so that the effect of separating protein and lipid in negative plasma is better, the influence of matrix effect on clinical detection results is eliminated, and the prepared matrix serum has stronger stability.
In a preferred embodiment of the method of the present invention, in the steps S2 and S4, the mass concentration of the ammonium sulfate in the negative serum is 50% to 100%, and the mass ratio of the activated carbon to the dextran is (5-8): (1-2).
The active carbon and the dextran have better effect of separating protein and lipid from negative serum by setting a specific proportion, the influence of matrix effect on clinical detection results is eliminated, the prepared matrix serum has stronger stability, and the reliability and the accuracy of the clinical detection results are improved.
In a preferred embodiment of the method of the present invention, the concentration of sodium chloride in the physiological saline is 0.9% in steps S2 and S4.
In the technical scheme of the invention, in the process of forming the mixed precipitate A and the mixed precipitate B, 0.9% of normal saline is added for soaking the activated carbon/diatomite/dextran to achieve the effect of fully wetting.
As a preferred embodiment of the method of the present invention, in the step S2 and the step S4, the centrifugation speed is 4000-5000rpm, and the centrifugation time is 20-30 min.
The second object of the present invention is to provide a substrate serum, which is obtained by the above method for separating proteins and lipids from plasma or serum.
The matrix serum disclosed by the invention has a good detection result when being used for diluting an HCV positive serum sample, and has the minimum difference with a theoretical value concentration; the diluted HCV positive serum sample can be stably stored for 5 days when placed in an environment of 25 ℃, and nucleic acid degradation occurs in comparative examples 1-3, even the nucleic acid degradation cannot be detected. Therefore, the matrix serum provided by the invention is used for diluting HCV serum samples and has the advantages of good dilution effect and good stability.
The third purpose of the invention is to provide the application of the substrate serum in the preparation of HCV RNA quality control substances.
The fourth purpose of the invention is to provide the application of the substrate serum in preparing HBsAg quality control substances.
The fifth purpose of the invention is to provide the application of the substrate serum in the preparation of the saccharide antigen 125 quality control substance.
The sixth purpose of the invention is to provide the application of the substrate serum in diluting clinical samples.
Compared with the prior art, the invention has the following beneficial effects:
1) the invention provides a method for separating protein and lipid in blood plasma or blood serum, which can effectively remove most of protein and lipid substances in blood plasma and blood serum, eliminate the influence of matrix effect on clinical detection results and improve the stability of matrix blood serum;
2) the invention provides a matrix serum, which is consistent with a matrix of a clinical sample and is mainly used for preparing a quality control substance and diluting the clinical sample.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 76.8g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative plasma, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4000rpm for 20-30min, and taking supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.5g of activated carbon and 0.1g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of the tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.5g of activated carbon and 0.1g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of the tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4000rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a filter membrane of 0.45 mu m to obtain the matrix serum.
Example 2A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 100g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative plasma, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 5000rpm for 20-30min, and taking supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.5g of activated carbon and 0.2g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature by using a shaker, centrifuging at 4500rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of the tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.5g of activated carbon and 0.2g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature by using a shaker, centrifuging at 4500rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of the tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4000rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a filter membrane of 0.45 mu m to obtain the matrix serum.
Example 3A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 50g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative plasma, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4500rpm for 20-30min, and reserving supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.8g of activated carbon and 0.1g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of the tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.8g of activated carbon and 0.1g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of the tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4000rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a filter membrane of 0.45 mu m to obtain the matrix serum.
Example 4A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 100g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative plasma, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4500rpm for 20-30min, and reserving supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.8g of activated carbon and 0.2g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 5000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of a tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.8g of activated carbon and 0.2g of diatomite, adding 20mL of the physiological saline prepared in the step S2, shaking and uniformly mixing, shaking for 30 minutes in a room-temperature shaking table, centrifuging at 5000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of a tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4500rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a 0.45-micrometer filter membrane to obtain the matrix serum.
Example 5A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 38.4g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative serum, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4000rpm for 20-30min, and reserving supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.5g of activated carbon and 0.1g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes in a room temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of a tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.5g of activated carbon and 0.1g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes in a room temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of a tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4000rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a filter membrane of 0.45 mu m to obtain the matrix serum.
Example 6A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 50g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative serum, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4000rpm for 20-30min, and reserving supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.5g of activated carbon and 0.2g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes in a room temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of a tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.5g of activated carbon and 0.2g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes in a room temperature shaking table, centrifuging at 4000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of a tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4000rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a filter membrane of 0.45 mu m to obtain the matrix serum.
Example 7A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 100g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative serum, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 5000rpm for 20-30min, and reserving supernatant I;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.8g of activated carbon and 0.1g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature by using a shaker, centrifuging at 4500rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of a tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.8g of activated carbon and 0.1g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature by using a shaker, centrifuging at 4500rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of a tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4500rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a 0.45-micrometer filter membrane to obtain the matrix serum.
Example 8A method for separating proteins and lipids from plasma or serum
A method for separating proteins and lipids from plasma or serum comprising the steps of:
s1, weighing 50g of ammonium sulfate powder, adding the ammonium sulfate powder into 100mL of negative serum, slowly adding the ammonium sulfate powder into the solution while stirring, standing for 30min, centrifuging at 4500rpm, and keeping supernatant I after 20-30 min;
s2, weighing 0.9g of sodium chloride, dissolving the sodium chloride in 40mL of sterilized pure water, and finally fixing the volume to 100mL to form 0.9% physiological saline.
S3, weighing 0.8g of activated carbon and 0.2g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature by using a shaker, centrifuging at 5000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate A at the bottom of a tube;
s4, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S3, uniformly mixing and centrifuging, and taking a supernatant II;
s5, weighing 0.8g of activated carbon and 0.2g of dextran, adding 20mL of the physiological saline prepared in the step S2, shaking and mixing uniformly, shaking for 30 minutes at room temperature, centrifuging at 5000rpm for 20-30 minutes, removing supernatant, and leaving mixed precipitate B at the bottom of a tube;
s6, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S5, shaking and uniformly mixing, shaking for 3 hours at room temperature, centrifuging at 4500rpm for 30 minutes, collecting the supernatant III, and filtering the supernatant III by using a 0.45-micrometer filter membrane to obtain the matrix serum.
Test example I measurement of proteins and lipids before and after treatment with human negative plasma and human negative serum
Human negative plasma and human negative serum were treated by the methods of example 1 and example 5, and the results of the detection were statistically analyzed using the SPSS19.0 chi-square test, and the results are shown in tables 1 and 2.
Table 1 plasma/serum protein measurements before and after treatment (N ═ 5)
Table 2 plasma/serum lipid measurements before and after treatment (N ═ 5)
The results show that: the method can effectively remove most of protein and lipid substances in blood plasma and blood serum, eliminate the influence of matrix effect on clinical detection results and improve the stability of matrix blood serum.
Second test example, evaluation experiment of substrate Effect and interchangeability in preparing HCV RNA quality control substance Using the substrate serum of the present invention
1. Experimental Material
(1)25 different types of fresh clinical patient samples: selecting samples with concentration values covering high, medium and low concentration ranges of prepared samples and no known stabilizer and preservative interferents;
(2) preparation to be tested: HCV RNA quality controls S1, S2, S3 prepared from the negative serum after the treatment of example 5, the calf serum after the treatment, and the negative plasma after the treatment of example 1;
(3) and (3) comparing the kit: roche COBAS AmpliPrep/COBAS TaqMan HCV Test, version 2.0;
(4) evaluation kit: hepatitis C virus nucleic acid detection kit (PCR-fluorescent probe method) of Daan Gen-John GmbH of Zhongshan university
2. Experimental methods
And (3) operating the preparation to be detected and 25 fresh clinical patient samples according to corresponding kit instructions, measuring 25 clinical samples and the preparation to be detected by using three detection systems of Roche and Daann, repeatedly measuring for 3 times, and performing standard PCR amplification detection.
3. Experimental results (LoG LOG mean)
TABLE 3
As a result: evaluation kit for detecting mean values of to-be-detected preparations S1, S2 and S3
All are within 95% credible interval of clinical fresh serum samples, which indicates that the to-be-detected preparations S1, S2 and S3 have no matrix effect on the evaluation kit, and indicates that the substance has interchangeability between the comparison kit and the evaluation kit.
Test example III test on the stability of HCV RNA quality control substance (liquid) prepared by Using the serum of the substrate of the present invention
1. Instruments, reagents and samples
The main apparatus is as follows: ABI7500 PCR detector;
the main reagents are as follows: a hepatitis C virus nucleic acid quantitative detection kit (PCR-fluorescence quantitative method) of Daan GenBank of China, Zhongshan university;
sample preparation: HCV RNA quality control substances (liquid) prepared from the treated matrix serum, such as S1, S2 and S3;
2. inspection method
Stability profile of the reference substance under specific storage conditions. Extracting a proper amount of standard substance samples, marking, respectively placing in a specified environment, carrying out inspection according to a specified period, extracting 5 tubes of standard substance each time, and calculating the average value. The experimental plan is detailed in table 4. The detection results were analyzed using SPSS19.0 statistical software.
TABLE 4 Experimental schedules
3. The results of the experiments are shown in tables 5-16.
TABLE 5 HCV quality control S1 stability test results [ 80 deg.C, control group, IU/mL ]
TABLE 6 stability test results of HCV quality control substance S1 (-20 + -5) deg.C, IU/mL)
TABLE 7 stability test results of HCV quality control substance S1 [ 2-8 deg.C, IU/mL ]
TABLE 8 stability test results of HCV quality control substance S1 [ 20-25 deg.C, IU/mL ]
TABLE 9 stability test results of HCV quality control S2 [ 80 deg.C, control group, IU/mL ]
TABLE 10 stability test results of HCV quality control substance S2 (-20. + -. 5 ℃ C., IU/mL)
TABLE 11 stability test results of HCV quality control substance S2 [ 2-8 ℃ C., IU/mL ]
TABLE 12 stability test results of HCV quality control substance S2 [ 20-25 ℃ C., [ IU/mL ]
TABLE 13 HCV quality control S3 stability test results [ 80 deg.C, control group, IU/mL ]
TABLE 14 stability test results of HCV quality control substance S3 (-20. + -. 5 ℃ C., IU/mL)
TABLE 15 stability test results of HCV quality control substance S3 [ 2-8 ℃ C., IU/mL ]
TABLE 16 stability test results of HCV quality control substance S3 [ 20-25 deg.C, IU/mL ]
4. Conclusion of the experiment
HCV RNA in blood plasma is easily degraded by RNase, and protein and lipid components in the blood plasma influence the stability of the RNA; experimental results show that the HCV quality control substance diluted by the matrix serum is stored in the environments of minus 20 +/-5 ℃, 2-8 ℃ and 20-25 ℃, and is subjected to T test statistical analysis with the HCV quality control substance stored in the environment of minus 80 ℃, wherein P values are all larger than 0.05, so that the HCV quality control substance diluted by the matrix serum can be stable for 14 months in the environment of minus 20 +/-5 ℃, can be stable for 2 months in the environment of (2-8) ℃ and can be stable for 6 days in the environment of (20-25), the stability meets requirements, and the actual measurement needs can be met.
The stability of the HCV quality control substances diluted with the matrix serum prepared in the other examples is similar to that of the matrix serum prepared in example 1 and example 5.
EXAMPLE four results of different substrate serum dilutions on HBsAg detection by ELISA
Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used immunoassay methods for serological markers of infectious diseases, and is currently and generally applied to blood screening of blood donors for free blood collection institutions in China. The factors such as lipid, bilirubin, hemoglobin and blood viscosity in clinical samples can interfere with the ELISA assay results. For example, the blood lipid sample has high viscosity and is combined with lipophilic components to generate a shielding effect, and the lipid can replace water in cells, so that the blood lipid sample has a certain dilution effect on a sample and has a large influence on a detection result. Besides, hemoglobin contains substance similar to horse radish peroxidase, and can be colored with color-developing agent. The detection linear range of the HBsAg is narrow, the content of the HBsAg in serum of a patient often exceeds the detection range of the kit, and the patient needs to be diluted and then measured to obtain an accurate and reliable result. Therefore, the selection of the diluent directly affects the accuracy and reliability of the result.
A semi-automatic Victor31420 fluorescence detector of Perkinelmer company and HBsAg positive serum with the concentration of 1000IU/mL (wherein HBsAb, anti-HIV, anti-HCV and anti-TP are all negative) are selected, the HBsAg positive serum diluted by the matrix serum prepared in the example 5 and the negative serum are respectively detected by adopting a hepatitis B virus surface antigen diagnostic kit of Shanghai Kehua bioengineering GmbH, and each sample is subjected to gradient dilution according to 2000 times, 3000 times, 4000 times, 5000 times, 6000 times and 7000 times, and is detected for 10 times. The S/CO values of HBsAg positive samples with the same concentration in different diluents are analyzed by t-test.
TABLE 17 results of two S/CO value sets
As is clear from the results in Table 17, the S/CO values of HBsAg-positive serum diluted with the matrix serum of the present invention were higher than those of HBsAg-positive serum diluted with the negative serum at the same concentration, and the difference therebetween was statistically significant (P < 0.05). And the standard deviation of the detection result of the HBsAg positive serum diluted by the negative serum is larger than that of the detection result of the substrate serum diluted by the negative serum, which indicates that the components of the negative serum may influence the ELISA reaction environment except the dilution effect on the HBsAg positive serum.
The S/CO values of the HBsAg-positive serum diluted with the substrate serum prepared in the other examples were similar to those of the substrate serum prepared in example 5.
EXAMPLE V preparation of HBsAg quality control substance (liquid) stability experiment 1, Instrument, reagent and sample Using the matrix serum of the invention
The main apparatus is as follows: semi-automatic Victor31420 fluorescence detector from PerkinElmer;
the main reagents are as follows: hepatitis B virus surface antigen diagnostic kit (enzyme linked immunosorbent assay) from Zhuhaizhu reagent GmbH;
sample preparation: HBsAg quality control prepared from the treated matrix serum of example 5 (liquid);
2. inspection method
Stability profile of the quality control material under specific storage conditions. And (3) extracting a proper amount of quality control samples, marking, respectively placing the samples into a specified environment, checking according to a specified period, extracting 5 tubes of quality control substances each time, and calculating a mean value. The experimental plan is detailed in table 18. The detection results were analyzed using SPSS19.0 statistical software.
TABLE 18 Experimental schedules
3. The results of the experiments are shown in tables 19-22.
TABLE 19 HBsAg quality control stability test results [ 80 deg.C, control group, IU/mL ]
TABLE 20 HBsAg quality control substance stability experiment results [ 20 + -5 deg.C, IU/mL ]
TABLE 21 HBsAg quality control substance stability experiment results [ 2-8 deg.C, IU/mL ]
TABLE 22 HBsAg quality control substance stability experiment results [ 20-25 deg.C, IU/mL ]
4. Conclusion of the experiment
Experimental results show that HBsAg quality control substances diluted by matrix serum are stored in the environment of minus 20 +/-5 ℃, 2-8 ℃ and 20-25 ℃, and are subjected to T test statistical analysis with the HBsAg quality control substances stored in the environment of minus 80 ℃, wherein P values are all larger than 0.05, so that the HBsAg quality control substances diluted by the matrix serum can be stable for 14 months in the environment of minus 20 +/-5 ℃, can be stable for 4 months in the environment of (2-8) ℃ and can be stable for 15 days in the environment of (20-25) ℃, and the stability meets requirements and can meet the actual measurement requirements.
The stability of the HBsAg quality control substance diluted with the matrix serum prepared in the rest examples is similar to that of the matrix serum prepared in example 5.
EXAMPLE six assay of matrix serum dilution of beta-HCG according to the invention
The β -HCG values are often high in concentration, outside the detection range, and require multiple dilutions of the sample. In 2016, Yangchang et al reported that the difference between the detection result of beta-HCG and the theoretical value is large due to the matrix effect when a patient sample is diluted by physiological saline and deionized water.
Fresh high-value serum (8255mIU/mL) is selected, the substrate serum, the negative serum (untreated), normal saline and deionized water of example 5 are respectively adopted to dilute the sample by 20 times and 50 times, the diluted sample is repeatedly detected for 3 times, an electrochemiluminescence method is adopted to detect, and statistical software SPSS19.0 is used to analyze the result.
TABLE 23 test results (mIU/mL) of high value fresh samples diluted with different dilutions
TABLE 24 chi fang test
a.0 expected count of cells (0.0%) is less than 5. The minimum expected count is 54.90.
As is clear from Table 23, when the results of the 20-fold and 50-fold dilutions were compared with the theoretical values, the bias of the results of the dilution with the matrix serum was minimal.
As can be seen from table 24, the desired frequency of 0 grid is less than 5, the minimum desired frequency is 54.90, and N is 2439>40，x2588.842, degree of freedom df 4, P<0.001, the difference is statistically significant, so that it can be considered as fourThe dilution effect of different dilutions is not all equal.
The serum dilution of the matrix prepared in the other examples showed similar effects to those of the matrix prepared in example 5.
EXAMPLE seventhly, stability experiment of CA125 quality control substance (lyophilized powder) prepared from matrix serum of the invention
1. Instruments, reagents and samples
The main apparatus is as follows: siemens Centaur;
the main reagents are as follows: a Siemens mating reagent;
sample preparation: CA125 quality control (lyophilized) prepared from the treated matrix serum of example 5;
2. inspection method
Stability profile of the quality control material under specific storage conditions. And (3) extracting a proper amount of quality control samples, marking, respectively placing the samples into a specified environment, checking according to a specified period, extracting 5 tubes of quality control substances each time, and calculating a mean value. The experimental plan is detailed in table 25. The detection results were analyzed using SPSS19.0 statistical software.
TABLE 25 Experimental schedules
3. The results of the experiments are shown in tables 26-29.
TABLE 26 CA125 quality control substance stability test results [ 80 deg.C, control group, U/mL ]
TABLE 27 CA125 quality control substance stability experiment results [ 20 ℃ below zero +/-5 ℃, U/mL ]
TABLE 28 CA125 quality control substance stability experiment results [ 2-8 deg.C, U/mL ]
TABLE 29 CA125 quality control substance stability experiment results [ 20-25 deg.C, U/mL ]
4. Conclusion of the experiment
Experimental results show that the CA125 quality control substance prepared from the matrix serum is stored in the environment of (-20 +/-5) DEG C, ((2-8) DEG C) and (20-25) DEG C, and is subjected to T-test statistical analysis with the CA125 quality control substance stored in the environment of-80 ℃, wherein P values are all larger than 0.05, so that the CA125 quality control substance diluted by the matrix serum can be stable for 14 months in the environment of (-20 +/-5) DEG C, can be stable for 4 months in the environment of (2-8) DEG C and can be stable for 15 days in the environment of (20-25) DEG C, the stability meets requirements, and the actual measurement needs can be met.
The stability of the CA125 quality control substance prepared by the matrix serum prepared in the other examples is similar to the effect of the matrix serum prepared in the example 5.
Example eight, comparative experiment
Comparative example 1: negative plasma not treated by the method of the invention;
comparative example 2: negative sera not treated by the method of the invention;
comparative example 3: commercially available calf serum;
the substrate serum of the present invention and the above comparative examples 1 to 3 were used to treat HCV seropositive samples (concentration value of 2.5X 10)5IU/mL) were diluted in multiple times, and the samples diluted 10 times were placed at-80 deg.c (control) and 25 deg.c, respectively, and examined 1 time per day. The detection results are as follows:
TABLE 30 test results of dilution by multiple
| Dilution factor | Theoretical concentration | Comparative example 1 | Comparative example 2 | Comparative example 3 | Example 5 |
| Original multiple | 2.5×105 | 2.84×105 | 2.26×105 | 2.41×105 | 2.47×105 |
| 10 | 2.5×104 | 8.69×103 | 1.02×104 | 2.68×104 | 2.36×104 |
| 100 | 2.5×103 | 2.35×102 | 2.64×103 | 3.77×103 | 2.61×103 |
| 1000 | 2.5×102 | Negative of | 1.93×102 | 3.03×102 | 2.33×102 |
Table 31 stability test results of 10-fold diluted samples
As can be seen from Table 30, the matrix serum of the present invention has a good detection result for diluting HCV positive serum samples, and the difference from the theoretical concentration is minimal; as can be seen from Table 31, the diluted HCV samples of the present invention were stored stably at 25 ℃ for 5 days, whereas the nucleic acid degradation in comparative examples 1 to 3 was observed, even though it could not be detected. Therefore, the invention is used for diluting HCV serum samples and has the advantages of good dilution effect and good stability.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A method for separating proteins and lipids from plasma or serum, comprising the steps of:
s1, weighing ammonium sulfate, adding the ammonium sulfate into negative plasma or negative serum, mixing, and centrifuging to obtain a supernatant I;
s2, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate A;
s3, adding the supernatant I obtained in the step S1 into the mixed precipitate A obtained in the step S2, uniformly mixing and centrifuging, and taking a supernatant II;
s4, weighing activated carbon and diatomite or dextran, adding normal saline, uniformly mixing, centrifuging, removing supernatant, and leaving mixed precipitate B;
s5, adding the supernatant II obtained in the step S3 into the mixed precipitate B obtained in the step S4, uniformly mixing and centrifuging, taking the supernatant III, and filtering to obtain the matrix serum.
2. The method of claim 1, wherein in the steps S2 and S4, the mass concentration of the ammonium sulfate in the negative plasma is 50-100%, and the mass ratio of the activated carbon to the diatomite is (5-8): (1-2).
3. The method of claim 1, wherein in steps S2 and S4, the mass concentration of ammonium sulfate in negative serum is 50-100%, and the mass ratio of the activated carbon to the dextran is (5-8) to (1-2).
4. The method of claim 1, wherein the concentration of NaCl in the physiological saline is 0.9% in the steps S2 and S4.
5. The method as set forth in claim 1, wherein the centrifugation speed is 4000-5000rpm and the centrifugation time is 20-30min in the steps S2 and S4.
6. A matrix serum obtained by the method for separating proteins and lipids from plasma or serum according to any one of claims 1 to 5.
7. The use of the matrix serum according to claim 6 for the preparation of HCV RNA quality control agents.
8. The use of the matrix serum according to claim 6 for the preparation of an HBsAg quality control substance.
9. The use of the matrix serum according to claim 6 for the preparation of a quality control agent for saccharide antigen 125.
10. Use of the matrix serum according to claim 6 for diluting clinical samples.
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