CN107603955B

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

Info

Publication number: CN107603955B
Application number: CN201710797562.7A
Authority: CN
Inventors: 王宏; 余娟; 李文丽
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2017-09-06
Filing date: 2017-09-06
Publication date: 2020-06-16
Anticipated expiration: 2037-09-06
Also published as: CN107603955A

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 strain 7H4, monoclonal antibody and application

Technical Field

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

Background

The cTnI is an inhibitory subunit of actin, and regulates the interaction of actin and myosin by inhibiting the activity of adenosine triphosphate (ATPase) of actin. the molecular weight of cTnI is about 24kDa, consists of 209 amino acids, is a protein rich in α helices with a theoretical isoelectric point of 9.87. TnI (troponin I) has three subtypes, namely, a fast skeletal muscle type and a slow skeletal muscle type, which have similar molecular weights (20 KD) but have a 40% difference in amino acid sequence therebetween, and a third is a phosphorylated form of troponin I (cTnI) also has a 40% difference in amino acid sequence with the skeletal muscle type, and the cTnI has a phosphorylation amino acid at the terminal of 40% and thus has a phosphorylation specificity at the terminal of 23. the four different amino acid types of myocardial TnI, cTnI is phosphorylated at the terminal of 23. the cardiac muscle type, and cTnI is phosphorylated at the terminal of four different amino acid types.

Acute Myocardial Infarction (AMI) is one of the most common cardiovascular diseases, and is characterized in that on the basis of coronary artery lesion, blood flow of coronary artery is sharply reduced or interrupted, so that coronary artery lesion caused by severe and persistent Acute ischemia of corresponding myocardium occurs, and finally ischemic necrosis of myocardium occurs. In severe cases, the heart fails to pump blood, which leads to sudden death of the patient. 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 AMI diagnosis, and since it appears in blood only when the myocardium is damaged, and it appears early, lasts long, and has strong specificity, cTnI has been regarded as the "gold standard" for AMI diagnosis by authorities such as the international association of clinical chemistry (IFCC), the american society for clinical biochemistry (NACB), and the chinese medical testing council.

Among the markers for myocardial infarction, cTnI is considered to be a "gold marker" that is more sensitive and more specific than other markers. When the membrane of the cardiomyocytes is intact, cTnI is not able to permeate the membrane into the blood. Only when the myocardial cells are damaged, the cTnI is rapidly released from the myocardial cells into the blood, the cTnI begins to rise within 2-8 hours, the peak is reached within 1-2 days, the amount of the cTnI can still be measured to rise after 3-8 days, theoretically, the cTnI is maintained in serum for more than 10 days, and a longer diagnosis window period is very important for the diagnosis of myocardial infarction. Research proves that the critical value of cTnI in the serum of normal people and myocardial infarction patients is 50pg/mL, and the blood concentration can reach 100-300 mug/L during disease attack. The majority of cTnI in blood exists as cTnI-T-C or as a complex of cTnI-C. In the research, it was found that cTnI is initially released in the form of monomers into blood, but as the degree of myocardial damage increases, cTnI exists in the form of a complex in order to prevent it from being degraded, and then the complex is decomposed into monomers due to various factors, so that an antibody for detecting cTnI should recognize various complex forms of cTnI in blood with the same efficiency, and such a monoclonal antibody resource is seriously lacking in the prior art.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a human cardiac troponin I hybridoma cell strain 7H 4.

The monoclonal antibody secreted by the hybridoma cell strain 7H4 is named as human cardiac troponin I monoclonal antibody 7H4, and the monoclonal antibody 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 monoclonal antibody can be used for detecting the content of cTnI in serum, and lays a foundation for developing a kit for rapidly detecting myocardial infarction.

The third purpose of the invention is to provide the application of the human cardiac troponin I monoclonal antibody 7H4 in detecting free cTnI, dimer compound cTnI-C and trimer compound cTnI-T-C as three forms of cTnI.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a hybridoma cell strain 7H4 of human cardiac troponin I, wherein 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 address of the depository is: wuhan university Collection in Wuchang district, Wuhan city, Hubei province.

The human cardiac troponin I monoclonal antibody 7H4 secreted by the hybridoma cell strain 7H 4. The variable region genes of the human cardiac troponin I monoclonal antibody 7H4 comprise a heavy chain variable region gene and a light chain variable region gene, and the light and heavy chain amino acid sequence of the monoclonal antibody 7H4 is shown in figure 2.

The human cardiac troponin I monoclonal antibody 7H4 is applied in detecting free cTnI, dimer compound cTnI-C and trimer compound cTnI-T-C.

The monoclonal antibody 7H4 is prepared by utilizing immune cTnI-T-C protein, and the specific preparation method comprises the following steps: immunizing a Balb/C mouse by using holoprotein cTnI-T-C, fusing spleen cells of the mouse with myeloma cells, screening out positive hybridoma cell strains through indirect ELISA, obtaining hybridoma cell strains capable of secreting stable antibodies through a three-time limiting dilution cloning technology, preparing a ascites monoclonal antibody, and purifying to obtain the monoclonal antibody.

Compared with the prior art, the invention has the following beneficial effects: the human cardiac troponin I monoclonal antibody 7H4 provided by the invention 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 human cardiac troponin I monoclonal antibody can be used for detecting the content of cTnI in serum, and lays a foundation for developing a kit for rapidly detecting myocardial infarction.

Drawings

FIG. 1 shows the 3 CDR (complementarity determining region) regions of the light and heavy chain variable region of antibody 7D 2.

FIG. 2 shows the 3 CDR (complementarity determining region) regions of the light and heavy chain variable region of antibody 7H 4.

FIG. 3 is a SDS-PAGE image of purified antibodies 7D2 and 7H4, wherein 1 is protein Marker, 2 is 7D2, and 3 is 7H 4.

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

FIG. 5 is a graph of affinity constant detection ELISA for antibodies 7D2 and 7H4, wherein panel A is 7D2 and panel B is 7H 4.

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

FIG. 7 shows the detection curves of 7D2/HRP-7H4 and 7H4/HRP-7D 2.

FIG. 8 is a graph showing the standard detection curve of 7D2/HRP-7H 4.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

The preparation of hybridoma cell strain and monoclonal antibody secreted by the hybridoma cell strain comprises the following steps:

antigen, antigen

The monoclonal antibody 7D2 is a monoclonal antibody prepared by immunizing Balb/c mice with complete antigen obtained by coupling amino acids at positions 13-24 on cTnI with BSA, and the preparation method of the complete antigen containing amino acids at positions 13-24 on cTnI comprises the following steps: simulating the structure of cTnI-T-C by using Discovery studio4.0 software, analyzing the epitope on cTnI subunit, and selecting the 13 th to 24 th amino acids as epitope antigen. Amino acids 13-24 of cTnI were synthesized and conjugated with BSA to form complete antigen, a procedure synthesized by Hangzhou midpeptide.

The monoclonal antibody 7H4 was a monoclonal antibody prepared by immunizing Balb/C mice with the holoprotein complex cTnI-T-C, which was purchased from Hytest, Finland.

Secondly, preparing hybridoma cell strains: the preparation method of the hybridoma cell line 7D2 is the same as that of the hybridoma cell line 7H4, the hybridoma cell line and the hybridoma cell line only use different antigens, and the preparation method comprises the following steps:

1. animal immunization

(1) Primary immunization: emulsifying 50 mu g of antigen and the volume of the complete Freund adjuvant with the same volume, and performing subcutaneous multi-point injection;

(2) and (3) boosting immunity: 2 weeks apart, the same antigen amount as the primary immunization is emulsified with incomplete Freund's adjuvant in the same volume, and is injected subcutaneously in multiple points; collecting blood 10 days after 3-5 times of immunization to measure the titer;

(3) 25 μ g of antigen without adjuvant was injected intraperitoneally 72 hours prior to fusion.

2. Cell fusion

(1) Feeding of myeloma cells SP 2/0. The frozen SP2/0 cells were thawed ten days before fusion and expanded. A portion of SP2/0 was cultured in HAT medium for 24 hours to examine whether or not the cells were apoptotic.

(2) Preparing feeder cells: the day before fusion, feeder cells of healthy Balb/c mice are taken for plating, and the cell concentration is cellTo 1X 10⁵one/mL, 100. mu.L per well.

(3) Preparation of SP 2/0. Gently blowing down the cells, collecting the cells into a centrifuge tube, recording the total volume of the solution, uniformly mixing the solution, taking a small amount of cells, properly diluting the cells, and counting the cells. Preparation of splenocytes. The spleen of the fusion mouse was removed, the spleen was washed with 1640 basic medium with the spleen in a humid environment, the spleen was ground, spleen cells remaining on the needle core and the filter screen were washed with basic medium, the ground solution was collected, and the total volume was recorded. And taking part of the grinding fluid for counting.

(4) Uniformly mixing SP2/0 cells and splenocytes in a centrifuge tube according to the ratio of 1: 5-1: 10, centrifuging at 1000rpm for 7min, and discarding the supernatant. Adding 1mL of PEG which is preheated in water at 37 ℃, then dripping 15mL of 1640 basic culture medium which is preheated to terminate the reaction, centrifuging at 1000rpm/min for 7min, discarding the supernatant, adding HAT complete culture medium for resuspension, adding 100 mu L of HAT complete culture medium into each hole, placing the mixture in a cell incubator for culture, and observing the fusion result after five days.

3. Positive cell selection and cloning

(1) Observing cells on the fifth day after fusion, changing the liquid of the hole with larger cell mass, taking the cell supernatant to a plate coated with 50 ng/hole antigen on the third day, carrying out ELISA test detection, changing the liquid of the positive hole once again, carrying out detection again every other day, and cloning the hole which is detected to be positive twice.

(2) Positive cells were cloned by limiting dilution, typically three times.

After positive cells are screened and cloned, a hybridoma cell line 7D2 and a hybridoma cell line 7H4 are screened, the hybridoma cell line and the hybridoma cell line are respectively preserved in China Center for Type Culture Collection (CCTCC) in 2017, 8 and 23 months, and the preservation number of the hybridoma cell line 7D2 is CCTCC NO: c2017120, the preservation number of the hybridoma cell strain 7H4 is CCTCC NO: C2017121. the address of the depository is: wuhan university Collection in Wuchang district, Wuhan city, Hubei province.

Thirdly, preparing the monoclonal antibody:

the method for preparing the monoclonal antibody 7D2 from the hybridoma cell strain 7D2 is the same as the method for preparing the monoclonal antibody 7H4 from the hybridoma cell strain 7H4, and comprises the following specific steps:

1. preparation of ascites type antibody

(1) And injecting 500 mu L of incomplete Freund's adjuvant into the abdominal cavity of the inoculated Balb/c mice 7-14 days before the hybridoma cell strain is injected.

(2) Intraperitoneal injection of 2X 10 per mouse⁵～1×10⁶The injection volume of each hybridoma cell strain is 500 mu L. Generally, the abdomen of the mouse is obviously raised after 7 to 14 days, and ascites can be extracted when the mouse moves slowly.

(3) Centrifuging the collected ascites at 4 deg.C and 12000rpm/min for 30min, collecting supernatant, and packaging at-20 deg.C.

2. Purification of antibodies

(1) Ammonium sulfate precipitation method, ① ascites is taken out from-20 deg.C, thawed at 4 deg.C, centrifuged at 12000rpm/min at ② 4 deg.C for 30min, the supernatant is taken, the volume of the supernatant is recorded, ③ ascites is stirred in an ice water mixing environment at 4 deg.C, an equal volume of saturated ammonium sulfate solution is slowly added dropwise while stirring, white precipitate is observed to be generated, stirring is continued for 15min, standing is carried out overnight at 4 deg.C, ④ ascites after saturated ammonium sulfate precipitation is centrifuged at 7500 rpm for 30min at 4 deg.C, the supernatant is discarded, and the supernatant is resuspended in 1.5mL of PBS (suction filtered through a 0.015M filter membrane).

(2) The desalting column separates the salt ions in the protein ① equilibrium: the column is connected (preventing air from entering the column), the filtered PBS with 5 times the column volume is used to wash out 20% of the ethanol for preservation in the column, ② sample loading is carried out with slow speed, ③ sample collection is carried out, because the protein molecules are bigger than the salt ions, the protein molecules can not pass through the narrow gap in the filler in the column, and only can come out from the big gap nearby, therefore only the sample with the first absorption peak is collected, ④ equilibrium: the filtered PBS with more than 5 times the column volume is used to wash out the column, ⑤ preservation is carried out, the column is sealed with 20% of the filtered ethanol to prevent pollution, and the storage is carried out at 4 ℃.

(3) Affinity chromatography column Protein G for separating hybrid Protein ① equilibrium: the column is connected (preventing air from entering the column), 5 times column volume of filtered PBS is used to wash off 20% of ethanol for storage in the column, ② sample is loaded with slow speed, ③ sample is collected, the first absorption peak is the hybrid Protein and is not collected, after the hybrid Protein is washed out by PBS, the target Protein is eluted by eluent (pH2.7 glycine solution), Protein is collected as soon as the peak value rises, and 100 uL of Tris-HCL is added to 1mL of eluted Protein, ④ equilibrium is that the column is washed by PBS with more than 5 times column volume of filtered PBS, ⑤ storage is that the column is sealed by 20% filtered ethanol to prevent pollution, and the column is stored at 4 ℃.

(4) And (3) ultrafiltration: a50 KD ultrafiltration tube was filled with filtered PBS, centrifuged at 5000 rpm/min for 10min, and repeated three times. So that the inside and the outside of the membrane are balanced. Protein samples were added and centrifuged at 5000 rpm/min for 10 min. And collecting the concentrated solution collected on the membrane. Adding PBS, centrifuging at 5000 rpm/min for 10min, repeating for three times, adding 20% ethanol, and storing at 4 deg.C.

Fourthly, detecting the performance of the monoclonal antibody

Respectively extracting total RNA of hybridoma cell strains 7D2 and 7H4, carrying out reverse transcription on the RNA to form cDNA, carrying out PCR amplification on a target gene, connecting a pMD-18T vector, finally introducing into competent escherichia coli for large-scale amplification, extracting plasmids and carrying out sequencing. The amino acid sequences of the light and heavy chains of monoclonal antibody 7D2 are shown in FIG. 1, and the amino acid sequences of the light and heavy chains of monoclonal antibody 7H4 are shown in FIG. 2.

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

Indirect ELISA assays were used to identify binding of 2 antibodies to two other forms of cTnI: cTnI monomer (purchased from Hytest corporation, Finland) and cTnI-C complex (self-expressed in the laboratory), 2 antibodies from FIG. 6 were able to bind to cTnI and cTnI-C, indicating that 2 antibodies of the present invention were able to recognize different forms of cTnI. According to the existing research (prokaryotic expression and identification of cTnI-linker-TnC fusion protein, J.Bioengineering, China 2015,35(4): 48-53) and this example, 7D2 and 7H4 can generate specific binding reaction with cTnI, cTnI-C or cTnI-T-C, which shows that it can be combined with cTnI in different forms

Example 2

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

1. selection of partner antibodies

(1) Coating: antibody 7D2 was diluted to 2. mu.g/mL with coating solution and 100. mu.L was added to a 96-well plate. 4 ℃ overnight. Three washes with PBS-T buffer were performed for 3min each.

(2) And (3) sealing: add 5% skim milk to the ELISA plates at 200. mu.L/well and block for one hour at 37 ℃. Three washes with PBS-T buffer were performed for 3min each.

(3) Adding an antigen cTnI: the antigen was diluted to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL with PBS buffer, respectively. Add 100. mu.L to each well. Incubate at 37 ℃ for one hour. Three washes with PBS-T buffer were performed for 3min each. PBS alone was added as a negative control.

(4) Labeling HRP antibody 7H 4: HRP was diluted to the appropriate fold with PBS-T buffer (OD in direct ELISA)₄₅₀Dilution fold corresponding to around 2.0) per well, and incubated at 37 ℃ for one hour. Wash five times for 3min in PBS-T buffer.

(5) Color development: adding 100 mu LTMB developing solution, and keeping away from light for 10min at room temperature.

(6) Add 50. mu.L of stop buffer to each well.

(7) Reading absorbance value OD of 450nm by enzyme-linked immunosorbent assay₄₅₀And analyzing the data.

2. The established 7D2 is a capture antibody, the established 7H4 is the establishment of a detection curve and the establishment of a detection range of a double-antibody sandwich ELISA detection method for detecting the antibody: antibody 7D2 was diluted to 2. mu.g/mL with 0.05M, pH 9.6 carbonate coating buffer, 100. mu.L per well, overnight at 4 ℃. 5% skimmed milk powder was sealed for one hour. cTnI as antigen (1000 ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25 ng/mL) was added to each of the samples at different concentrations, which were obtained from Hytest corporation in Finland diluted in PBS-T buffer solutionng/mL, 15.625 ng/mL), 50. mu.L per well, incubated at 37 ℃ for 1 hour. 100 μ L of each was added as 1: HRP-7H4 at 2000 dilution was incubated for one hour. Adding 100 μ L of color developing solution TMB, developing for 10min, adding 50 μ L of stop solution (10% H)₂SO₄). Reading absorbance value OD of 450nm by enzyme-linked immunosorbent assay₄₅₀And analyzing the data and making a standard curve according to the data. In the range of cTnI concentration from 30ng/mL to 500ng/mL, the OD of the ELISA reaction increased with the increase of the cTnI concentration₄₅₀Increased value, cTnI concentration and OD₄₅₀The values are linearly related, so the range of detection of the method is: 30 ng/mL-500 ng/mL (FIG. 7). And a standard curve for the double antibody sandwich ELISA detection method with 7D2 as capture antibody and 7H4 as detection antibody was plotted (fig. 8).

The range of detection was confirmed.

3. Detection of clinical samples: 30 clinical serum samples are detected by a self-made double-antibody sandwich ELISA method, wherein 22 positive samples are detected, and 8 negative samples are detected. The statistical analysis result shows that the positive detection rate is 77.3 percent and the negative detection rate is 100 percent.

Comparative example 1

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

1. selection of partner antibodies

(1) Coating: antibody 7H4 was diluted to 2. mu.g/mL with coating solution and 100. mu.L was added to a 96-well plate. 4 ℃ overnight. Three washes with PBS-T buffer were performed for 3min each.

(3) Adding an antigen cTnI: the antigen was diluted to 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL with PBS buffer, respectively. Add 100. mu.L to each well. Incubate at 37 ℃ for one hour. Washed three times with PBS-T buffer, 3min each time. PBS alone was added as a negative control.

(4) Labeled HRP antibody7D 2: HRP was diluted to the appropriate fold with PBS-T buffer (OD in direct ELISA)₄₅₀Dilution fold corresponding to around 2.0) per well, and incubated at 37 ℃ for one hour. Wash five times for 3min in PBS-T buffer.

(6) Add 50. mu.L of stop buffer to each well.

2. The established 7H4 is a capture antibody, the 7D2 is the establishment of a detection curve and the establishment of a detection range of a double-antibody sandwich ELISA detection method for detecting the antibody: antibody 7H2 was diluted to 2. mu.g/mL with coating buffer using 0.05M carbonate coating buffer, pH 9.6, 100. mu.L per well, overnight at 4 ℃. 5% skimmed milk powder was sealed for one hour. Different concentrations of the standard antigen cTnI (0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 ng/mL) diluted in PBS-T buffer were added to each well at 50. mu.L/well and incubated at 37 ℃ for 1 hour. mu.L of HRP-7D2 diluted at 1:2000 was added and incubated for one hour. 100. mu.L of color developing solution TMB was added, and color development was carried out for 10 minutes, and 50. mu.L of stop solution was added. Reading absorbance value OD of 450nm by enzyme-linked immunosorbent assay₄₅₀From the analysis of the data, it was found that the OD of the ELISA reaction increased with the increase of the cTnI concentration in the range of 60ng/mL to 500ng/mL of the cTnI concentration₄₅₀Increased value, cTnI concentration and OD₄₅₀The values are linearly related, but the slope of the correlation is small. Therefore, the detection range of the method is as follows: 60 ng/mL-500 ng/mL (FIG. 7).

Comparing example 2 with comparative example 1, it can be found that:

the detection ranges of the detection curves of the example 7D2/HRP-7H4 and the comparative example 7H4/HRP-7D2 are determined, and the detection curve of the 7D2/HRP-7H4 is larger in change rate, wide in detection range and high in sensitivity than the detection curve of the 7H4/HRP-7D 2.

Claims

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.