Soluble CD39 protein content detection kit and application thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a soluble CD39 protein content detection kit and its application. Background Technology

[0002] CD39, also known as ectonucleoside triphosphate diphosphate hydrolase-1, is encoded by the ENTPD1 gene. CD39 is a transmembrane protein with two transmembrane domains and one extracellular domain, existing in both membrane-bound and soluble forms. Functionally, CD39 binds to exogenous ATP (eATP) and hydrolyzes it to AMP, accompanied by the generation of inorganic phosphate (Pi). Another extracellular nuclease, CD73, hydrolyzes AMP to 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 the bladder, brain, breast, colon, uterus, stomach, and prostate, and is mainly expressed in endothelial cells and immune cells. Significant differences in CD39 expression also exist among different cell types. The expression rate of CD4+ T cells (including memory T cells and regulatory T cells) is approximately 20%–30%, while the expression rate of CD8+ T cells is less than 5%, and the expression rate of natural killer cells (NK cells) is 2%–5%. CD39 is expressed on tumor-infiltrating regulatory T cells (Tregs), myeloid cells, and some NK cells. Compared with normal cells, tumor cells overexpress CD39, and almost all high CD39 expression is considered a marker of poor prognosis and disease progression. Studies have found that CD39+CD8+ T cells in breast cancer specimens are expressed in tumors or metastatic lymph nodes, and their expression differs from that in peripheral blood and non-metastatic lymph nodes. Another study indicated that the CD39 content in breast cancer tissue is higher than in normal tissue, and analysis suggests that CD39 expression is associated with the survival of patients with lumens breast cancer, indicating that CD39 may be an effective therapeutic target for breast cancer. Currently, there are few studies on soluble CD39, and the detection value of soluble CD39 in the peripheral serum of cancer patients deserves further exploration. Summary of the Invention

[0003] One of the objectives of this application is to provide a kit for detecting the content of soluble CD39 protein; The second objective of this application is to provide the application of the soluble CD39 protein content detection kit described in the first aspect of this application in detecting the soluble CD39 protein content in patients with immune diseases; The third objective of this application is to provide the application of the soluble CD39 protein content detection kit described in the first aspect of this application in the clinical detection of breast cancer.

[0004] To achieve the objectives of this invention, the following technical solution is adopted: The first aspect of this application provides a soluble CD39 protein content detection kit, the kit comprising CD39-19A4 anti-human monoclonal antibody and 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.

[0005] According to some embodiments of the soluble CD39 protein content detection kit described in this application, the CD39-19A4 anti-human monoclonal antibody includes 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.

[0006] 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.

[0007] The CD39-5H11 anti-human monoclonal antibody comprises heavy and light chains; 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.

[0008] 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.

[0009] According to some embodiments of the soluble CD39 protein content detection kit described in this application, the kit is used to detect the content of soluble CD39 protein in human serum, plasma or tissue fluid.

[0010] The second aspect of this application provides the application of the soluble CD39 protein content detection kit described in the first aspect of this application in detecting the soluble CD39 protein content in patients with immune diseases.

[0011] The third aspect of this application provides an application of the soluble CD39 protein content detection kit described in the first aspect of this application in the clinical detection of breast cancer.

[0012] The beneficial effects of this invention include: the soluble CD39 protein content detection kit described in this application can effectively detect the soluble CD39 content. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the standard curve for the soluble CD39 protein content detection kit described in this application; Figure 2 This is a schematic diagram illustrating the specificity assessment of the soluble CD39 protein content detection kit described in this application. Figure 3 A schematic diagram comparing the expression levels of soluble CD39 protein in the serum of normal individuals, patients with benign breast diseases, and patients with malignant breast cancer; Figure 4 A schematic diagram illustrating the clinical correlation between the expression level of soluble CD39 protein and various pathological parameters in patients with malignant breast cancer. Detailed Implementation

[0014] The embodiments of the present invention are described in detail below. These embodiments are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0015] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0016] Example 1 Determination of the light and heavy chain variable region sequence of anti-human CD39 monoclonal antibody: The method for determining the variable regions of the heavy and light chains of anti-human CD39 monoclonal antibodies includes the following steps: 1.1 Obtaining cDNA from hybridoma cells mRNA was extracted from hybridoma cell lines secreting anti-human CD39 monoclonal antibody, and cDNA was synthesized by enzymatic reverse transcription. Sequence information of the antibody heavy chain variable region (mVH) and light chain variable region (mVL) was obtained by molecular cloning and PCR sequencing.

[0017] 1.2 The heavy chain variable region (mVH) and light chain variable region (mVL) were ligated to the pJET cloning vector, respectively. Subsequently, the ligation products were transformed into DH5a competent cells, and the transformed bacterial culture was evenly spread on LB solid medium.

[0018] 1.3 Select colonies with clear edges and good growth on LB solid medium for sequencing identification.

[0019] 1.4 Based on the sequencing results, candidate light and heavy chain variable region sequences were retained. The variable region sequences of the light and heavy chains that could be ligated to the expression vector were cloned again by PCR. The variable region sequences were then ligated to the expression vector, and the ligation product was transformed into DH5α. The transformed bacterial culture was evenly spread on LB solid medium and incubated overnight.

[0020] 1.5 Select well-growing bacteria for sequencing and compare the results of two sequencing studies to obtain transformed bacteria with the correct sequence. After expansion culture, plasmid extraction is performed.

[0021] 1.6 The expression vector containing the heavy and light chain variable region genes of the monoclonal antibody was co-transfected into eukaryotic expression cells 293.

[0022] 1.7 293 cells were cultured in suspension using serum-free SFM4Transfx-293 medium without L-Glutamine. During transfection, the medium was replaced with serum-free Gibco medium. ® FreeStyle™ 293 Expression Medium.

[0023] 1.8 The ELISA kit was used to detect the supernatant containing the target antibody, and the results were satisfactory.

[0024] The heavy chain variable region of CD39-19A4 anti-human monoclonal antibody was obtained using the method described above. EVQLQQSGAELARPGASVKMSCKTSGYTFTSYTIHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAVYYCAREYYRYGYAVDYWGQGTSVTVSS; CD39-19A4 anti-human monoclonal antibody light chain variable region: DIQMTQSPSSMYASLGERVTITCKASQDIKNYLSWYQQKPWKSPKTLIYYATNLADGVPSRFSGSGSGQDYSLTISSLESDDTATYYCLQHGESPYTFGGGTKLEIK; CD39-5H11 anti-human monoclonal antibody heavy chain variable region: EVQLKESGPELVKPGASVKVSCKASGYSFTDYNIYWVKQSHRESLEWIGYFDPYNGGTRYNQKFEGKATLTADKSSSTAFMHLNSLTSEDSAVYYCARGLRFRFVYWGQGTLVTVSS; CD39-5H11 anti-human monoclonal antibody light chain variable region: DIVLTQSPAIMSTSPGEKVTMTCRASSSVSSTYFHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDVATYYCQQFSGYPLTFGAGTKLELK.

[0025] This invention extracts the variable regions of the heavy and light chains of an anti-human CD39 monoclonal antibody from hybridoma cells. Based on sequencing results, candidate light and heavy chain variable region sequences are retained, and the light and heavy chain variable region sequences matching the expression vector are amplified by PCR. The PCR product is then ligated into the expression vector pretreated with double enzyme digestion. The expression vector containing the target monoclonal antibody's light and heavy chain variable regions is co-transfected into eukaryotic expression cell line 293. The supernatant collected after culture contains the target antibody, indicating that the obtained heavy and light chain variable region sequences are correct.

[0026] Example 2 An ELISA kit for detecting the content of soluble CD39 protein (sCD39): 2.1 Components of the reagent kit The ELISA kit of the present invention comprises the CD39 coated antibody (CD39-19A4) of Example 1 coated on an enzyme-linked immunosorbent assay (ELISA) plate, the HRP-labeled CD39 detection antibody (CD39-5H11-HRP), soluble CD39 protein standard (Shanghai Baiying Biotechnology Co., Ltd.), sample diluent, washing buffer (PBST), chromogenic solution (TMB) and stop solution.

[0027] 2.2 Sample Collection and Processing 2.2.1 Collect a batch of serum from normal individuals, patients with benign breast diseases, and patients with malignant breast cancer in the hospital. After aliquoting, store the serum at -80°C and avoid repeated freeze-thaw cycles.

[0028] 2.2.2 Before testing serum samples, allow the samples to equilibrate at room temperature for half an hour and shake them to mix. Take an appropriate amount of patient serum sample and dilute it 5 times with diluent.

[0029] 2.3 Method for determining the content of soluble CD39 protein 2.3.1 Dilute the coating antibody CD39-19A4 (1µg / ml) with coating buffer (Na2CO3 and NaHCO3), then add the coating antibody to a 96-well microplate (100 µl / well) and incubate overnight at 4°C.

[0030] 2.3.2 On the second day, wash the plate three times with washing buffer (PBST), then add 100 µl of 3% bovine serum blocking buffer to each well and block at 37°C for 1 h.

[0031] 2.3.3 Remove the blocking solution, add 100µl of diluted plasma sample to be tested and serially diluted soluble CD39 protein standard (starting from 1ng / ml, serially diluted to a total of 8 concentrations, (1ng / ml, 0.5ng / ml, 0.25ng / ml, 0.125ng / ml, 0.0625ng / ml, 0.03125ng / ml, 0.015625ng / ml, 0ng / ml), perform 3 replicates for each gradient, and incubate at 37℃ for 2h.

[0032] 2.3.4 Wash the plate three times with PBST washing buffer, add HRP-labeled detection antibody CD39-5H11 (100µl / well) at a ratio of 1:2000, and incubate at 37°C for 1 hour.

[0033] 2.3.5 Wash the plate six times with PBST washing buffer, add TMB chromogenic solution (100 µl / well), incubate at room temperature in the dark for 10 min, then add stop solution (50 µl / well) to stop the color reaction, and measure the OD450 value of each well using a microplate reader.

[0034] 2.3.6 Plot a standard curve with the CD39 standard concentration multiplied by the sample dilution factor (×5) on the ordinate and the corresponding measured OD450 value on the abscissa (e.g., Figure 1 The calculation formula is obtained, and the content of soluble CD39 in the sample is calculated based on the OD450 value of the sample to be tested.

[0035] The reagent kit of this invention, according to methodological verification, can achieve the following indicators: The equation of the curve is: y = -0.1024x 2 +1.0914x+0.0758 Standard curve linearity: R 2 =0.9993; Limit of detection ≤78.1 pg / ml.

[0036] 2.4 Specificity assessment PD-L1 1g, B7-H3 1g, OX40L 1g, and CD39 1g were serially diluted to different concentrations. Anti-human CD39 monoclonal antibody (19A4) was adjusted to 5µg / ml with carbonate buffer (0.01M CBS, pH 9.3) and coated onto the ELISA plate overnight at 4°C. The plate was washed three times with PBS (containing 0.1% Tween 20) and blocked for 1 h at room temperature with 3% BSA. After three washes with PBS, the diluted commercial protein was added, and the plate was reacted for 2 h at room temperature, followed by three washes with PBS. Then, HRP-labeled monoclonal antibody HRP-5H11 (1:2000, 100µl / well) was added, and the plate was reacted for another 1 h at room temperature. After six washes with PBST, HRP reaction substrate TMB (100µl / well) was added, and the plate was reacted for 15 min at room temperature. The reaction was terminated with 2mol / L H2SO4, and the OD was measured at 450nm using a microplate reader. Each sample was tested in triplicate. Specificity analysis results as follows Figure 2 As shown.

[0037] 2.5 Accuracy and stability assessment: 1) In-slot 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 replicates for sCD39 detection, and the accuracy of the kit was analyzed (Table 1).

[0038] 2) Interplate accuracy analysis: In different batches of 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 replicates for sCD39 detection. The accuracy of the kit was analyzed, and the results are shown in Table 1.

[0039] Table 1

[0040] As can be seen from Table 1, the coefficient of variation (CV) for both intra-plate and inter-plate detection is <6%, confirming that the detection method has good accuracy.

[0041] 3) Stability analysis: The above ELISA kit was used to detect the stability of three CD39 proteins at 5 ng / ml, 2.5 ng / ml and 1.25 ng / ml on day 0, day 30 and day 90 respectively. The results are shown in Table 2.

[0042] Table 2

[0043] As can be seen from Table 2, the CV values ​​of all detection groups are <5%, confirming that the detection method has good stability.

[0044] Example 3 Expression and significance of soluble CD39 in the serum of breast cancer patients Peripheral blood serum was collected from healthy individuals (ND), patients with benign breast disease (BT), and patients with malignant breast cancer (MT), including 30 healthy controls (ND), 40 BT patients, and 101 MT patients. The expression of sCD39 in peripheral blood serum was detected using the ELISA kit described in Example 2. Changes in serum sCD39 expression levels were compared among the groups, and the results are as follows: Figure 3 As shown.

[0045] from Figure 3 It can be seen that there was no significant statistical difference in sCD39 expression in peripheral blood serum of MT patients compared with BT group and ND group (P>0.05).

[0046] Further analysis of intra-group differences in breast cancer patients revealed that, within the breast cancer group, for invasive ductal carcinoma grades I-II compared to grade III, the worse the tumor histological grade, the higher the expression level of soluble CD39 (P<0.05). Figure 4 (A). Breast cancer patients were classified according to the presence or absence of lymph node metastasis. It was found that patients with lymph node metastasis had higher levels of soluble CD39 in platelet-depleted plasma than those without lymph node metastasis (P<0.05). Figure 4 (B) Furthermore, even though over 80% of breast cancer patients are initially diagnosed as stage I-II, stage III-IV patients still showed higher levels of soluble CD39 expression compared to early-stage patients (P<0.05). Figure 4 (C). Ki-67 is an indicator of tumor cell proliferation. Data showed that the content of soluble CD39 in patients with Ki-67 ≥ 20% was significantly higher than that in patients with Ki-67 < 20% (P < 0.001). Figure 4 (D).

[0047] from Figure 4 It can be seen that patients with poorer ductal carcinoma tumor histological grade (IDBC grade III vs IDBC grade I-II), lymph node metastasis (N1+N2+N3 vs N0), higher tumor grade (Stage III-IV vs Stage I-II), and stronger proliferative capacity (Ki67>=20% vs Ki67<20%) had higher soluble CD39 content (P<0.05 for all groups).

[0048] Although the above embodiments have been shown and described, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made to the above embodiments by those skilled in the art are within the protection scope of the present invention.

Claims

1. A soluble CD39 protein content detection kit, characterized in that: The kit includes CD39-19A4 anti-human monoclonal antibody and 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.

2. The soluble CD39 protein content detection kit according to claim 1, characterized in that: The CD39-19A4 anti-human monoclonal antibody comprises heavy and light chains; 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 heavy and light chains; 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.

3. The soluble CD39 protein content detection kit according to claim 1, characterized in that: The kit is used to detect the content of soluble CD39 protein in human serum, plasma or tissue fluid.

4. The use of the soluble CD39 protein content detection kit according to any one of claims 1-3 in detecting the soluble CD39 protein content in patients with immune diseases.

5. The application of the soluble CD39 protein content detection kit according to any one of claims 1-3 in clinical breast cancer detection.

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