A soluble cd39 protein content detection kit and application thereof

Abstract

The application belongs to the technical field of biomedicine, and relates to a soluble CD39 protein content detection kit and application thereof. The kit comprises two anti-human CD39 monoclonal antibodies, CD39-19A4 and CD39-5H11. The amino acid sequence of the heavy chain variable region of the CD39-19A4 is shown in SEQ ID NO. 1, the amino acid sequence of the light chain variable region of the CD39-19A4 is shown in SEQ ID NO. 2, the amino acid sequence of the heavy chain variable region of the CD39-5H11 is shown in SEQ ID NO. 3, and the amino acid sequence of the light chain variable region of the CD39-5H11 is shown in SEQ ID NO. 4. The soluble CD39 protein content detection kit can effectively detect the content of soluble CD39, and has high clinical value in the prognosis judgment of clinical breast cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedicine, and particularly relates to a soluble CD39 protein content detection kit and application thereof. BACKGROUND

[0002] CD39 is also known as ectonucleoside triphosphate diphosphohydrolase-1, which is encoded by the ENTPD1 gene. CD39 is a transmembrane protein with two transmembrane domains and an extracellular domain, and exists in two forms of membrane type and solubility. Functionally, CD39 can bind to extracellular ATP (eATP) and hydrolyze it into AMP, accompanied by the generation of inorganic phosphate (Pi); another extracellular nucleic acid enzyme CD73 hydrolyzes AMP into adenosine (ADO). In the ATP-ADO pathway, CD39 is the rate-limiting enzyme for eATP hydrolysis. In addition, CD39 can also hydrolyze ADP, but with lower efficiency. Studies have shown that CD39 is widely expressed in various tissues and organs, such as bladder, brain, breast, colon, uterus, stomach, prostate, etc., and is mainly expressed in endothelial cells and immune cells. The expression of CD39 in different types of cells also varies significantly. The expression rate of CD4+ T cells (including memory T cells and regulatory T cells) is about 20%~30%, while the expression rate of CD8+ T cells is less than 5%, and the expression rate of natural killing cell (NK) is 2%~5%. CD39 is expressed on tumor-infiltrating regulatory T cells (Treg), myeloid cells and some NK cells. Compared with normal cells, tumor cells overexpress CD39, and almost all high CD39 expression is considered to be a marker of poor prognosis and disease progression. Studies have found that CD39+CD8+ T cells are expressed in tumor or metastatic lymph nodes in breast cancer specimens, and there is differential expression in peripheral blood and non-metastatic lymph nodes. Another study pointed out that the content of CD39 in breast cancer tissue is higher than that in normal tissue, and analysis showed that CD39 expression was related to the survival of Luminal type breast cancer patients, indicating that CD39 may be an effective therapeutic target for breast cancer. At present, there are few studies on soluble CD39, and the detection value of soluble CD39 in the peripheral serum of tumor patients is worth further exploring. SUMMARY

[0003] One of the purposes of the present application is to provide a soluble CD39 protein content detection kit;

[0004] The second object of the present application is to provide an application of the soluble CD39 protein content detection kit of the first aspect of the present application in detecting the soluble CD39 protein content in patients with immune diseases.

[0005] The third object of the present application is to provide an application of the soluble CD39 protein content detection kit of the first aspect of the present application in clinical breast cancer detection.

[0006] In order to achieve the object of the present application, the technical solution adopted is as follows:

[0007] The first aspect of the present application provides a soluble CD39 protein content detection kit, which comprises a CD39-19A4 anti-human monoclonal antibody and a CD39-5H11 anti-human monoclonal antibody, wherein the CD39-19A4 anti-human monoclonal antibody is used as a coating antibody, and the CD39-5H11 anti-human monoclonal antibody is used as a detection antibody.

[0008] According to some embodiments of the soluble CD39 protein content detection kit of the present application, the CD39-19A4 anti-human monoclonal antibody comprises a heavy chain and a light chain.

[0009] The amino acid sequence of the heavy chain variable region (mVH) of the CD39-19A4 anti-human monoclonal antibody is shown in SEQ ID NO. 1:

[0010] EVQLQQSGAELARPGASVKMSCKTSGYTFTSYTIHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCAREYYRYGYAVDYWGQGTSVTVSS.

[0011] The amino acid sequence of the light chain variable region (mVL) of the CD39-19A4 anti-human monoclonal antibody is shown in SEQ ID NO. 2:

[0012] DIQMTQSPSSMYASLGERVTITCKASQDIKNYLSWYQQKPWKSPKTLIYYATNLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPYTFGGGTKLEIK.

[0013] The CD39-5H11 anti-human monoclonal antibody comprises a heavy chain and a light chain.

[0014] The amino acid sequence of the heavy chain variable region (mVH) of the CD39-5H11 anti-human monoclonal antibody is shown in SEQ ID NO. 3:

[0015] EVQLKESGPELVKPGASVKVSCKASGYSFTDYNIYWVKQSHRESLEWIGYFDPYNGGTRYNQKFEGKATLTADKSSSTAFMHLNSLTSEDSAVYYCARGLRFRFVYWGQGTLVTVSS.

[0016] The amino acid sequence of the light chain variable region (mVL) of the CD39-5H11 anti-human monoclonal antibody is shown as SEQ ID NO. 4:

[0017] DIVLTQSPAIMSTSPGEKVTMTCRASSSVSSTYFHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDVATYYCQQFSGYPLTFGAGTKLELK.

[0018] According to some embodiments of the soluble CD39 protein content detection kit provided in the present application, the kit is used for detecting the soluble CD39 protein content in human serum, plasma or tissue fluid.

[0019] The second aspect of the present application provides an application of the soluble CD39 protein content detection kit of the first aspect of the present application in detecting the soluble CD39 protein content of patients with immune diseases.

[0020] The third aspect of the present application provides an application of the soluble CD39 protein content detection kit of the first aspect of the present application in clinical breast cancer detection.

[0021] The beneficial effects of the present application include that the soluble CD39 protein content detection kit provided in the present application can effectively detect the soluble CD39 content. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 A standard curve diagram of the soluble CD39 protein content detection kit provided in the present application;

[0023] Figure 2 A specificity investigation diagram of the soluble CD39 protein content detection kit provided in the present application;

[0024] Figure 3 A comparison diagram of the expression levels of soluble CD39 protein in serum of normal people, patients with benign breast diseases and patients with malignant breast cancer;

[0025] Figure 4 A diagram of clinical correlation analysis of the expression levels of soluble CD39 protein and pathological parameters of patients with malignant breast cancer. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below, examples of which are intended to be illustrative only and are not intended to be limiting.

[0027] In the present application, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the particular feature, structure, material, or characteristic being described is included in at least one embodiment or example of the present application. The illustrative appearances of the above-mentioned terms in this specification are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. In addition, the terms "comprising", "including", "containing", and "consisting" and their derivatives, are not intended to mean that the present application excludes other features, structures, materials, or characteristics not specifically listed. In addition, the terms "comprising", "including", "containing", and "consisting" and their derivatives are intended to mean that the present application includes the features, structures, materials, or characteristics specifically listed, and that the possibility of including other features, structures, materials, or characteristics is not excluded.

[0028] Example 1

[0029] Determination of the sequences of the light and heavy chain variable regions of the anti-human CD39 monoclonal antibody:

[0030] The method for determining the sequences of the heavy and light chain variable regions of the anti-human CD39 monoclonal antibody comprises the following steps:

[0031] 1.1 Obtaining of the cDNA of the hybridoma cells

[0032] The mRNA is extracted from the hybridoma cell strain secreting the anti-human CD39 monoclonal antibody, and the cDNA is synthesized by enzymatic reverse transcription. The sequence information of the heavy chain variable region (mVH) and the light chain variable region (mVL) of the antibody is obtained by molecular cloning and PCR sequencing.

[0033] 1.2 The heavy chain variable region (mVH) and the light chain variable region (mVL) are respectively connected with the pJET cloning vector. Subsequently, the connected product is transformed into the DH5a competent bacteria, and the transformed bacterial liquid is uniformly coated on the LB solid medium.

[0034] 1.3 The colonies with clear edges and good growth on the LB solid medium are selected for sequencing identification.

[0035] 1.4 The candidate light and heavy chain variable region sequences are retained according to the sequencing results. The light and heavy chain variable region sequences that can be connected with the expression vector are cloned again by PCR, and the variable region sequences are connected with the expression vector. Subsequently, the connected product is transformed into DH5a. The transformed bacterial liquid is uniformly coated on the LB solid medium and cultured overnight.

[0036] 1.5 Pick well grown bacteria for sequencing and compare the results of the two sequencing runs to obtain the transformed bacteria with the correct sequence, which is then expanded and plasmid extracted.

[0037] 1.6 Co-transfect eukaryotic expression cell 293 with expression vector containing the heavy and light chain variable region genes of the monoclonal antibody.

[0038] 1.7 293 cells are grown in suspension in serum free medium SFM4Transfx-293 without L-Glutamine and the medium is replaced with serum free medium Gibco ® FreeStyle™ 293 Expression Medium.

[0039] 1.8 The supernatant containing the antibody of interest is tested using an ELISA kit and the results are good.

[0040] The CD39-19A4 anti-human monoclonal antibody heavy chain variable region is obtained by the above method:

[0041] EVQLQQSGAELARPGASVKMSCKTSGYTFTSYTIHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCAREYYRYGYAVDYWGQGTSVTVSS;

[0042] The CD39-19A4 anti-human monoclonal antibody light chain variable region is:

[0043] DIQMTQSPSSMYASLGERVTITCKASQDIKNYLSWYQQKPWKSPKTLIYYATNLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPYTFGGGTKLEIK;

[0044] The CD39-5H11 anti-human monoclonal antibody heavy chain variable region is:

[0045] EVQLKESGPELVKPGASVKVSCKASGYSFTDYNIYWVKQSHRESLEWIGYFDPYNGGTRYNQKFEGKATLTADKSSSTAFMHLNSLTSEDSAVYYCARGLRFRFVYWGQGTLVTVSS;

[0046] The CD39-5H11 anti-human monoclonal antibody light chain variable region is:

[0047] DIVLTQSPAIMSTSPGEKVTMTCRASSSVSSTYFHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDVATYYCQQFSGYPLTFGAGTKLELK.

[0048] The variable region of the heavy chain and the light chain of the antibody is extracted from the hybridoma cell secreting the anti-human CD39 monoclonal antibody, the candidate light chain and heavy chain variable region sequences are reserved according to the sequencing result, the light chain and heavy chain variable region sequences matched with the expression vector are amplified by PCR, and the PCR product is connected with the double enzyme pretreated expression vector. The expression vector connected with the light chain and heavy chain variable region of the monoclonal antibody is co-transfected into the eukaryotic expression cell strain 293, and the supernatant after culture contains the target antibody, which indicates that the obtained light chain and heavy chain variable region sequences are correct.

[0049] Example 2

[0050] An ELISA kit for detecting the content of soluble CD39 protein (sCD39):

[0051] 2.1 Composition of the kit

[0052] The ELISA kit of the present application comprises the CD39 coating antibody (CD39-19A4) in Example 1 coated on the enzyme-labeled plate, the HRP-labeled CD39 detection antibody (CD39-5H11-HRP), the soluble CD39 protein standard (Shanghai Baiying Biotechnology Co., Ltd.), the sample diluent, the washing solution (PBST), the color developing solution (TMB) and the termination solution.

[0053] 2.2 Collection and processing of samples

[0054] 2.2.1 Collect a batch of serum of normal people, benign breast disease and malignant breast cancer patients in the hospital, store them in the-80℃ refrigerator after being divided into small bottles, and avoid repeated freezing and thawing.

[0055] 2.2.2 Before detecting the serum sample, pre-equilibrate the sample at room temperature for half an hour, shake and mix, take an appropriate amount of patient serum sample, dilute it with diluent for 5 times dilution.

[0056] 2.3 Determination method of the content of soluble CD39 protein

[0057] 2.3.1 Dilute the coating antibody CD39-19A4 (1 µg / ml) with the coating solution (Na2CO3 and NaHCO3), then add the coating antibody into the 96-well enzyme-labeled plate (100 µl / well), and incubate at 4℃ overnight.

[0058] 2.3.2 The next day, wash the plate with washing solution (PBST) 3 times, then add 100 μl of 3% bovine serum blocking solution to each well, and block at 37°C for 1 h.

[0059] 2.3.3 Remove the blocking solution, add 100 μl of diluted plasma sample to be tested and gradient-diluted soluble CD39 protein standard (start with 1 ng / ml, and dilute by 2 times, a total of 8 concentrations (1 ng / ml, 0.5 ng / ml, 0.25 ng / ml, 0.125 ng / ml, 0.0625 ng / ml, 0.03125 ng / ml, 0.015625 ng / ml, 0 ng / ml), 3 repeats for each gradient, and incubate in a 37°C incubator for 2 h.

[0060] 2.3.4 Wash the plate with PBST solution 3 times, add HRP-labeled detection antibody CD39-5H11 (100 μl / well) at a ratio of 1:2000, and incubate in a 37°C incubator for 1 h.

[0061] 2.3.5 Wash the plate with PBST solution 6 times, add color developing solution TMB (100 μl / well), and incubate at room temperature in the dark for 10 min, then add stop solution (50 μl / well) to stop the color reaction, and determine the OD450 value of each well by means of an enzyme-labeled instrument.

[0062] 2.3.6 Take the concentration of CD39 standard multiplied by the value of sample dilution times (5) as the ordinate, and the corresponding measured OD450 value as the abscissa to draw a standard curve (as shown in Figure 1 ), and obtain a calculation formula, according to which the content of soluble CD39 in the sample to be tested is calculated.

[0063] The kit of the present application can achieve the following indicators according to methodological identification:

[0064] Curve equation: y = -0.1024x + 1.0914x + 0.0758 2

[0065] Standard curve linearity: R 2 = 0.9993; minimum detection limit ≤ 78.1 pg / ml.

[0066] 2.4 Specificity investigation

[0067] ​Serially dilute PD-L1 1g, B7-H3 1g, OX40L 1g and CD39 1g into different concentrations. Adjust anti-human CD39 monoclonal antibody (19A4) to 5 pg / ml with carbonate buffer (0.01M CBS, pH 9.3) to coat ELISA detection plate at 4°C overnight. Wash 3 times with PBS (containing 0.1% Tween 20), and block with 3% BSA at room temperature for 1 h. After washing 3 times with PBS, add the above-mentioned diluted commercial proteins, and react at room temperature for 2 h, and wash 3 times with PBS. Then, add HRP-labeled monoclonal antibody HRP-5H11 (1:2000, 100 pl / well), and continue to react at room temperature for 1 h, wash 6 times with PBST, and then add HRP reaction substrate TMB (100 pl / well), and react at room temperature for 15 min, stop the reaction with 2 mol / L H2SO4, and determine OD450nm with an enzyme-labeled instrument. Set 3 replicate wells for each sample. The specificity analysis results are shown in Table 1. Figure 2

[0068] 2.5 Accuracy and stability investigation:

[0069] 1) Intra-plate accuracy analysis: In the same experiment, five samples with known concentrations (500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL) were set up in 10 replicate wells for sCD39 detection to analyze the accuracy of the kit (Table 1).

[0070] 2) Inter-plate accuracy analysis: In different batch experiments, five samples with known concentrations (500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, and 31.25 pg / mL) were set up in 3 replicate wells for sCD39 detection to analyze the accuracy of the kit, and the results are shown in Table 1.

[0071] Table 1

[0072]

[0073] As can be seen from Table 1, the coefficients of variation CV of intra-plate and inter-plate detection are both <6%, confirming that the detection method has good accuracy.

[0074] 3) Stability analysis: The above-mentioned ELISA kit was subjected to ELISA detection of 5 ng / ml, 2.5 ng / ml, and 1.25 ng / ml of CD39 protein at day 0, day 30, and day 90, respectively, to analyze the stability of the kit, and the results are shown in Table 2.

[0075] Table 2

[0076] ​

[0077] From Table 2, it can be seen that the CV values of all detection groups are <5%, which confirms that the detection method has good stability.

[0078] Example 3

[0079] Expression and significance of soluble CD39 in serum of breast cancer patients

[0080] Peripheral blood serum of normal healthy people (ND), benign breast disease patients (BT) and malignant breast cancer patients (MT) was collected, including 30 cases of healthy control group (ND), 40 cases of BT patients and 101 cases of MT patients. The expression of sCD39 in peripheral blood serum was detected by the ELISA kit described in Example 2. The changes of sCD39 expression levels in serum of patients in each group were compared, and the results are shown in Figure 3

[0081] From Figure 3 it can be seen that the expression of sCD39 in peripheral blood serum of MT patients has no significant statistical difference compared with BT group and ND group (P>0.05).

[0082] Further analysis of differences in breast cancer patient group, in the breast cancer group, the worse the histological grade of invasive ductal carcinoma I-II grade and III grade, the higher the expression level of soluble CD39 (P<0.05, Figure 4 Fig. A). According to whether there is lymph node metastasis, it was found that the content of soluble CD39 in platelet-poor plasma of patients with lymph node metastasis was higher than that of patients without lymph node metastasis (P<0.05, Figure 4 Fig. B). In addition, even if more than 80% of breast cancer patients are early stage I-II patients at the initial diagnosis, it is still found that the expression amount of soluble CD39 in patients with III-IV stage is higher than that in early stage patients (P<0.05, Figure 4 Fig. C). As a tumor cell proliferation index, the data show that the content of soluble CD39 in patients with Ki-67>=20% is significantly higher than that in patients with Ki-67<20% (P<0.001, Figure 4 Fig. D).

[0083] From Figure 4 it can be seen that the higher the histological grade of ductal carcinoma (IDBC grade III vs IDBC grade I-II), the presence of lymph node metastasis (N1+N2+N3 vs N0), the higher the tumor grade (Stage III-IV vs Stage I-II) and the stronger the proliferation ability (Ki67>=20% vs Ki67<20%) of the patients, the higher the content of soluble CD39 (P<0.05 for each group comparison). ​

[0084] Although the above embodiments have been shown and described, it is to be understood that the above embodiments are exemplary, and are not to be construed as limiting the present application, and any changes, modifications, replacements, and variations of the above embodiments made by those skilled in the art are within the scope of the present application.

Claims

1. A soluble CD39 protein content test kit, characterized by: The kit comprises a CD39-19A4 anti-human monoclonal antibody and a CD39-5H11 anti-human monoclonal antibody, wherein the CD39-19A4 anti-human monoclonal antibody is used as a coating antibody, and the CD39-5H11 anti-human monoclonal antibody is used as a detection antibody; The CD39-19A4 anti-human monoclonal antibody comprises a heavy chain and a light chain; The amino acid sequence of the heavy chain variable region mVH of the CD39-19A4 anti-human monoclonal antibody is shown in SEQ ID NO. 1: EVQLQQSGAELARPGASVKMSCKTSGYTFTSYTIHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCAREYYRYGYAVDYWGQGTSVTVSS; The amino acid sequence of the light chain variable region mVL of the CD39-19A4 anti-human monoclonal antibody is shown in SEQ ID NO. 2: DIQMTQSPSSMYASLGERVTITCKASQDIKNYLSWYQQKPWKSPKTLIYYATNLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPYTFGGGTKLEIK; The CD39-5H11 anti-human monoclonal antibody comprises a heavy chain and a light chain; The amino acid sequence of the heavy chain variable region mVH of the CD39-5H11 anti-human monoclonal antibody is shown in SEQ ID NO. 3: EVQLKESGPELVKPGASVKVSCKASGYSFTDYNIYWVKQSHRESLEWIGYFDPYNGGTRYNQKFEGKATLTADKSSSTAFMHLNSLTSEDSAVYYCARGLRFRFVYWGQGTLVTVSS; The amino acid sequence of the light chain variable region mVL of the CD39-5H11 anti-human monoclonal antibody is shown in SEQ ID NO. 4: DIVLTQSPAIMSTSPGEKVTMTCRASSSVSSTYFHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDVATYYCQQFSGYPLTFGAGTKLELK.

2. The soluble CD39 protein content assay kit of claim 1, wherein: The kit is used for detecting the content of soluble CD39 protein in human serum, plasma or tissue fluid.

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