CN107603955B - Human cardiac troponin I hybridoma cell strain 7H4, monoclonal antibody and application - Google Patents

Abstract

The invention discloses a human cardiac troponin I hybridoma cell strain 7H4, a monoclonal antibody and application. The hybridoma cell strain 7H4 is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 8 and 23 months, and the preservation number is CCTCC NO: C2017121. the monoclonal antibody 7H4 secreted by the hybridoma cell strain 7H4 is a cTnI murine monoclonal antibody with high affinity and high specificity, and can be combined with free cTnI, a dimer compound cTnI-C and a trimer compound cTnI-T-C to form cTnI, so that the method can be used for detecting the content of cTnI in serum and lays a foundation for developing a kit for quickly detecting myocardial infarction.

Description

Human cardiac troponin I hybridoma cell line 7H4 and monoclonal antibody and application

technical field

The present invention relates to the technical field of cellular immunity, more particularly, to the hybridoma cell line 7H4 of human cardiac troponin I, monoclonal antibody and application thereof.

Background technique

Cardiac troponin is a key protein in the regulation of cardiac contraction. A trimeric complex consisting of three subunits of cardiac troponin I (cTnI), cardiac troponin C (cTnC) and cardiac troponin T (cTnT). cTnI is the inhibitory subunit of actin, which regulates the interaction between actin and myosin by inhibiting the activity of adenosine triphosphate (ATPase) of actin. The molecular weight of cTnI is about 24kDa and consists of 209 amino acids. It is an α-helix-rich protein with a theoretical isoelectric point of 9.87. There are three isoforms of TnI (troponin I): fast skeletal muscle type and slow skeletal muscle type exist in skeletal muscle troponin I (sTnI), which have similar molecular weights (20KD), but the amino acids between the two The sequences differ by about 40%; the third is the myocardial type. There is also a 40% difference in the amino acid sequence of cardiac troponin I (cTnI) and skeletal muscle type. The terminal of cTnI is 31 amino acids more than the terminal of sTnI, and cTnI only exists in one isoform in cardiomyocytes, which determines that cTnI is in cardiac muscle. specificity in cells. Positions 22 and 23 of the cTnI molecule are serines. Both of these amino acids can be phosphorylated by protein kinase A in vivo, so there are four different phosphorylation types of cTnI in vivo.

Acute myocardial infarction (AMI) is one of the most common cardiovascular diseases. On the basis of coronary artery disease, the blood flow of the coronary arteries is drastically reduced or interrupted, resulting in severe and persistent acute myocardial infarction. Coronary artery disease caused by blood, and eventually lead to ischemic necrosis of the myocardium. In severe cases, the heart can no longer perform its pumping function, resulting in sudden death. Therefore, early diagnosis of acute myocardial infarction is very important for its prevention and treatment. cTnI is considered by researchers to be one of the best biomarkers for the diagnosis of AMI. Because it only appears in the blood when the myocardium is damaged, it appears early, lasts for a long time, and has strong specificity. Therefore, the International Federation of Clinical Chemistry (IFCC) Authoritative institutions such as the American Society of Clinical Biochemistry (NACB) and the Chinese Medical Laboratory Branch have regarded cTnI as the "gold standard" for the diagnosis of AMI.

Among myocardial infarction markers, cTnI is considered to be a more sensitive and specific "gold standard" than other markers. When the cell membrane of cardiomyocytes is intact, cTnI cannot penetrate the cell membrane and enter the blood. Only when the cardiomyocytes are damaged, cTnI is rapidly released from the cardiomyocytes into the blood, starts to increase within 2-8 hours, peaks within 1-2 days, and the amount of cTnI can still be measured after 3-8 days. High, theoretically, cTnI can be maintained in serum for more than 10 days, and a longer diagnostic window period is very important for the diagnosis of myocardial infarction. Studies have shown that the critical value of cTnI in the serum of normal people and patients with myocardial infarction is 50 pg/mL, and the blood concentration can reach 100-300 μg/L at the time of onset. Most of the cTnI in blood is present in the form of cTnI-T-C or cTnI-C complex. In the study, it was found that cTnI was initially released into the blood in the form of a monomer, but as the degree of myocardial damage increased, in order to prevent cTnI from being degraded, cTnI would exist in the form of a complex, and then due to the influence of various factors, the complex It is decomposed into monomers, so the antibody used to detect cTnI should be able to recognize various complex forms of cTnI in blood with the same efficiency, but there is a serious lack of such monoclonal antibody resources in the prior art.

SUMMARY OF THE INVENTION

In order to overcome the above deficiencies in the prior art, the present invention provides a hybridoma cell line 7H4 of human cardiac troponin I.

Another object of the present invention is to provide the monoclonal antibody secreted by the above-mentioned hybridoma cell line 7H4, the monoclonal antibody is named as human cardiac troponin I monoclonal antibody 7H4, and the monoclonal antibody 7H4 is a kind of cTnI with high affinity and high specificity. Murine monoclonal antibody can bind to free cTnI, dimer complex cTnI-C, trimer complex cTnI-T-C three forms of cTnI, so it can be used to detect cTnI content in serum, for the development of rapid detection The Myocardial Infarction Kit lays the foundation.

The third object of the present invention is to provide the application of human cardiac troponin I monoclonal antibody 7H4 in the detection of three forms of cTnI in free cTnI, dimer complex cTnI-C and trimer complex cTnI-T-C .

In order to achieve the above object, the present invention is achieved through the following technical solutions:

A hybridoma cell line 7H4 of human cardiac troponin I, the hybridoma cell line 7H4 was deposited in the China Center for Type Culture Collection (CCTCC) on August 23, 2017, and the deposit number is CCTCC NO: C2017121. The address of the collection center is: Collection Center of Wuhan University, Wuchang District, Wuhan City, Hubei Province.

The human cardiac troponin I monoclonal antibody 7H4 secreted by the hybridoma cell line 7H4 as described above. The variable region gene of human cardiac troponin I monoclonal antibody 7H4 includes heavy chain variable region gene and light chain variable region gene. The amino acid sequence of light and heavy chain of monoclonal antibody 7H4 is shown in Figure 2.

The application of human cardiac troponin I monoclonal antibody 7H4 in the detection of free cTnI, dimer complex cTnI-C and trimer complex cTnI-T-C three forms of cTnI as described above.

Monoclonal antibody 7H4 is a monoclonal antibody prepared by immunizing cTnI-T-C protein. The specific preparation method is as follows: immunize Balb/c mice with whole protein cTnI-T-C, and spleen cells of mice are fused with myeloma cells. Positive hybridoma cell lines were screened by ELISA, and hybridoma cell lines capable of secreting stable antibodies were obtained through three limited dilution cloning techniques, ascites-type monoclonal antibodies were prepared, and monoclonal antibodies were obtained after purification.

Compared with the prior art, the present invention has the following beneficial effects: the human cardiac troponin I monoclonal antibody 7H4 provided by the present invention is a high-affinity, high-specificity cTnI murine monoclonal antibody, which can bind free Three forms of cTnI, cTnI, dimer complex cTnI-C, and trimer complex cTnI-T-C, can be used to detect the content of cTnI in serum, laying the foundation for the development of a rapid detection kit for myocardial infarction.

Description of drawings

Figure 1 shows the three CDR (complementarity determinants) regions of the variable region of the light and heavy chains of antibody 7D2.

Figure 2 shows the three CDR (complementarity determinants) regions of the variable region of the light and heavy chains of antibody 7H4.

Figure 3 is the SDS-PAGE detection chart after purification of antibodies 7D2 and 7H4, wherein 1 is a protein marker, 2 is 7D2, and 3 is 7H4.

Figure 4 is a graph showing the titer detection ELISA results of antibodies 7D2 and 7H4, wherein, Figure A is 7D2, and Figure B is 7H4.

Figure 5 is an ELISA chart for the detection of affinity constants of antibodies 7D2 and 7H4, wherein, Figure A is 7D2, and Figure B is 7H4.

Fig. 6 is a graph showing the reactivity of antibodies 7D2 and 7H4 with cTnI and cTnI-C, wherein, Fig. A is the cTnI monomer, and Fig. B is the cTnI-C complex.

Figure 7 is the detection curve of 7D2/HRP-7H4 and 7H4/HRP-7D2.

Figure 8 is the standard curve of 7D2/HRP-7H4 detection.

Detailed ways

The present invention will be further elaborated below with reference to the accompanying drawings and specific embodiments of the description, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

Example 1

The preparation of the hybridoma cell line and the monoclonal antibody it secretes includes the following steps:

1. Antigen

Monoclonal antibody 7D2 is a monoclonal antibody prepared by immunizing Balb/c mice with a complete antigen conjugated with amino acids 13-24 on cTnI and BSA. The preparation method of the complete antigen containing amino acids 13-24 on cTnI is as follows: : Use Discovery Studio4.0 software to simulate the cTnI-T-C structure, analyze the epitope on the cTnI subunit, and select the 13-24th amino acid as the epitope antigen. The amino acids 13-24 on cTnI were synthesized and coupled with BSA to form a complete antigen. This step was synthesized by Hangzhou Zhongpeptide Company.

The monoclonal antibody 7H4 antibody was prepared by immunizing Balb/c mice with the whole protein complex cTnI-T-C, wherein cTnI-T-C was purchased from Hytest Company in Finland.

2. Preparation of hybridoma cell lines: The preparation methods of hybridoma cell line 7D2 and hybridoma cell line 7H4 are the same, but they only use different antigens. The preparation methods are as follows:

1. Animal immunity

(1) Primary immunization: 50 μg of antigen was emulsified with an equal volume of complete Freund's adjuvant, and injected subcutaneously at multiple points;

(2) Booster immunization: 2 weeks apart, the same amount of antigen as the initial immunization was emulsified with an equal volume of incomplete Freund's adjuvant, subcutaneously injected at multiple points; blood was collected 10 days after the 3rd to 5th immunization to measure its titer;

(3) 72 hours before fusion, 25 μg of antigen without adjuvant was intraperitoneally injected.

2. Cell fusion

(1) Feeding of myeloma cells SP2/0. The cryopreserved SP2/0 cells were recovered ten days before fusion and expanded. A part of SP2/0 was cultured in HAT medium for 24 hours to observe whether the cells would be apoptotic.

(2) Feeder cell preparation: The feeder cells of healthy Balb/c mice were plated the day before fusion, and the cell concentration was 1×10 ⁵ cells/mL, 100 μL per well.

(3) Preparation of SP2/0. Gently blow down the cells, collect them into a centrifuge tube, record the total volume of the solution, mix well and take a small amount of cells to properly dilute them for counting. Preparation of splenocytes. Remove the spleen of the fused mouse, rinse the spleen with 1640 basal medium, and keep the spleen in a moist environment, grind the spleen, rinse the spleen cells remaining on the needle core and the filter with the basal medium, collect the grinding fluid, and record the total volume . Take part of the grinding liquid for counting.

(4) SP2/0 cells and splenocytes were mixed in a centrifuge tube at a ratio of 1:5 to 1:10, centrifuged at 1000 rpm/min for 7 min, and the supernatant was discarded. Add 1 mL of PEG that has been preheated in 37°C water, then drop 15 mL of pre-warmed 1640 basal medium to stop the reaction, centrifuge at 1000 rpm/min for 7 min, discard the supernatant, add HAT complete medium to resuspend, each Add 100 μL to the well, place it in a cell culture incubator, and observe the fusion result after five days.

3. Screening and cloning of positive cells

(1) Observe the cells on the fifth day after fusion, and change the medium for the larger wells of the cell mass. On the third day, take the cell supernatant to a plate containing 50ng/well of antigen, and detect by ELISA test. Change the medium once, test again the next day, and clone the wells that are positive both times.

(2) The positive cells are cloned by limiting dilution method, generally three clones are required.

After screening and cloning of positive cells, the hybridoma cell line 7D2 and the hybridoma cell line 7H4 were screened, which were respectively stored in the China Center for Type Culture Collection (CCTCC) on August 23, 2017. The hybridoma cell line 7D2 The deposit number is CCTCC NO: C2017120, and the deposit number of the hybridoma cell line 7H4 is CCTCC NO: C2017121. The address of the collection center is: Collection Center of Wuhan University, Wuchang District, Wuhan City, Hubei Province.

3. Preparation of monoclonal antibodies:

The method for preparing monoclonal antibody 7D2 from hybridoma cell line 7D2 is the same as the method for preparing monoclonal antibody 7H4 from hybridoma cell line 7H4, as follows:

1. Preparation of ascites-type antibodies

(1) 500 μL of incomplete Freund's adjuvant was intraperitoneally injected into the inoculated BaLb/c mice 7 to 14 days before the injection of the hybridoma cell line.

(2) Each mouse was intraperitoneally injected with 2×10 ⁵ to 1×10 ⁶ hybridoma cell lines, and the injection volume was 500 μL. Generally, after 7 to 14 days, the abdomen of the mouse is obviously bulging, and the ascites can be extracted when the movement is slow.

(3) Centrifuge the collected ascites at 4°C and 12000 rpm/min for 30 min, take the supernatant, and store it in aliquots at -20°C.

2. Antibody purification

(1) Ammonium sulfate precipitation method: ① Take out the ascites fluid from -20°C and thaw at 4°C. ② Centrifuge at 12000rpm/min at 4°C for 30min, take the supernatant, and record the volume of the supernatant. ③Put the ascites in a mixed environment of ice and water at 4°C for stirring, slowly add an equal volume of saturated ammonium sulfate solution dropwise while stirring, and a white precipitate can be observed. ④ Centrifuge the ascites after saturated ammonium sulfate precipitation at 4 °C and 7500 rpm for 30 min, discard the supernatant, and resuspend with 1.5 mL of PBS (filtered with a 0.015M filter).

(2) Desalting column to separate salt ions in protein: ① Equilibration: Connect the column (to prevent air from entering the column), and wash off the 20% ethanol in the column for storage with 5 times the column volume of suction-filtered PBS. ②Sampling: The speed should be slow when loading. ③ Sample collection: Since the protein molecule is larger than the salt ion, the protein molecule cannot pass through the narrow gap in the column filler, but can only come out first from the large gap next to it, so only the sample with the first absorption peak is collected. ④Equilibrium: Rinse the column with PBS filtered more than 5 times the column volume. ⑤Storage: Seal the column with 20% filtered ethanol to prevent contamination, and store at 4°C.

(3) Separation of impurity proteins by affinity chromatography column Protein G: ① Equilibrium: connect the column (to prevent air from entering the column), wash off the 20% ethanol in the column for preservation with 5 times the column volume of PBS filtered by suction . ②Sampling: The speed should be slow when loading. ③ Sample collection: The first absorption peak is the impurity protein, which is not collected. After the impurity protein was washed out by PBS, the target protein was eluted with the eluent (pH 2.7 glycine solution), and the protein was collected immediately when the peak increased, and 100 μL of neutralization solution (pH 9) was added to each 1 mL of eluted protein. .0Tris-HCL). ④Equilibrium: Rinse the column with PBS filtered more than 5 times the column volume. ⑤Storage: Seal the column with 20% filtered ethanol to prevent contamination, and store at 4°C.

(4) Ultrafiltration: take a 50KD ultrafiltration tube, fill it with filtered PBS, centrifuge at 5000 rpm/min for 10 min, repeat three times. Balance the inside and outside of the membrane. Protein samples were added and centrifuged at 5000 rpm/min for 10 min. Collect the concentrate collected on the membrane. Top up with PBS, centrifuge at 5000 rpm/min for 10 min, repeat three times, top up with 20% ethanol, and store the ultrafiltration tube at 4°C.

4. Performance testing of monoclonal antibodies

The total RNA of the hybridoma cell lines 7D2 and 7H4 was extracted respectively, the RNA was reverse transcribed into cDNA, the target gene was amplified by PCR, connected to the pMD-18T vector, and finally introduced into competent E. coli for large-scale amplification, and the plasmid was extracted and sent to Sequencing. The light and heavy chain amino acid sequences of the known monoclonal antibody 7D2 are shown in Figure 1, and the light and heavy chain amino acid sequences of the monoclonal antibody 7H4 are shown in Figure 2.

The purity of the purified monoclonal antibodies 7D2 and 7H4 was identified by SDS-PAGE test, and the results are shown in FIG. 3 . The titers of the purified monoclonal antibodies 7D2 and 7H4 were detected by ELISA, and the results are shown in FIG. 4 . The affinity constants of the purified monoclonal antibodies 7D2 and 7H4 were detected by ELISA, and the results are shown in FIG. 5 .

The indirect ELISA test was used to identify the binding of the two antibodies to the other two forms of cTnI: cTnI monomer (purchased by Hytest, Finland) and cTnI-C complex (self-expression in the laboratory). Binding to cTnI and cTnI-C indicates that the two antibodies of the present invention can recognize different forms of cTnI. According to the existing research (prokaryotic expression and identification of cTnI-linker-TnC fusion protein, Chinese Journal of Biotechnology, 2015, 35(4):48-53) and this example, 7D2 and 7H4 can interact with cTnI, cTnI- C or cTnI-T-C undergoes a specific binding reaction, indicating that it can bind to different forms of cTnI

Example 2

The monoclonal antibody 7D2 is used as the capture antibody and 7H4 is used as the detection antibody to establish a double-antibody sandwich ELISA detection method, which can be used to develop a rapid detection kit for myocardial infarction. The establishment process of the double antibody sandwich ELISA method is as follows:

1. Selection of paired antibodies

(1) Coating: Dilute antibody 7D2 to 2 μg/mL with coating solution, and add 100 μL to 96-well enzyme-linked plate. 4°C overnight. Wash three times with PBS-T buffer for 3 min each.

(2) Blocking: add 5% nonfat milk powder to the enzyme-linked plate, add 200 μL to each well, and block at 37°C for one hour. Wash three times with PBS-T buffer for 3 min each.

(3) Add antigen cTnI: Dilute the antigen to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL with PBS buffer, respectively. Add 100 μL to each well. Incubate at 37°C for one hour. Wash three times with PBS-T buffer for 3 min each. Only PBS was added as a negative control.

(4) Add labeled HRP antibody 7H4: Dilute HRP with PBS-T buffer to an appropriate multiple (the corresponding dilution multiple of OD ₄₅₀ in direct ELISA is around 2.0) 100 μL per well, incubate at 37°C for one hour. Wash five times with PBS-T buffer, 3 min each time.

(5) Color development: add 100 μL TMB color developing solution, and protect from light at room temperature for 10 min.

(6) Add 50 μL of stop solution to each well.

(7) The microplate reader reads the absorbance value OD ₄₅₀ at 450 nm and analyzes the data.

2. Establishment of double-antibody sandwich ELISA detection method with 7D2 as capture antibody and 7H4 as detection antibody Establishment of detection curve and detection range: Dilute antibody 7D2 with 0.05 M, pH 9.6 carbonate coating buffer to 2 μg/ mL, add 100 μL to each well, overnight at 4°C. 5% nonfat dry milk was blocked for one hour. Different concentrations of cTnI purchased from Hytest in Finland diluted with PBS-T buffer solution were added as antigen (1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625 ng/mL), 50 μL per well, incubated at 37°C for 1 hour. 100 μL of HRP-7H4 diluted 1:2000 was added and incubated for one hour. Add 100 μL of color developing solution TMB, develop color for 10 minutes, and add 50 μL of stop solution (10% H ₂ SO ₄ ). The microplate reader reads the absorbance value _OD450 at 450nm, analyzes the data, and makes a standard curve according to the data. In the range of cTnI concentration from 30ng/mL to 500ng/mL, with the increase of cTnI concentration, the OD ₄₅₀ value of ELISA reaction increases, and the cTnI concentration is linearly correlated with the OD ₄₅₀ value, so the detection range of this method is: 30ng /mL～500ng/mL (Figure 7). The standard curve of the double-antibody sandwich ELISA detection method with 7D2 as the capture antibody and 7H4 as the detection antibody was drawn (Fig. 8).

Confirm the detection range.

3. Detection of clinical samples: 30 clinical serum samples were tested by self-made double antibody sandwich ELISA method, of which 22 were positive and 8 were negative. Statistical analysis showed that the positive detection rate was 77.3%, and the negative detection rate was 100%.

Comparative Example 1

The monoclonal antibody 7H4 is used as the capture antibody and 7D2 is used as the detection antibody to establish a double-antibody sandwich ELISA detection method, which can be used to develop a rapid detection kit for myocardial infarction. The establishment process of the double antibody sandwich ELISA method is as follows:

1. Selection of paired antibodies

(1) Coating: Dilute antibody 7H4 to 2 μg/mL with coating solution, and add 100 μL to 96-well enzyme-linked plate. 4°C overnight. Wash three times with PBS-T buffer for 3 min each.

(2) Blocking: add 5% nonfat milk powder to the enzyme-linked plate, add 200 μL to each well, and block at 37°C for one hour. Wash three times with PBS-T buffer for 3 min each.

(3) Add antigen cTnI: Dilute the antigen to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL with PBS buffer, respectively. Add 100 μL to each well. Incubate at 37°C for one hour. Wash three times with PBS-T buffer for 3 min each. Only PBS was added as a negative control.

(4) Add labeled HRP antibody 7D2: Dilute HRP with PBS-T buffer to a suitable multiple (the corresponding dilution multiple of OD ₄₅₀ in direct ELISA is around 2.0) 100 μL per well, incubate at 37°C for one hour. Wash five times with PBS-T buffer, 3 min each time.

(5) Color development: add 100 μL TMB color developing solution, and protect from light at room temperature for 10 min.

(6) Add 50 μL of stop solution to each well.

(7) The microplate reader reads the absorbance value OD ₄₅₀ at 450 nm and analyzes the data.

2. The established 7H4 is the capture antibody and 7D2 is the detection antibody. The establishment of the detection curve and the establishment of the detection range of the double-antibody sandwich ELISA detection method: use the coating buffer to dilute with 0.05 M, pH 9.6 carbonate coating buffer Antibody 7H2 to 2 μg/mL, add 100 μL to each well, overnight at 4°C. 5% nonfat dry milk was blocked for one hour. Different concentrations of standard antigen cTnI diluted with PBS-T buffer (0, 15.625, 31.25, 62.5, 125, 250, 500, 1000ng/mL) were added, 50 μL per well, and incubated at 37°C for 1 hour. 100 μL of HRP-7D2 diluted 1:2000 was added and incubated for one hour. Add 100 μL of color developing solution TMB, develop color for 10 minutes, and add 50 μL of stop solution. The microplate reader reads the absorbance value _OD450 at 450nm, and analyzes the data. It can be seen that in the range of cTnI concentration from 60ng/mL to 500ng/mL, with the increase of cTnI concentration, the _OD450 value of ELISA reaction increases, and cTnI concentration and OD The ₄₅₀ values are linearly correlated, but the slope of this correlation is small. Therefore, the detection range of this method is: 60ng/mL～500ng/mL (Fig. 7).

Comparing Example 2 and Comparative Example 1, it can be found that:

According to the detection curves of Example 7D2/HRP-7H4 and Comparative Example 7H4/HRP-7D2, the respective detection ranges were determined. According to the figure, the detection curves of 7D2/HRP-7H4 were larger than those of 7H4/HRP-7D2. Wide and high sensitivity.

Claims (2)

1. A hybridoma cell strain 7H4 of human cardiac troponin I is characterized in that the hybridoma cell strain 7H4 is preserved in China Center for Type Culture Collection (CCTCC) in 2017, 8 and 23 months, and the preservation number is CCTCC NO: C2017121.

2. the hybridoma cell line 7H4 of claim 1 secreting human cardiac troponin I monoclonal antibody 7H 4.

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