WO2021164109A1

WO2021164109A1 - Enzyme-linked immunoassay kit for detecting content of soluble st2, and use thereof

Info

Publication number: WO2021164109A1
Application number: PCT/CN2020/083144
Authority: WO
Inventors: 张学光
Original assignee: 苏州旭光科星抗体生物科技有限公司
Priority date: 2020-02-21
Filing date: 2020-04-03
Publication date: 2021-08-26
Also published as: CN111308086A; CN111308086B

Abstract

Disclosed are an enzyme-linked immunoassay kit for detecting the content of soluble ST2, and the use thereof. The kit comprises two anti-human ST2 monoclonal antibodies, wherein the first anti-human ST2 monoclonal antibody is an ST2-4F12 antibody and comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of ST2-4F12 is identical to SEQ ID NO. 1 and the amino acid sequence of the light chain variable region (mVL) of ST2-4F12 is identical to SEQ ID NO. 2; the second anti-human ST2 monoclonal antibody is an ST2-2D1 antibody and comprises a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of ST2-2D1 is identical to SEQ ID NO. 3 and the amino acid sequence of the light chain variable region (mVL) of ST2-2D1 is identical to SEQ ID NO. 4. The kit can effectively detect the content of soluble ST2 and has the effect of guaranteeing subsequent detection quality.

Description

Enzyme-linked immunoassay kit for detecting soluble ST2 content and use

Technical field

The invention belongs to the field of biomedical technology, and specifically relates to an enzyme-linked immunoassay kit for detecting the content of soluble ST2 and its use.

Background technique

Heart failure (Heart Failure, HF) is abbreviated as heart failure. It mainly refers to the failure of the systolic and diastolic function of the heart when the myocardium is damaged due to various reasons. A clinical syndrome of blood stasis and insufficient blood filling in the arterial system, which in turn causes cardiac circulatory disorders. In recent years, with the discovery of IL-33/ST2 signal transduction pathway, growth-stimulating expression gene 2 protein ST2 has attracted people's attention as a new indicator of heart failure detection.

Growth stimulation expression gene 2 protein ST2 (IL1RL1 or IL-1R) is a member of the interleukin 1 receptor family. At present, ST2 related to heart disease is soluble (sST2) and cross-model (ST2L). Studies have shown that when the concentration of sST2 is low, ST2L can bind to the functional ligand IL-33 of ST2 to have a protective effect on the heart. When the concentration of sST2 is high, sST2 can competitively bind to IL-33, resulting in ST2L and IL-33. The combination is reduced, thereby blocking the ST2L/IL-33 signal transduction pathway, thereby inhibiting IL-33's anti-cardiomyocyte hypertrophy, anti-atherosclerosis and anti-tissue fibrosis, reducing the function of the heart and increasing the risk of heart disease, such as Myocardial hypertrophy, decreased cardiac ejection fraction and atrial and ventricular dilatation are similar to myocardial remodeling after acute myocardial infarction or severe heart failure.

In addition, it is reported in the literature that ST2 can also play an important role in the occurrence and development of acute coronary syndrome as an inflammatory marker reflecting the degree of coronary artery vulnerability.

Summary of the invention

In order to overcome the above-mentioned defects of the prior art, one of the objectives of the present invention is to provide an enzyme-linked immunoassay kit for detecting the content of soluble ST2.

The second objective of the present invention is the detection method of the kit, and the detection method is mainly to detect the soluble ST2 content.

The third object of the present invention is the use of the kit.

In order to achieve one of the objectives of the present invention, the technical solution adopted is:

An enzyme-linked immunoassay kit for detecting the content of soluble ST2, wherein the kit includes two anti-human ST2 monoclonal antibodies:

The first anti-human ST2 monoclonal antibody is the ST2-4F12 antibody, which includes a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region (mVH) of the ST2-4F12 is the same as SEQ ID NO. 1, specifically as follows :

The amino acid sequence of the light chain variable region (mVL) of ST2-4F12 is the same as SEQ ID NO. 2, and the details are as follows:

The second anti-human ST2 monoclonal antibody is an ST2-2D1 antibody, which includes a heavy chain and a light chain, and the amino acid sequence of the heavy chain variable region (mVH) of the ST2-2D1 is the same as SEQ ID NO. 3, specifically as follows :

The amino acid sequence of the light chain variable region (mVL) of ST2-2D1 is the same as SEQ ID NO. 4, specifically as follows:

In order to achieve the second objective of the present invention, the technical solution adopted is:

A use of an enzyme-linked immunoassay kit for detecting the content of soluble ST2, wherein the use is for detecting the content of soluble ST2.

The beneficial effects of the present invention are:

The kit of the present invention can effectively detect the content of soluble ST2 and play a role in subsequent quality.

Description of the drawings

Figure 1 is a schematic diagram showing the content of soluble ST2 in a sample detected by the kit of the present invention.

Figure 2 is a schematic diagram of the correlation of the test results of the commercially available kits.

Detailed ways

Example 1:

Preparation of anti-human ST2 monoclonal antibody hybridoma cells:

1.1 Immunization of mice

The fusion recombinant protein was used to immunize BALB/c mice four times with an interval of 21 days. After 5-7 days after the fourth immunization, the mouse's orbital blood was taken and the titer was determined. Strengthen immunity.

1.2 Cultivation of trophoblast cells

1.2.1 On the day before the start of the fusion experiment, take BALB/c mice over 6 weeks old, put them into a beaker filled with 75% alcohol and soak for 2 minutes after being sacrificed by spinal dislocation.

1.2.2 Take out the mouse spleen in a sterile ultra-clean bench and place it in a 200-mesh sterile stainless steel screen, and obtain a single cell suspension by grinding. After washing twice by centrifugation and resuspension in 1640 basal medium, resuspend the cells with an appropriate amount of 1640 medium containing 15% FBS. Use a sterile Pasteur glass pipette to evenly drop the cell liquid into a 96-well culture plate. The volume is about 80ul, and finally the culture plate is placed in an incubator for overnight culture, and the culture conditions are 37 degrees Celsius and 5% CO2 concentration.

1.3 Cell fusion and screening

1.3.1 The immunized mice were sacrificed by cervical dislocation and placed in a beaker filled with 75% alcohol for 2 minutes.

1.3.2 Take out the mouse spleen in a sterile operating table, grind it in a 200-mesh sterile sieve to obtain a single cell suspension, and then use 1640 basal medium preheated at 37°C under the conditions of 1400rpm/5min Centrifugal resuspension and wash 2 times.

1.3.3 Collect sp2/0 cells that grow well and are in the logarithmic phase of growth, and also use pre-warmed 1640 basal medium under the conditions of 1400rpm/5min by centrifugation and resuspension to wash twice.

1.3.4 Mix the washed spleen cells and sp2/0 cells in a centrifuge tube at a ratio of approximately 5:1, and add the preheated 1640 basal medium to mix thoroughly, and then centrifuge once at 1400rpm/5min. After discarding the supernatant, flick the bottom of the tube with your fingers to mix the two kinds of cells.

1.3.5 Put the centrifuge tube containing the mixed cells in a water bath at 37°C and heat it, and add 1ml of the preheated PEG solution dropwise. During this time, gently shake the centrifuge tube to make the fusion process more effective.

1.3.6 After adding the PEG solution, drop 14ml 1640 basal medium at a constant speed to stop the fusion process. Place the centrifuge tube in a 37°C incubator for 10 min, then centrifuge at 800 rpm/5 min, and discard the supernatant.

1.3.7 Add preheated 1640 full medium and gently resuspend the fused cells, and use a sterile 5ml Pasteur glass pipette to evenly drop them into a 96-well culture plate containing trophoblast cells. Each well is about 80ul/well. After that, it was cultured in a 37°C, 5% CO2 incubator, and the medium was exchanged with HT medium every 3-4 days. The medium was exchanged twice, and then a small amount of supernatant was taken from each well for ELISA detection.

1.3.8 Select the wells with higher detection value for subcloning, and perform ELISA test after each subcloning, usually after 3-4 times of subcloning can cells with stable secreting antibody phenotype can be obtained, and after the last subcloning The cells were cryopreserved.

Example 2:

Determination of the variable region sequence of the light and heavy chain of the anti-human ST2 monoclonal antibody:

The method for determining the variable regions of the heavy and light chains of an anti-human ST2 monoclonal antibody includes the following steps:

2.1 Obtaining cDNA of Hybridoma Cells

RNA is extracted from the obtained hybridoma cells, and the obtained RNA is reverse transcribed into cDNA by RT-PCR technology. The heavy chain variable region (mVH) and light chain variable region (mVL) of the hybridoma cells were cloned by PCR using specially designed upstream and downstream primers.

2.2 The heavy chain variable region (mVH) and light chain variable region (mVL) are respectively connected to the cloning vector (pJET cloning vector), and the connected product is transformed into competent bacteria (DH5a), because the pJET cloning vector contains the ampicillin resistance gene (Amp+), so the transformed bacterial solution can be evenly spread on Amp-resistant LB solid medium, placed in an incubator, and cultivated overnight at 37°C.

2.3 Select the colonies with clear edges and good growth on the culture medium for sequencing and identification.

2.4 According to the sequencing results, the candidate light and heavy chain variable region sequences are retained, and the light chain and heavy chain variable region sequences that can be connected to the expression vector are cloned again by PCR, and the PCR product is linearly expressed with the double enzyme digestion treatment in advance The vector is ligated, and the ligated product is transformed into DH5a. Since the expression vector contains the kanamycin resistance gene (Kana+), the transformed bacterial solution can be evenly spread on the Kana-resistant LB solid medium, and placed Incubate overnight at 37°C in an incubator.

2.5 Select well-growing bacteria for sequencing, and compare the two sequencing results to obtain transformed bacteria with the correct sequence, and carry out plasmid extraction after expanding the culture.

2.6 Co-transfect the eukaryotic expression cell 293 with the expression vector linked to the heavy chain and light chain variable region genes of the cloned antibody. 293 cells are suspended culture in serum-free medium SFM4Transfx-293 without L-Glutamine, during transfection Replace the medium with serum-free medium

FreeStyle ^TM 293 Expression Medium. After one week of continuous cultivation, the supernatant culture solution was collected and centrifuged (5000rpm for 20min), and the obtained supernatant was filtered and sterilized with a 0.22um filter membrane.

2.7 Using ELISA kit to detect the supernatant containing the target antibody, the results showed good results.

Obtain the heavy chain variable region of the anti-human ST2-4F12 monoclonal antibody by the above method:

ST2-4F12 monoclonal antibody light chain variable region

ST2-2D1 monoclonal antibody heavy chain variable region

ST2-2D1 monoclonal antibody light chain variable region

The present invention extracts the variable regions of the heavy and light chains of the antibody from the hybridoma cells expressing the anti-human ST2 monoclonal antibody, retains the candidate light and heavy chain variable region sequences according to the sequencing results, and amplifies and expresses them through PCR The light and heavy chain variable region sequences matched by the vector are ligated with the PCR product and the expression vector pretreated by double enzyme digestion, and then transformed into competent bacteria DH5a. The expression vector linked to the light chain and heavy chain variable region of the target monoclonal antibody was co-transfected into the eukaryotic expression cell line 293, and the supernatant after culture was collected and contained the target antibody, indicating that the obtained heavy chain and light chain variable region sequences were correct.

Example 3:

An ELISA kit for detecting the content of soluble ST2:

3.1 The composition of the kit

The ELISA kit of the present invention includes the ST2 coating antibody (ST2-2D1) prepared in Example 1 coated on an enzyme-labeled plate, a biotin-labeled ST2 detection antibody (ST2-4F12), and a soluble ST2 protein standard product (R&D Systems), Horseradish Peroxidase (HRP), Sample Diluent, Washing Solution (PBST), Chromogenic Solution (TMB) and Stop Solution.

3.2 Sample collection and processing

Use correct medical technology to collect serum from a batch of hospital heart failure patients. If the collected serum samples cannot be detected within 8 hours, they should be stored in a refrigerator at 4°C. If storage or transportation is required for more than 72 hours, the samples should be stored at -20°C. Freeze storage and avoid repeated freezing and thawing. Before the test, place the sample at room temperature for half an hour in advance, shake and mix, and take an appropriate amount of patient serum and dilute it to 50 times with the sample diluent.

3.3 Determination method of soluble ST2 content

3.3.1 The coating solution composed of Na2CO3 and NaHCO3 dilutes the coating antibody ST2-2D1 (1ug/ml), and adds 100ul each to the 96-well microtiter plate at 4°C overnight to make the coating antibody tightly bind to the microtiter plate.

3.3.2 Wash the plate with washing solution (PBST) 3 times after coating, add 100ul of 3% bovine serum blocking solution to each well and block for 1 hour at 37°C. After the blocking is finished, spin dry, and add the diluted serum sample to be tested and the soluble ST2 protein standard diluted in gradients (contrast dilution from 2ng/ml, set 7 concentrations in total, and add the sample diluent in the last blank as a blank control. Do 3 replicates for each gradient), add liquid with a volume of 100ul/well, and incubate with shaking at 75rpm for 1h at room temperature.

3.3.3 Wash the plate with PBST three times and spin dry, then add biotin-labeled detection antibody ST2-4F12, 100ul/well, and incubate for 1h at room temperature with shaking at 75rpm.

3.3.4 Wash the plate three times with PBST and spin dry, add horseradish peroxidase (HRP), 100ul/well, and incubate for 1h with shaking at 75rpm at room temperature.

3.3.5 Wash the plate with PBST six times and spin dry, add color developing solution TMB, 100ul/well, incubate with shaking at room temperature for 10min, then add stop solution (100ul/well) to stop the color reaction, and measure the OD450 value of each well with a microplate reader.

3.3.6 Multiply the ST2 standard product concentration by the sample dilution factor (×50) as the ordinate, and use the corresponding measured OD450 value as the abscissa to make a standard curve (as shown in Figure 1), and obtain the calculation formula, according to the test The OD450 value of the sample calculates the content of soluble ST2 in the sample.

According to methodological identification, the kit of the present invention can reach the following indicators:

Standard curve linearity: R ² >0.9990

Accuracy: 82% addition rate

The lowest detection limit≤2.14pg/ml

Repeatability: Coefficient of variation <5%.

3.3.7 The measured values of 14 serum samples were compared with the commercially available soluble growth-stimulating expression gene 2 protein detection kit A, and the correlation between the detection results of the kit of the present invention and the commercially available kit was compared, and the results are shown in Figure 2.

It can be seen from the data of the present invention and Figure 2 that the standard curve of the present invention has a linear R ² > 0.9990, while the standard curve of the commercially available kit in the prior art is only R ² = 0.9312. It can be seen that the kit of the present invention is different from the existing standard curve. Compared with the commercially available kits, the detection accuracy is higher, and it is more supportive for later data collection and other tasks.

Claims

An enzyme-linked immunoassay kit for detecting the content of soluble ST2, characterized in that the kit includes two anti-human ST2 monoclonal antibodies:

The first anti-human ST2 monoclonal antibody is the ST2-4F12 antibody, which includes a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region (mVH) of the ST2-4F12 is the same as SEQ ID NO. 1, specifically as follows :

EVQLQQSGAELARPGASVKLSCKASGYTFTSYWMQWLKQRPGQGLEWIGAIYPGDGVTTYTQKFKGKATLTADKSSSTAYMQLSSLAFEDSAVYYCARRGLTEDHWGQGTTLTVSS;

The amino acid sequence of the light chain variable region (mVL) of ST2-4F12 is the same as SEQ ID NO. 2, and the details are as follows:

DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTHFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK;

The second anti-human ST2 monoclonal antibody is an ST2-2D1 antibody, which includes a heavy chain and a light chain. The amino acid sequence of the heavy chain variable region (mVH) of the ST2-2D1 is the same as SEQ ID NO. 3, which is specifically as follows :

EVQLKQSGPGLVKPSQSLSLTCTVTGYSITSDYAWHWHWIRQFPGNKLEWMGYITYSGSTSYNPSLKSRISITRDTSNNLFFLQLNSVTTEDTATYYCAYRYDWDFDVWGAGTTVTVSS;

The amino acid sequence of the light chain variable region (mVL) of ST2-2D1 is the same as SEQ ID NO. 4, specifically as follows:

DIVMTQSPSSLAVSVGEKVTMSCKSSQSLFNSRTRKNHLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLTVYYCKQSYILYTFGGGTKLEIK.
The use of an enzyme-linked immunoassay kit for detecting the content of soluble ST2 according to claim 1, wherein the use is for detecting the content of soluble ST2.