CN112851801A - Preparation method of antigen for chemiluminescence immune analysis calibrator or quality control material - Google Patents

Abstract

The invention discloses a preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control product, which is used for purifying natural porcine myoglobin and comprises the following steps in sequence: the pig heart tissue homogenate, the ammonium sulfate precipitation, the ion exchange chromatography and the hydrophobic chromatography are adopted, the pig myoglobin is adopted to replace the human myoglobin, the problem of raw material source can be solved, in addition, the preparation method has simple steps, the obtained myoglobin has high yield and high effective active concentration, and can meet the requirement of large-scale production; meanwhile, the requirements of a chemiluminescence immunoassay calibrator or a quality control product are met, the linear correlation is good, the stability is more stable than that of recombinant myoglobin, and the amplitude reduction is less than or equal to 5 percent after 7 days of acceleration at 37 ℃.

Description

Preparation method of antigen for chemiluminescence immune analysis calibrator or quality control material

Technical Field

The invention relates to the field of protein purification, in particular to a preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control material.

Background

Myoglobin (Mb) is an oxygen binding globulin, consisting of a peptide chain and a heme prosthetic group, widely found in cardiac and skeletal muscle of humans and other mammals. The heme prosthetic group is a complex formed by an iron atom and a porphyrin compound, 4 pyrroles are connected through 4 methyl alkynyl groups to form a large ring, and Fe2+ is positioned in the center of the pyrrole ring. The human myoglobin and the pig myoglobin consist of 154 amino acid residues, and the molecular weight is 16.7 kDa; the arrangement of myoglobin molecules is compact, one molecule of protein binds to one molecule of oxygen, and the structure enables the myoglobin to play a role in the processes of storing and delivering oxygen more quickly.

In the case of acute myocardial injury, myoglobin is firstly released in blood, Mb in blood exceeds the upper limit of normal after 2-4h, and peak is reached after 6-12 h; thus detection of Mb levels can aid in rapid diagnosis of disease and timely treatment, thereby reducing mortality. The Mb detection methods in the market include ELISA, RIA, chemiluminescence immunoassay, fluorescence immunoassay, immunoturbidimetry, and the like; mb raw materials are mainly used as a calibrator or a quality control product in a diagnostic kit and are used for improving the accuracy and precision of detection; at present, most of the domestic detection kits use imported Mb raw materials, the price is high, and the popularization and the application of Mb detection are limited to a great extent. Therefore, a high-performance Mb raw material is urgently needed to be developed, so that the dependence of imported raw materials is eliminated, and the domestic detection kit is assisted.

In the prior art, recombinant myoglobin antigen or naturally extracted human myoglobin antigen is mainly used as a raw material of a diagnostic reagent, the recombinant myoglobin antigen has poor stability, and after a calibrator or a quality control product is prepared, the accelerated stability at 37 ℃ can only be stabilized for 3-4 days; myoglobin from human cardiac muscle is difficult to be obtained, and is difficult to be produced on a large scale.

Disclosure of Invention

The invention aims to provide a preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control product, which solves the problems of poor stability of a recombinant myoglobin antigen and the like, obtains a high-activity and high-stability natural porcine myoglobin antigen, provides a diagnostic reagent raw material for clinical detection of myocardial damage and prognosis, and lays a foundation for research and development of a rapid diagnostic kit for cardiac diseases such as acute myocardial infarction and the like.

The technical scheme adopted by the invention is as follows: a preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control product is disclosed, wherein the antigen is myoglobin, and the preparation of the myoglobin adopts purified natural porcine myoglobin, and the preparation method sequentially comprises the following steps: homogenizing pig heart tissue, precipitating with ammonium sulfate, performing ion exchange chromatography and hydrophobic chromatography.

Preferably, the reagents used for equilibration and elution during ion exchange chromatography are both Tris-EDTA buffer, more preferably 10-100mM Tris, 1-5mM EDTA, pH 7.0-9.0.

Preferably, the crude protein obtained by ammonium sulfate precipitation is subjected to thawing, dialysis and membrane filtration before ion exchange chromatography.

Preferably, the reagents used for equilibration and elution by hydrophobic chromatography consist of Tris-EDTA buffer and ammonium sulphate, the concentration of sulphate of the reagent used for elution being 0.25-0.75 times the concentration of sulphate of the reagent used for equilibration. Specifically, it is preferable that the reagent for hydrophobic chromatographic equilibration is 10-100mM Tris, 1-5mM EDTA, 2-3M ammonium sulfate, pH7.0-9.0, and the reagent for hydrophobic chromatographic elution is 10-100mM Tris, 1-5mM EDTA, 0.5-1.5M ammonium sulfate, pH 7.0-9.0.

Preferably, prior to the hydrophobic chromatography, a relatively pure myoglobin sample obtained by ion exchange chromatography is dialyzed.

Preferably, the ammonium sulphate precipitation comprises the steps of:

s21, homogenizing the pig heart tissue to obtain a crude extract, precipitating with 70% ammonium sulfate, and removing the precipitate to obtain a supernatant;

s22, precipitating the supernatant obtained in the step S21 by 100% ammonium sulfate, removing the supernatant, and taking the precipitate to obtain crude protein.

Preferably, the pig heart tissue homogenate comprises the following specific steps: taking the pig heart tissue, preparing into slurry, centrifuging, and taking the supernatant to obtain the pig heart tissue homogenate supernatant.

The invention has the advantages that:

1. the invention adopts the porcine myoglobin to replace the human myoglobin, can solve the problem of raw material source, and in addition, the preparation method has simple steps, the obtained myoglobin has high yield and high effective active concentration, and can meet the requirement of large-scale production; meanwhile, the requirements of a chemiluminescence immunoassay calibrator or a quality control product are met, the linear correlation is good, the stability is more stable than that of recombinant myoglobin, and the accelerated stability at 37 ℃ is more than or equal to 7 days.

2. Different from two-step purification by ammonium sulfate precipitation and gel filtration, the myoglobin can be obtained with high purity after being purified by ion exchange chromatography and hydrophobic chromatography; most of the foreign protein can be removed by ion exchange chromatography, and then purified by hydrophobic chromatography.

3. The myoglobin prepared by the invention is prepared into a calibration product or a quality control product for chemiluminescence immunoassay, and is mainly used for calibration during the quantitative determination of Myo concentration in a sample. The amplitude reduction is within +/-10% after opening the bottle for 30 days; and the consistency with the standard product is better.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious to those skilled in the art that other drawings can be obtained according to the drawings without any inventive exercise.

FIG. 1 is an electrophoretogram of myoglobin sample purified by Sepharose Fast Flow chromatography column;

1. precipitating and dialyzing the supernatant sample by ammonium sulfate; 2-7, eluting the myoglobin sample (adopting a buffer solution A to elute to obtain a sample); 8-9, eluting the mixed protein sample (the sample is obtained by eluting with a buffer solution A containing 100mM NaCl); m, 14.4-116 kDa;

FIG. 2Q Sepharose Fast Flow chromatography column NaCl isocratic elution electrophoresis chart;

1: sampling before sample loading; 2: flowing through the sample; 3: elution with 100mM NaCl; 4: elution with 100mM NaCl; 5: elution with 100mM NaCl; 6: elution with 100mM NaCl; 7: elution with 100mM NaCl; m: 14-116kDa

FIG. 3 shows the electrophoresis of the myoglobin sample purified by Octyl-Sepharose High Performance column chromatography FIG. 1, sample before loading; 2-4, eluting the myoglobin sample (eluting by using buffer solution C containing ammonium sulfate to obtain a sample); 5. eluting the heteroprotein sample (using buffer C to elute to obtain a sample); m, 14.4-116kDa

FIG. 4 myoglobin antigen linear correlation assay;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention and the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the accompanying drawings.

A preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control product is disclosed, wherein the antigen is myoglobin, and the preparation of the myoglobin adopts purified natural porcine myoglobin, and the preparation method sequentially comprises the following steps: homogenizing pig heart tissue, precipitating with ammonium sulfate, performing ion exchange chromatography and hydrophobic chromatography.

In the present application, the pig heart tissue homogenate comprises the specific steps of: taking the pig heart tissue, preparing into slurry, centrifuging, and taking the supernatant to obtain the pig heart tissue homogenate supernatant.

The ammonium sulfate precipitation specifically comprises the following steps:

s21, homogenizing the pig heart tissue to obtain supernatant, precipitating with 70% ammonium sulfate, and removing the precipitate to obtain supernatant;

s22, precipitating the supernatant obtained in the step S21 by 100% ammonium sulfate, removing the supernatant, and taking the precipitate to obtain crude protein.

Preferably, the first and second electrodes are formed of a metal,

the ion exchange chromatography specifically comprises the following steps:

s31, carrying out re-melting and dialysis on the crude protein obtained by ammonium sulfate precipitation to obtain a crude protein sample;

s32, balancing a Q Sepharose Fast Flow chromatographic column by using a buffer solution A, filtering a crude protein sample by using a filter membrane, and purifying the crude protein sample by using the column; eluting with buffer solution A, collecting when the eluate is light red, and stopping collecting when the eluate is colorless to obtain relatively pure myoglobin sample; wherein the buffer solution A is 10-100mM Tris, 1-5mM EDTA, pH7.0-9.0.

The re-melting liquid adopted in the step S31 is 10-100mM Tris, 50-100mM NaCl, 1-5mM EDTA, pH7.0-9.0.

The specific process of dialysis in the step S31 is to precipitate ammonium sulfate to obtain crude protein, to melt, to put into dialysis bag with molecular weight cutoff of 3.5kDa, to put into buffer solution A, to stir and dialyze for 4-8h at 0-4 deg.C, while to replace buffer solution for 1-3 times.

The hydrophobic chromatography specifically comprises the following steps:

s41, dialysis: dialyzing the purer myoglobin sample obtained in the step 3);

s42, protein purification: after equilibration of the Octyl-Sepharose High Performance column with buffer B containing ammonium sulfate, the purer myoglobin sample dialyzed by step S41 was column purified; eluting with buffer solution C containing ammonium sulfate, and collecting eluate with light absorption value greater than 400mAU wavelength to obtain high purity myoglobin sample;

wherein the buffer solution B containing ammonium sulfate is 10-100mM Tris, 1-5mM EDTA, 2-3M ammonium sulfate, pH7.0-9.0, and the buffer solution C containing ammonium sulfate is 10-100mM Tris, 1-5mM EDTA, 0.5-1.5M ammonium sulfate, pH 7.0-9.0.

The specific process of dialysis in the step S41 is to put the obtained relatively pure myoglobin sample into a dialysis bag with molecular weight cut-off of 3.5kDa, put into buffer B, and stir and dialyze for 4-8h at 4 ℃ while replacing the buffer 1-3 times.

In order to facilitate the technical solution better understood by those skilled in the art, the following will further describe the preparation method of the polystyrene hydrophilic fluorescent microsphere with an example.

1) Crude tissue extract sample:

taking 99.4g of pig heart tissue, removing connective tissue and fat tissue by using surgical scissors, and then cutting the tissue into a minced meat shape; adding 50mM Tris, 100mM NaCl, 5mM EDTA and pH8.0 buffer solution, homogenizing for 4-5 times at intervals of 10s for 30s by using a portable homogenizer until the tissue is homogenized until no obvious tissue block is observed by naked eyes; centrifuging at 10000rpm and 4 deg.C for 30min, and collecting supernatant;

2) ammonium sulfate precipitation

S21, 70% ammonium sulfate precipitation: accurately weighing an appropriate amount of ammonium sulfate powder, slowly adding the ammonium sulfate powder into the supernatant solution obtained in the step (1) in a small amount for multiple times, and stirring while adding; when the concentration of the ammonium sulfate in the solution reaches 70%, stirring for 30min in an ice-water bath until no obvious ammonium sulfate particles exist; the homogenate is divided into two centrifuge bottles and centrifuged for 30min at 10000rpm and 4 ℃; after the centrifugation is finished, taking the supernatant, and discarding the precipitate;

s22, 100% ammonium sulfate precipitation: accurately weighing ammonium sulfate powder, slowly adding the ammonium sulfate powder into the supernatant solution obtained in the step (1) in a small amount for multiple times, and stirring the ammonium sulfate powder while adding; when the concentration of the ammonium sulfate in the solution reaches 100%, stirring for 30min in an ice-water bath until no obvious ammonium sulfate particles exist; the homogenate is divided into two centrifuge bottles and centrifuged for 30min at 10000rpm and 4 ℃; and after the centrifugation is finished, removing the supernatant, and taking the precipitate to obtain crude protein.

3) Ion exchange chromatography

S31 re-melting dialysis: adding the crude protein into 6-8mL dialysis buffer (50mM Tris, 100mM NaCl, 5mM EDTA, pH8.0) to resuspend the precipitate, placing the precipitate into a dialysis bag with the molecular weight cutoff of 3.5kDa, placing the dialysis bag into 50mM Tris, 5mM EDTA, pH8.0 buffer, and stirring and dialyzing for 4 hours at 4 ℃ while replacing the buffer for 1 time;

purifying the S32 protein: the Q Sepharose Fast Flow chromatographic column is equilibrated by 50mM Tris, 5mM EDTA, pH8.0 buffer solution, and the sample is filtered by a 0.22 μm filter membrane and then purified by column loading at a Flow rate of 2 mL/min; eluting the sample by using 50mM Tris, 5mM EDTA and pH8.0 buffer solution, starting to collect the sample when the effluent sample is light red, and stopping collecting the sample when the effluent sample is colorless to obtain a purer myoglobin sample;

4) hydrophobic chromatography

S41 dialysis: placing the obtained relatively pure myoglobin sample in a dialysis bag with the molecular weight cutoff of 3.5kDa, putting the dialysis bag in 50mM Tris, 3mM EDTA, 2M ammonium sulfate and pH8.5 buffer solution, and carrying out stirring dialysis for 4 hours at the temperature of 4 ℃ while replacing the buffer solution for 1 time;

purifying the S42 protein: conducting column purification on an Octyl-Sepharose High Performance column at a flow rate of 2mL/min while equilibrating the column with 50mM Tris, 3mM EDTA, 2M ammonium sulfate, pH8.5 buffer; the sample is eluted by 50mM Tris, 3mM EDTA, 0.8M ammonium sulfate and pH8.5 buffer solution, 280nm ultraviolet absorption values are monitored on an AkTa primer protein purification system (GE Healthcare Life Science), the sample is collected by adopting a 10mL EP multi-tube, high absorption peak samples with absorption values larger than 400mAU are combined to obtain 31.56mg of high-purity myoglobin sample, and the concentration of the myoglobin is 1.67mg/mL, the effective activity is 3.64mg/mL and the purity is 92 percent.

In order to show that the application has good linear correlation and more stable stability than recombinant myoglobin, and the accelerated stability at 37 ℃ is more than or equal to 7 days, the application is specifically described by test data.

(1) Carrying out electrophoresis detection on a myoglobin sample purified by a Q Sepharose Fast Flow chromatographic column, and dialyzing a supernatant sample by ammonium sulfate precipitation; eluting myoglobin sample by Q Sepharose Fast Flow chromatographic column (obtaining sample by elution); a hetero-protein sample eluted by a Q Sepharose Fast Flow chromatographic column (a sample is obtained by eluting by a buffer solution A containing 100mM NaCl); the standard protein with molecular weight of 14.4-116kDa was subjected to electrophoresis respectively, and the results are shown in FIG. 1, and it can be seen that myoglobin and hetero-protein in the dialyzed supernatant sample after precipitation with 70% and 100% ammonium sulfate were well separated, mainly myoglobin with molecular weight of 16.7kDa and cytochrome C with molecular weight of 11-13kDa, and myoglobin sample was eluted by using buffer A (20mM Tris, 1mM EDTA, pH9.0 buffer), while hetero-protein cytochrome C was eluted by adding 100mM NaCl to buffer A.

Meanwhile, for further verification, the myo protein was purified by loading the buffer solution containing 100mM NaCl (Q Sepharose Fast Flow column chromatography), and as shown in FIG. 2, the elution was performed using the buffer solution containing 100mM NaCl, and the protein was passed through the column, so that almost all the protein was hetero-protein, and myoglobin could not be obtained.

(2) Electrophoretic detection of sample of myoglobin purified by Octyl-Sepharose High Performance chromatographic column

Sampling a relatively pure myoglobin; myoglobin sample eluted from Octyl-Sepharose High Performance column); (ii) a sample of heteroprotein eluted from the Octyl-Sepharose High Performance column; the protein with the molecular weight of 14.4-116kDa is subjected to electrophoresis, and the result is shown in figure 3, only 1 band is eluted by buffer C, and the molecular weight is 16.7kDa, which indicates that purer myoglobin is obtained.

(3) Myoglobin antigen linear correlation assay

As shown in FIG. 4, it can be seen from the data that the myoglobin obtained by the present invention has excellent linearity, R²0.9992 can be reached.

(4) Stability test

The obtained sample is subpackaged in 1.5mL of EP tubes, 1 mL/tube, 5 tubes in total, and the sealing film is used for sealing; wherein 1 of the samples is placed at 4 ℃ as a control, the other 4 samples are placed at 37 ℃ for one week of acceleration test, the samples are respectively sampled and measured for 0, 1, 3, 5 and 7 days, the luminescence value and the protein concentration are shown in tables 1 and 2, the samples are accelerated for 1, 3, 5 and 7 days at 37 ℃, and compared with the 0 th day, the activity deviation is less than 5 percent; the concentration of the protein is not changed obviously, and the deviation of the protein concentration is less than or equal to 5 percent

TABLE 1

TABLE 2

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (9)

1. A preparation method of an antigen for a chemiluminescence immunoassay calibrator or a quality control product is characterized in that the antigen is myoglobin, and the preparation of the myoglobin adopts purified natural porcine myoglobin, and the preparation method sequentially comprises the following steps: homogenizing pig heart tissue, precipitating with ammonium sulfate, performing ion exchange chromatography and hydrophobic chromatography.

2. The method of claim 1, wherein the reagents used for equilibration and elution during ion exchange chromatography are Tris-EDTA buffer.

3. The method of claim 2, wherein the equilibration and elution reagents used in the ion exchange chromatography are 10-100mM Tris, 1-5mM EDTA, pH 7.0-9.0.

4. The method of claim 1, wherein the crude protein obtained by precipitation with ammonium sulfate is subjected to thawing, dialysis and membrane filtration before ion exchange chromatography.

5. The method of claim 1, wherein the reagents used for the hydrophobic chromatographic equilibration and elution are Tris-EDTA buffer and ammonium sulfate, and the concentration of the sulfate salt used for the elution is 0.25 to 0.75 times the concentration of the sulfate salt used for the equilibration reagent.

6. The method of claim 5, wherein the reagent for hydrophobic chromatography equilibration is 10-100mM Tris, 1-5mM EDTA, 2-3M ammonium sulfate, pH7.0-9.0, and the reagent for hydrophobic chromatography elution is 10-100mM Tris, 1-5mM EDTA, 0.5-1.5M ammonium sulfate, pH 7.0-9.0.

7. The method of claim 1, wherein the relatively pure myoglobin sample obtained by ion exchange chromatography is dialyzed before hydrophobic chromatography.

8. The method of claim 1, wherein the ammonium sulfate precipitation comprises the following steps:

s21, homogenizing the pig heart tissue to obtain supernatant, precipitating with 70% ammonium sulfate, and removing the precipitate to obtain supernatant;

s22, precipitating the supernatant obtained in the step S21 by 100% ammonium sulfate, removing the supernatant, and taking the precipitate to obtain crude protein.

9. The method of claim 1, wherein the step of homogenizing the porcine heart tissue comprises the steps of: taking the pig heart tissue, preparing into slurry, centrifuging, and taking the supernatant to obtain the pig heart tissue homogenate supernatant.

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