CN1165548C - Purification method of human cardiac troponin Ⅰ - Google Patents

Abstract

The invention discloses a method for purifying human cardiac troponin I, a preparation method of a monoclonal antibody thereof and a method for measuring the content of the human cardiac troponin I by a one-step method. The method is characterized in that a human cardiac troponin I is purified by adopting an affinity chromatography method, a monoclonal antibody of the cardiac troponin I is prepared by utilizing the purified cardiac troponin I and BALB/c mouse immunization, and a one-step method for determining the content of the cardiac troponin I is established. Its advantages are high homology, specificity and sensitivity. The detection method is simple to operate and low in cost. Can be widely used for the examination of heart diseases.

Description

Purification method of human cardiac troponin I

technical field

The invention relates to a method for purifying human cardiac troponin I by affinity chromatography, a method for preparing a monoclonal antibody of the cardiac troponin I and a method for determining its content by one-step method.

Background technique

Myocardial ischemic injury, especially acute myocardial infarction (AMI) is one of the major diseases that threaten human life. Clinically, efforts have been made to find serum indicators with good specificity and high sensitivity to diagnose it. At present, creatine kinase isoformic acid (CK-MB) has been used as an important indicator for the diagnosis of myocardial infarction, but CK-MB is not unique to the heart, and also exists in a small amount in skeletal muscle of normal people. Cardiac surgery or skeletal muscle injury patients often have increased CK-MB, which can easily lead to false positive diagnosis of AMI. Studies in recent years have shown that cardiac troponin I (cardiac troponin I, cTnI) has a high degree of myocardial specificity. When myocardial cells are damaged, cTnI in the blood appears early and lasts for a long time, which is closely related to the degree of myocardial injury and prognosis. relevant. It can be used as a specific indicator of myocardial injury and is of great significance for the diagnosis of heart disease.

Therefore, to purify human cardiac troponin I and find its corresponding antibody for the diagnosis of heart disease patients has become the frontier of heart disease diagnosis research. In 1999, the European and World Association for Clinical Chemistry (IFCC) proposed to use cardiac troponin I (cTnI) as an index for diagnosing AMI.

At present, the extraction of cardiac troponin I is mostly done by animals, and there is no report on the use of human cardiac muscle, such as Bodor GS (Bodor GS, Porter S, Landt Y, et al. The development of monoclonal antibodies and an assay for Cardiactroponin I with preliminary results in suspected myocardialinfarction. Chin Chem, 1992; 38: 2203-2214), Thulin E (Thulin E, VogelHJ.Purification of rabbit skeletal muscle troponin C. Clin Chem Acta, 1988Syska15H, 211) (Syska H, Perry SV, Trayer. A new method of preparation of troponin I using affinity chromatography. FEB S Letters, 1974; 40: 253-257), Pharmacia LKB Biotechnology Ltd Co: (Affinity chromatography principle and methods. 1988, ) and other documents have reported on the extraction of cardiac troponin I. However, the main disadvantage of using animal extraction is poor homology. It is used to cultivate monoclonal antibodies to produce cardiac troponin I, which has poor specificity and poor sensitivity. The diagnostic accuracy for heart disease still needs to be improved.

At present, cardiac troponin I is used to prepare its antibody, and cardiac troponin I is mostly extracted from animal myocardial tissue to prepare its monoclonal or polyclonal antibody. Cummins et al. (Cummins P, Young A, Auckand ML, et al.Comparison of serum cardiac specifictoponin I with creatine kinase, creatine kinase MB isoenzyme, tropomyosin, myoglobin and C reactive protein release in marathonrunners:cardiac orlin skeletal C ? Invest, 1987; 17:317) first injected rabbits and sheep with cardiac troponin I in 1987, cultivated and prepared polyclonal antibodies to cardiac troponin I, and labeled it with iodine. The quantitative measurement of cardiac troponin I by radioimmunoassay was established. Using animals to prepare monoclonal antibodies for detecting the content of human cardiac troponin I mainly has the defects of poor antibody homology, specificity, and poor sensitivity, and the accuracy of heart disease diagnosis cannot be further improved.

At present, there are three methods for detecting the content of cardiac troponin I in patients with heart disease: qualitative, semi-qualitative and quantitative. Due to the different sources of monoclonal antibodies, the reference values are not completely consistent. The principle of determination is mainly based on various immunoenzyme detection methods and radioimmunoassay. In foreign countries, the immunoenzyme detection method is mainly completed by using automatic analyzers (such as Status, Access and other automatic analyzers), and the results can be obtained within 1 hour, but it is necessary to purchase supporting reagents such as cellulose acetate membranes coated with antibodies, and this type of Reagents are expensive. There are also ELISA double-sandwich quantitative methods for detection, but this method is generally operated manually and takes a long time, which is not conducive to gaining valuable diagnosis and treatment time for disease patients.

Contents of the invention

The purpose of the present invention is to provide a method for purifying human cardiac troponin I by using affinity chromatography, and a method for preparing its monoclonal antibody by using the cardiac troponin I. At the same time, it provides a method for quickly measuring the content of cardiac troponin I in heart disease patients by using the cardiac troponin I obtained by the method and the monoclonal antibody to establish a one-step method.

The purpose of the present invention is achieved through the following technical solutions:

(1) According to the present invention, the purification method of human cardiac troponin I is as follows:

(1) Take human fresh or quick-frozen myocardial tissue, remove fat and fiber, after homogenization, centrifugation, precipitation, extraction, high-salt dissolution and other steps, the chromatographic medium is CMSephadex-C50, Cellulose-DE52, The buffer system was Tris-HCl, the pH value was 7.2-7.8, and the urea content was 6-8 mol/L. protein peak;

(2) Take the cardiac troponin C obtained in the above step (1), the coupling solution is 0.1-0.3mol/L NaHCO ₃ , the pH value is 7.8-8.5, 3-6mmol/L CaCl ₂ , and the equilibrium buffer is 45 ～55mmol/L Tris-HCl, 8～10mol/L urea, 0.8～1.2mmol/LCaCl ₂ , 12～18mmol/L β-mercaptoethanol, and Sepharose 4B affinity chromatography column under the condition of pH value 7.8～8.2 Coupling, prepared into Sepharose 4B affinity chromatography column;

(3) Take the cardiac troponin I peak collection solution of the crude extract after the above-mentioned step (1) after CM Sephadex-C50 chromatography, then add the obtained Sepharose 4B affinity chromatography column of step (2) to carry out affinity chromatography , to obtain purified cardiac troponin I.

(2) According to the present invention, the monoclonal antibody preparation method of human cardiac troponin I is as follows:

(1) Take the cardiac troponin I prepared in the above (1), and inject it into the peritoneal cavity of BALB/c mice several times to immunize the mice, with an interval of 2 to 3 weeks, until the mouse serum anti-cardiac troponin I antibody drops If the ratio is greater than 1:2000, mouse splenocytes and SP2/0 myeloma cells are taken;

(2) The two kinds of cells in the above step (1) are mixed evenly, and treated by adding DMEM serum-free culture solution, then adding HAT culture solution containing 15-20% fetal bovine serum, and culturing in an incubator for cell fusion;

(3) get the culture medium supernatant of above-mentioned step (2), detect the hybridoma cell in culture medium by ELISA method, i.e. the specific antibody of cardiac troponin I;

(4) The hybridoma cells detected in the above step (3) are cultured by limiting dilution method to obtain proliferating homologous cell clones, i.e. monoclonal cell lines;

(5) The monoclonal cell lines prepared in the above step (4) were compared with the standard monoclonal antibodies 2B1.9 and 2F6.6, and Western blot and ELISA methods were used to detect the anti-cardiac troponin I capture monoclonal Cloned antibody IgG ₁ (monoclonal antibody linked to horseradish peroxidase) and anti-cardiac troponin I labeled biotin antibody IgG _2b (monoclonal antibody linked to biotin), the above two monoclonal antibodies are the main The monoclonal antibody of cardiac troponin I obtained by the method has high specificity and high sensitivity.

(3) According to the present invention, the one-step rapid assay method of human cardiac troponin I is as follows:

(1) Coat a 96-well polystyrene enzyme-linked plate with 50-150 mmol/L, bicarbonate buffer solution with a pH value of 9.4-9.6, and streptavidin;

(2) Get the sample to be tested and the standard, contact with the monoclonal antibody against cardiac troponin I with biotin and the monoclonal antibody against cardiac troponin I with horseradish peroxidase respectively After that, add them to the coated wells of the enzyme-linked plate;

(3) Incubate the enzyme-linked plate of step (2);

(4) Take the incubated enzyme-linked plate, and add substrates hydrogen peroxide and 2,2'-oxygen-bis-3-ethylbenzothiazoline-6-sulfonate to the coated wells for enzyme promote color;

(5) Read the absorbance at a wavelength of 405 nm after enzymatic color development in the above step (4), compare the sample to be tested with the standard, and calculate the content of cardiac troponin I in the sample to be tested.

The present invention has the advantages of: using human cardiac muscle tissue to purify cardiac troponin I, and the obtained cardiac troponin I is used to cultivate and prepare its corresponding monoclonal antibody, which has good homology, high specificity and sensitivity of the obtained antibody The advantage is that the content of cardiac troponin I used to detect heart disease patients has high accuracy, which can further improve the diagnostic accuracy of heart disease. Moreover, the monoclonal antibody for cultivating cardiac troponin I can be produced in large quantities by using the present invention, the success rate is high, and it is suitable for industrial production. The preparation of the monoclonal antibody of cardiac troponin I by the invention can replace the import, greatly reduce the high cost of import and save the cost. In addition, using the method for measuring cardiac troponin I of the present invention, the operation is very simple, and the measurement time is fast, generally 30 minutes to obtain the result, which is convenient for popularization and application. On the one hand, it can save precious diagnosis time for patients; on the other hand, the assay method of the present invention does not need to purchase expensive automatic equipment, and does not need to use expensive imported reagents, which greatly reduces the detection cost and the burden on patients.

Detailed ways

Below in conjunction with embodiment above-mentioned method of the present invention is described in further detail:

(1) the present invention to the purification method of human cardiac troponin I:

1. Purification of human cardiac troponin C (cTnC)

(1) Take 100g of human ventricular muscle tissue fresh or frozen in liquid nitrogen and stored at -75°C, remove fat and fibers and shred, add 3 to 5 times the volume of homogenization buffer (Tris-HCl 50mmol/L, KCl 50mmol/L L, EDTA 1mmol/L, pH7.5) was fully homogenized, centrifuged for 30 minutes, the supernatant was removed, the precipitate was taken, and the above steps were repeated 10 times; fully homogenized each time. The precipitate was extracted more than 2 times with ethanol and ether, and dried into a dry powder. Dissolve the dry powder in buffer solution 1mol/L KCl for 12-24 hours, centrifuge at 10000g for 30 minutes, remove the precipitate, and take the supernatant;

(2) The above supernatant was precipitated with 40-60% (NH ₄ ) ₂ SO ₄ . Dissolve with 50ml of CMSephadex-C50 chromatography column equilibration buffer, dialyze in this buffer for 3 times, centrifuge at 10000g for 30 minutes, remove the precipitate, and take the supernatant;

(3) Pack the column (2.5×30cm) with well-balanced CM Sephadex-C50, equilibrate 5 column volumes with equilibration buffer, and load the supernatant of the above (2) (the amount of loaded protein is generally 200-400mg), After equilibrating 5 column volumes with the equilibration buffer, use NaCl 0-0.5mol/L gradient elution, collect in an automatic collector, 6mL per tube, speed 0.4mL/min;

(4) Collect the first protein peak of CM Sephadex-C50 chromatographic column NaCl gradient elution, balance fluid dialysis and then go through Cellulose-DE52 chromatographic column NaCl 0-0.3mol/L gradient elution, the latter protein peak is cTnC ;

2. Purification of human cardiac troponin I by affinity chromatography

(1) Preparation of cTnC Sepharose 4B affinity chromatography column: take 50 mg of human cTnC (final volume after concentration is 20 mL), dialyze in coupling solution (0.1 mol/L NaHCO ₃ , pH 8.3, 5 mmol/L CaCl ₂ ) 3 times. Take 7.5g of Sepharose 4B gel dry powder activated by cyanogen bromide, wash with 1mol/L HCl 1500mL within 15 minutes and drain it, then wash and drain it with the above coupling solution, mix with 20mL cTnC at 4°C Shake slowly for 20 hours. After taking it out, wash it repeatedly with the aforementioned coupling solution until the A280 value of the effluent is zero, then drain it. Suspend in 0.1 mol/L Tris-HCl buffer, pH 8.0, and shake slowly at 4°C for 12 hours. After taking it out and draining it, use 5 column volumes of 0.1mol/L acetate buffer (pH4.0, containing 0.5mol/L NaCl) and 0.1mol/L Tris-HCl buffer (pH8.0, containing 0.5 mol/L NaCl) for 3 rounds. Then wash with equilibration buffer (50mmol/L Tris-HCl, 9mol/L urea, 1mmol/L CaCl ₂ , 15mmol/L β-mercaptoethanol, pH8.0), and equilibrate on a column (1.2×15cm) for later use;

(2) Purification of human cTnI by affinity chromatography: take the supernatant of step 1 (1), put it into the coupling solution described in step 2 (1) and dialyze 3 times, and put it on cTnC Sepharose 4B chromatography column. Use 50 times column volume of equilibration buffer to wash away impurity proteins, and then wash with elution buffer (50mmol/L Tris-HCl, 9mol/L urea, 10mmol/L EGTA, 15mmol/L β-mercaptoethanol, pH8.0) Take off, collect by automatic collector, 2mL per tube, speed 0.1mL/min. The collected product is purified cardiac troponin I;

3. cTnI activity determination and protein quantification: cTnI activity was determined by double-antibody ELISA sandwich method, Coomassie Brilliant Blue method was used to determine the protein content of collected tubes, and SDS-PAGE electrophoresis was used to determine the cTnI molecular weight.

(2) Preparation of cardiac troponin I monoclonal antibody

1. Immune animals: Take BALB/c mice of the same strain as myeloma cells at the age of 8 to 12 weeks, mix thoroughly with cTnI antigen containing 100 μg of protein per mouse and the same amount of complete Freund’s adjuvant, and inject into the abdominal cavity of the mice Within two weeks, 100 μg/mouse of cTnI antigen was thoroughly mixed with the same amount of Freund's incomplete adjuvant, and injected into the abdominal cavity of mice several times for booster immunization. After the detection of mouse serum (indirect ELISA method), those whose titer is above 1:2000 can be used for fusion, and 3 days before the fusion, the mice are boosted again by intraperitoneal immunization, with a dose of 50 μg/mouse;

2. Cell Fusion

(1) Take 40mL of HAT culture solution, 15mL of DMEM serum-free culture solution and 1mL of 50% PEG (M ₁ 2000) and place them in a 37°C water bath for pre-warming;

(2) Take the SP2/0 myeloma cells (2-5×10 ⁷ ) and splenocytes (10 ⁸ ) suspensions of the above-mentioned immunized BALB/c mice and add them to a 50mL centrifuge tube to mix well, and add DMEM for serum-free culture liquid to 40mL. Centrifuge for 10 minutes, pour out the supernatant, and mix to form a paste;

(3) Place the centrifuge tube in a beaker filled with water pre-warmed at 37°C, take 0.7 mL of pre-warmed 50% PEG solution, add it within 1 minute, and let stand for 90 seconds. Immediately drop 15 mL of pre-warmed serum-free culture medium at 37°C to dilute the PEG and stop its effect. The dripping method is to add 1mL in the first 30 seconds; add 3mL in the last 30 seconds; then add it within 1 minute;

(4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and drain the supernatant. Add 40 mL of HAT medium containing 15% to 20% fetal bovine serum. Gently mix with a pipette, drop into the wells of four 96-well cell culture plates containing feeder cells, 2 drops per well, and culture in an incubator at 37°C and 7% _CO2 ;

3. Selection and culture of hybridoma cells

Immunized mouse splenocytes and mouse myeloma cells, after PEG treatment, form a mixture of various cell components, including unfused myeloma cells and immune splenocytes; myeloma cell synkaryocytes and immune splenocytes synkaryon, and the elicitor in myeloma cells and immune spleen cells. Only the latter form hybridoma cells. For this reason, unfused cells and allofused synkaryons must be removed from the various cell mixtures, and true hybrid cells must be selected. Therefore, on the 1st, 3rd, 5th, and 7th days after cell fusion, replace the medium with the aforementioned HAT medium;

4. Detection of specific antibodies and cloning of hybridoma cells

Aspirate the supernatant of each culture well, and use the indirect ELISA method to detect the culture wells containing the specific antibody of cTnI in the culture medium. Hybridoma cells were cloned by limiting dilution. After culture, single cells can proliferate into homogeneous cell clones;

5. Cryopreservation of Hybridoma Cells

Once the hybridoma cells are established, they should be frozen as soon as possible to prevent the hybridoma cells from being lost due to passage contamination or mutation. It is necessary to store batches of cells in liquid nitrogen early after the hybridoma cells secreting specific antibodies are obtained. The myeloma cell lines used for cell fusion are also preserved in the same way to obtain a stable and reliable source of parental cells.

Take vigorously growing and well-formed cTnI cloned cells to make a cell suspension, centrifuge for 5 minutes, remove the supernatant, add 4°C freezing solution (9 parts of complete culture solution plus 1 part of DMSO), and finally Make the cell density 3-5×10 ⁶ cells/mL, aliquot 1 mL of cell suspension into 2 mL cryopreservation tubes for cryopreservation or long-term storage in liquid nitrogen;

6. Mass Production of Monoclonal Antibodies

Before inoculating hybridoma cells, BALB/c mice were injected intraperitoneally with 0.5 mL pristane or liquid paraffin, and then each mouse was injected intraperitoneally with 5×10 ⁵ -10 ⁶ hybridoma cells. 7-10 days after inoculating the hybridoma cells, take the ascites, centrifuge for 10 minutes, absorb the supernatant, add 0.02% sodium azide, aliquot and store at -70°C;

7. Purification of Monoclonal Antibodies

The monoclonal antibody can bind to protein A in a high pH environment and can be eluted from protein A in a low pH environment. The operation steps are as follows:

(1) Add 5mL Protein A-Sepharose (Repligen, Cat.No.IPA-300) to the column. Wash with 50mL binding buffer;

(2) Mix 10-15 mL of ascitic fluid with an equal amount of PBS buffer (phosphate 100 mmol/L, pH 7.4) and add to the column. The hybridoma cell supernatant was adjusted to pH 9.0 with NaOH and added to the column. Wash with 50mL binding buffer;

(3) For elution with elution buffer, collect 1mL from each tube and mix with an equal amount of 0.1mol/L Tris, pH 9.0;

(4) A280 colorimetric. The collected peak tubes were merged, dialyzed in PBS solution, and stored at 4°C;

8. Screening of high specificity and high sensitivity cTnI monoclonal antibody

Through the above process, a total of 16 monoclonal antibody strains were finally obtained. The ELISA double-antibody sandwich method was used to replace the detection, and two monoclonal antibody strains were selected. Named JS09 (i.e. anti-cTnI monoclonal antibody linked with biotin) and JS05 (i.e. anti-cTnI monoclonal antibody linked with horseradish peroxidase), and two foreign anti-cTnI monoclonal antibodies 2B1.9, 2F6. 6 (these two antibodies are currently the only monoclonal antibody approved by the US FDA and the most widely used) comparison. Take a small amount of freshly homogenized human myocardium, skeletal muscle and smooth muscle samples, purified cTnI, cTnT (troponin T) and cTnC, and use Western Blot method and ELISA double antibody sandwich method to determine their association with JS09 and JS05 monoclonal antibody strains It was found that JS09 and JS05 monoclonal antibodies only bind to cTnI in human myocardial tissue, but have no cross-reaction with skeletal muscle, smooth muscle, cTnC and cTnT. The subtypes of JS09 and JS05 monoclonal antibodies were detected by immunocompetitive method, and they were IgG ₁ and IgG _2b respectively.

(3) One-step rapid determination of human cardiac troponin I

1. Detection kit composition

(1) Streptavidin pre-coated 96-well enzyme-linked plate, the preparation process is as follows:

a. Streptavidin 10 μg/mL was dissolved in bicarbonate buffer (100 mmol/L, pH 9.5), 100 μL per well, coated on a 96-well polystyrene enzyme-linked plate, 4°C, 18 hours.

b. Pour off the coating solution, block with 1% BSA, 37°C, 30min, or 4°C, 12 hours.

c. Pour off the blocking solution, wash twice with cleaning solution (containing Tween 0.04%), seal with plastic wrap and store at 4°C for later use or -20°C for later use.

(2) 5 copies of cTnI standard substance, the contents were 1, 2, 4, 8, 16 μg/L respectively.

(3) 1 copy of positive and negative quality control serum.

(4) One copy of cardiac troponin I monoclonal antibody linked with biotin (JS09-biotin) and one copy of cardiac troponin I monoclonal antibody linked with horseradish peroxidase (JS05-PODs).

(5) Antibody and sample dilution buffer PBS (phosphate 40mmol/L, pH7.2, NaCl 150mmol/L) 1 part.

(6) 1 part of washing buffer (Tween 0.04%, NaCl 150mmol/L).

(7) 1 part each of the substrates hydrogen peroxide (H ₂ O ₂ ) and 2,2'-oxy-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS).

(8) 1 part of substrate dilution buffer citrate phosphate buffer (100mmol/L, pH4.2).

2. Detection operation process

(1) Take cTnI standard (1, 2, 4, 8, 16 μg/L) or sample to be tested, quality control serum 50 μL/mL, take cardiac troponin I monoclonal antibody coupled with biotin (JS09-Bio 50 μL of cardiac troponin I monoclonal antibody (JS05-PODs) coupled with horseradish peroxidase, mixed evenly and added to the enzyme-linked plate for incubation at 37°C for 30 min, and the dilution buffer was PBS.

(2) Washing solution for 3 times, adding 100 μL of substrate hydrogen peroxide (H ₂ O ₂ ) and 2,2'-oxo-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS), The dilution buffer is citrate phosphate buffer, 37°C, 15min.

(3) Calculate the amount of cardiac troponin I contained in the sample to be tested by reading the absorbance at a wavelength of 405 nm and using the standard as a control.

3. Related parameters in detection

(1) When the coating concentration of streptavidin was 6 μg/mL, the reaction reached a plateau, and 10 μg/mL of streptavidin was taken as the fixed coating concentration, and the result was stable. Coating time is 18 hours at 4°C to reach a maximum amount. Coating buffer is most stable at pH 9.5. There is no statistical difference in the measurement results between the one-month storage at 4°C sealed with plastic wrap and the six-month storage at -20°C.

(2) When the cTnI standard contents were 1, 2, 4, 8, 16 μg/L, the linear correlation coefficient γ=0.994.

(3) When the reaction concentrations of JS09 and JS05 were 1 μg/mL and 2 μg/mL, the sensitivity was high and the results were stable. The antibody reacts best with sample dilution buffer PBS pH 7.2.

(4) The concentration range of the substrate hydrogen peroxide (H ₂ O ₂ ) is 0.01-0.03%, and the concentration of 2,2'-oxygen-bis-3-ethylbenzothiazoline-6-sulfonate (ABTS) is At 1.9mmol/L, the background is low and the sensitivity is high. The substrate dilution buffer pH4.0-4.4 is the best. When the color development time is 37°C for 15 minutes, the background is low, and the reaction is close to the plateau.

(5) cTnI standard substance or sample to be tested, quality control serum, mixed with JS09, JS05 and incubated at 37°C, reaching plateau after 30min.

(6) The difference between groups was 8%, and the difference between batches was 6%.

(7) The reference value of serum cTnI in 100 cases of normal people was 1.5 μg/L.

Claims (2)

1, the purification process of human body cardiac muscle troponin I is characterized in that comprising following steps:

(1) gets the cardiac muscular tissue of the fresh or quick freezing and cold preserving of human body, degrease and fiber, after step process such as homogenate, centrifugal, precipitation, extracting, high salt dissolving, at chromatography media is CMSephadex-C50, Cellulose-DE52, buffer system is Tris-HCl, the pH value is 7.2～7.8, and urea content is to carry out chromatography under 6～8mol/L condition successively, collects the protein peak that contains cardiac troponin C (cTnC) and cardiac muscle troponin I (cTnI) respectively;

(2) getting above-mentioned steps (1) gained cardiac troponin C, is 0.1～0.3mol/L NaHCO at coupling solution ₃, the pH value is 7.8～8.5,3～6mmol/L CaCl ₂, level pad is 45～55mmol/L Tris-HCl, 8～10mol/L urea, 0.8～1.2mmol/LCaCl ₂, 12～18mmol/L β mercaptoethanol, the pH value be under 7.8～8.2 the condition with the coupling mutually of Sepharose 4B affinity column, be prepared into Sepharose 4B affinity column;

(3) liquid is collected at the cardiac muscle troponin I peak of getting slightly the carry product of above-mentioned steps (1) behind CM Sephadex-C50 chromatography, adds step (2) gained Sepharose 4B affinity column again and carries out affinity chromatography, promptly gets the cardiac muscle troponin I of purifying.

2, method according to claim 1, cardiac troponin C, the I that it is characterized in that purifying derives from the people cardiac muscular tissue of-75 ℃ of storages behind fresh or the liquid nitrogen flash freezer.