CN111537726A - High-efficiency quantitative detection method, ELISA kit and method of PD-L1 level in extracellular vesicles - Google Patents

Abstract

The present invention relates to the field of molecular biology and biotechnology, in particular to a method for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles, an ELISA kit and a method of use, using anti-CD63 antibody, CD9 antibody or CD81 antibody to specifically capture Extracellular vesicles, using anti-PD-L1 antibody to detect PD-L1 protein level in extracellular vesicles, using the corresponding CD63-PD-L1, CD9-PD-L1 or CD81-PD-L1 fusion protein constructed as a standard This product realizes the quantitative detection of PD-L1 in extracellular vesicles. The invention realizes the quantitative detection of the PD-L1 level of the complex structure of extracellular vesicles; meanwhile, the self-constructed CD63-PD-L1, CD9-PD-L1, CD81-PD-L1 fusion proteins are used as standard proteins, The assay results are characterized in terms of specific protein concentrations.

Description

Method and ELISA kit for efficient quantitative detection of PD-L1 levels in extracellular vesicles and how to use it

technical field

The invention relates to the fields of molecular biology and biotechnology, in particular to a method for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles, an ELISA kit and a method for using the same.

Background technique

There are 4.3 million new malignant tumor patients and 2.8 million deaths in my country every year. Cancer has become the main cause of death in my country and one of the most important public health problems in China. What is even more worrying is that the prognosis of patients with malignant tumors has not been significantly improved with the improvement of surgery, radiotherapy and chemotherapy. Immunotherapy, which has emerged rapidly in recent years and won the 2018 Nobel Prize, has brought new hope to patients with malignant tumors. Among them, inhibitors targeting the immune checkpoint programmed apoptosis ligand-1/programmed death receptor-1 (PD-L1/PD-1) are the current representative drugs for tumor immunotherapy, and have been proved to be effective against a variety of malignant tumors. Has obvious curative effect. However, less than 30% of patients benefit from PD-L1/PD-1 immunotherapy. Patients who fail immunotherapy not only suffer from high treatment costs and side effects of immunotherapy, but also may miss the opportunity to receive other treatments. Therefore, there is an urgent need to find a biomarker that can accurately predict the efficacy of immunotherapy.

Extracellular vesicles (Evs) are lipid bilayer particles with a diameter of 50-1000 nm secreted by cells, which carry a variety of characteristic biological information molecules derived from mother cells, such as proteins, nucleic acids, lipids. classes, and even organelles. In recent years, extracellular vesicles in body fluids have gained extensive attention as an important branch of liquid biopsy.

Recent research by this project group has found that tumor cells can secrete PD-L1 ⁺ extracellular vesicles into peripheral blood, and the level of PD-L1 ⁺ extracellular vesicles in peripheral blood of tumor patients and its change trend and magnitude in the early stage of immunotherapy can effectively predict the patient's responsiveness to treatment. Based on the above results, we previously proposed an ELISA detection strategy based on the level of PD-L1 on extracellular vesicles in peripheral blood of tumor patients. In this ELISA strategy, the anti-PD-L1 antibody was used as the capture antibody, the extracellular vesicles were immobilized in the ELISA plate, and the anti-PD-L1 antibody was used to detect the PD-L1 level on the extracellular vesicles, using the PD-L1 standard Protein quantification of extracellular vesicle PD-L1 levels. However, both PD-L1 on extracellular vesicles and a large amount of free PD-L1 protein are present in the body fluids of patients. Obviously, direct ELISA detection of PD-L1 levels on body fluid samples will be interfered by free PD-L1 protein. Therefore, in order to avoid the interference of free PD-L1, this strategy requires the separation and purification of extracellular vesicles in body fluids by ultracentrifugation in advance. However, ultracentrifugation is not only time-consuming and labor-intensive, but also lacks standardized operation procedures and requires large-scale ultracentrifugation equipment, which is not conducive to clinical application; more importantly, the efficiency of ultracentrifugation to separate extracellular vesicles is less than 30%. Whether PD-L1 levels in these 30% EVs can accurately reflect PD-L1 levels in total EVs remains unclear.

Therefore, there is an urgent need to develop a reliable, rapid and economical method that can directly and efficiently quantify the level of PD-L1 on extracellular vesicles in body fluids.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a method for efficiently and quantitatively detecting the level of PD-L1 in extracellular vesicles, which avoids the cumbersome steps of centrifuging and purifying extracellular vesicles and the defects of low sample recovery rate, and also avoids free The interference of PD-L1 protein on the detection results can realize specific identification and quantitative detection of the protein level of PD-L1 in extracellular vesicles.

The second purpose of the present invention is to provide an ELISA kit for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles, which can rapidly and quantitatively detect the level of PD-L1 in extracellular vesicles.

The third purpose of the present invention is to provide a method for using an ELISA kit for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles.

The solution adopted by the present invention to achieve one of the objectives is: a method for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles, using the specific capture of anti-CD63 antibody, anti-CD9 antibody or anti-CD81 antibody to extracellular vesicles, The level of PD-L1 protein in extracellular vesicles was detected by anti-PD-L1 antibody, and the corresponding CD63-PD-L1 fusion protein, CD9-PD-L1 fusion protein or CD81-PD-L1 fusion protein was constructed as a standard. Quantitative detection of PD-L1 in extracellular vesicles.

Preferably, the nucleic acid sequence of the CD63-PD-L1 fusion protein is shown in SEQ ID No: 1, the nucleic acid sequence of the CD9-PD-L1 fusion protein is shown in SEQ ID No: 2, and the CD81-PD-L1 fusion protein is shown in SEQ ID No: 2. The nucleic acid sequence is shown in SEQ ID No:3.

Preferably, the extracellular vesicles are derived from any one of blood, saliva, urine, cerebrospinal fluid, ascites, pleural effusion, sweat, semen, lymph, and cell culture supernatant.

Except for blood for various types of cancers in the whole body, the other body fluids collected are for specific parts of the detected cancer, such as saliva for oral and adjacent cancers, urine for urothelial cancer, and pleural volume for lung cancer. fluid, cerebrospinal fluid for glioma, etc.

The scheme adopted by the present invention to achieve the second objective is: an ELISA kit for efficiently quantitatively detecting the level of PD-L1 in extracellular vesicles, including an ELISA plate, a sealing membrane, a standard protein, a standard protein diluent, a sample Diluent, concentrated washing solution, enzyme-labeled antibody A, enzyme-labeled antibody B, chromogenic reagent A solution, chromogenic reagent B solution and stop solution;

Wherein, the ELISA enzyme label plate is an enzyme label plate coated with anti-CD63 antibody, anti-CD9 antibody or anti-CD81 antibody in advance at a concentration of 1-5 μg/mL;

The standard protein corresponds to the constructed CD63-PD-L1, CD9-PD-L1 or CD81-PD-L1 fusion protein.

The ELISA plate is the Nunc type enzyme plate of Biolegend; the sealing film is the sealing film of Corning, the product number is UC-500, and the standard protein dilution solution includes buffer 100-300mmol/L, stabilizer 5-20g /L, preservatives 2-6g/L.

Preferably, the anti-CD63 antibody is an anti-CD63 antibody from Thermo Fisher, product number TJ26539346; the anti-CD9 antibody is an anti-CD9 antibody from Thermo Fisher, product number TL2686763; the anti-CD81 antibody is an anti-CD81 antibody from Novus company, Item No. 531413.

Preferably, the nucleic acid sequence of the CD63-PD-L1 fusion protein is shown in SEQ ID No: 1, the nucleic acid sequence of the CD9-PD-L1 fusion protein is shown in SEQ ID No: 2, and the CD81-PD-L1 fusion protein is shown in SEQ ID No: 2. The nucleic acid sequence is shown in SEQ ID No:3.

Preferably, the concentrated washing solution is an aqueous solution containing 0.01-0.05mol/L PBS and a mass percentage of 1‰-5‰ Tween-20; the enzyme-labeled antibody A is 1 μg/mL PD-L1 detection antibody, and the The enzyme-labeled antibody B was 1 μg/mL horseradish peroxidase-coupled color-developing antibody.

The enzyme-labeled antibody A provided in this kit is 1 μg/mL PD-L1 detection antibody. After screening various PD-L1 detection antibodies on the market, select the PD-L1 antibody from eBioscience, product number 13-5983-82. The enzyme-labeled antibody B provided in this kit is a 1 μg/mL horseradish peroxidase-coupled chromogenic antibody. After screening a variety of commercially available HRP chromogenic antibodies, the HRP chromogenic antibody of BD Company was selected, product number 554066.

Preferably, the color developer A solution includes 2.72% of sodium acetate, 0.32% of citric acid, and 0.02% of hydrogen peroxide by mass percentage, and is configured in distilled water; the color developer solution B includes, by mass percentage, ethylenediaminetetramine Disodium acetate 0.04%, citric acid 0.19%, glycerol 9%, 3,3',5,5'-tetramethylbenzidine 0.03%, were prepared in distilled water; the stop solution was 2mol/L dilute sulfuric acid.

The solution adopted in the third objective of the present invention is: a method for using the ELISA kit for efficient quantitative detection of PD-L1 levels in extracellular vesicles, comprising the following steps:

(1) After equilibrating at room temperature, take out the ELISA plate, set standard wells and sample wells on the plate, add standard proteins of different concentration gradients to the standard wells, and add the body fluid samples to be tested to the sample wells, 37 Incubate at ℃ for 1 hour;

(2) Discard the liquid in the well, inject 300 μL of washing solution, wash, repeat several times, discard the washing solution for the last time, put it on absorbent paper and pat it dry;

(3) Add 100 μL of pre-mixed enzyme-labeled antibody A solution and enzyme-labeled antibody B solution to the wells of the ELISA plate, seal the wells of the enzyme-labeled plate with a sealing film, and incubate at 37°C for 1 hour;

(4) Discard the liquid in the well, inject 300 μL of washing solution, wash, repeat several times, discard the washing solution for the last time, put it on absorbent paper and pat it dry;

(5) Mix the color developer A solution and the color developer solution B according to the volume ratio of 1:1, add 100 μL to each well, and develop the color at room temperature in the dark;

(6) After observing the color gradient change in the standard well for 10-30 minutes, add 50 μL of stop solution to stop the color development, shake gently until the well turns yellow, and measure the OD value of each well at the wavelengths of 450nm and 570nm within 15 minutes, respectively. Calculate the OD value at 450nm minus the OD value at 570nm to obtain the final OD value of the sample, and calculate the concentration of the sample to be tested.

Preferably, in the step (1), the standard concentration gradient is 16ng/mL, 12ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, 0ng/mL eight Concentration gradient, add 100 μL to each standard well.

The body fluid samples mentioned in step 1 of the present invention need to undergo preliminary centrifugation. There are mainly three centrifugation conditions for different body fluids: 1. For saliva and other viscous body fluids, the centrifugal force is 2600g, the centrifugation temperature is 4°C, and the centrifugation time is 15 minutes. Collect the supernatant and centrifuge twice in total; 2. For blood, the centrifugal force is 1550g, the centrifugation temperature is 25°C, and the centrifugation time is 20 minutes, and the clear plasma above is collected, and centrifuged twice in total; 3. For relatively clear body fluids such as urine, the centrifugal force is 1550g, The centrifugation temperature was 4°C, and the centrifugation time was 15 minutes. The supernatant was collected and centrifuged twice in total.

The incubation temperature set in steps 1 and 3 of the present invention is 37° C., and the incubation time is 1 hour.

The washing method mentioned in step 2 and step 4 of the present invention is as follows: suction the reaction solution in the well, inject 300 μL of the washing solution, shake gently for 2 minutes, and suck the washing solution in the well for the last time, and put it on the absorbent paper and pat Dry.

In the present invention, 8 or 12-channel pipettes are used to add liquid in steps 2 to 6 to reduce operation errors and sample loading errors.

The OD value mentioned in step 6 of the present invention is the absorbance value, which needs to be corrected by subtracting the OD value at the wavelength of 570 nm from the OD value at the wavelength of 450 nm, and finally calculate the sample concentration according to the linear relationship of the standard product.

The present invention has the following advantages and beneficial effects:

Studies have shown that almost all extracellular vesicles express the tetraspanin family molecules CD63, CD9, and CD81. Therefore, CD63, CD9, and CD81 molecules are used as markers for the identification of extracellular vesicles. Based on this, the present invention proposes a new method for directly detecting the level of extracellular vesicles-PD-L1 in human fluid or cell culture supernatant. Antibody or anti-CD81 antibody is used as capture antibody for extracellular vesicles, anti-PD-L1 antibody is used as detection antibody, and self-constructed CD63-PD-L1, CD9-PD-L1 or CD81-PD-L1 fusion protein is used as standard , so as to achieve quantitative and rapid detection of PD-L1 content in extracellular vesicles in body fluids or cell culture supernatants. Compared with the existing ELISA detection strategy that uses anti-PD-L1 antibody as the capture antibody, this method avoids the cumbersome step of centrifugation of body fluids and the defects of low sample recovery rate, and also avoids the detection of free PD-L1 protein. The interference of the results achieved the specific identification and quantitative detection of the protein level of extracellular vesicle-PD-L1. In addition, since not every extracellular vesicle expresses CD63, CD9 and CD81 at the same time, by combining and matching different marker molecules, the capture efficiency of total extracellular vesicles can be greatly improved, and the high efficiency of total extracellular vesicles can be achieved capture. The method will have broad application prospects in the early diagnosis, prognosis evaluation and immunotherapy efficacy prediction of malignant tumors.

The existing quantitative detection of PD-L1 ⁺ extracellular vesicles requires ultracentrifugation and purification of body fluids or cell culture supernatants to obtain extracellular vesicles, and then quantitative detection by ELISA. The invention breaks through the traditional mode of "AAA sandwich" ELISA for detecting free single protein molecules, uses the "A-AB-B" strategy to upgrade the original detection method, and realizes the quantification of the complex structure of extracellular vesicles Detection; at the same time, the self-constructed CD63-PD-L1, CD9-PD-L1, CD81-PD-L1 fusion proteins are used as standard proteins, so that the detection result is no longer a percentage, but is characterized by a specific protein concentration .

Description of drawings

Fig. 1 is the particle size distribution (a-c) of extracellular vesicles in normal cell line (HUVEC) and cancer cell line (Cal27, H1975) in Example 4 of the present invention;

Figure 2 is the standard curve of CD63-PD-L1 recombinant protein in Example 4 of the present invention;

Figure 3 shows the supernatant (Supernatant), extracellular vesicles (EVs), and supernatant (EVs-Free supernatant) of normal cell line (HUVEC) and cancer cell line (Cal27, H1975) in Example 4 of the present invention ) ELISA test results;

Figure 4 is the change trend of PD-L1 protein level and tumor volume in extracellular vesicles of WM164 xenografted nude mice in Example 5 of the present invention;

Figure 5 is the plasma (Plasma), extracellular vesicles (EVs), and ultracentrifugation of normal subjects (HD), non-small cell lung cancer patients (NSCLC), and malignant melanoma patients (MP) in Example 6 of the present invention. Supernatant (EVs-Free plasma) ELISA test results;

6 is the ELISA detection result after the saliva of oral cancer patients is captured by anti-CD63 antibody, anti-CD9 antibody, anti-CD81 antibody, and anti-CD63/9/81 mixed antibody in Example 7 of the present invention.

Detailed ways

For better understanding of the present invention, the following examples are further descriptions of the present invention, but the content of the present invention is not limited to the following examples.

Example 1: Construction of CD63-PD-L1 fusion protein

1. According to the Pubmed Gene query, the human CD63 gene sequence (Gene ID: 967) was selected, and the Ala103 to Val203 translation fragment was selected; the PD-L1 gene sequence (Gene ID: 29126), the Phe19 to Thr239 translation fragment was selected; A segment of connexin sequence (GGTGGTGGTGGTAGCGGTGGTGGCGGT AGTGGTGGTGGTGGTAGC) was inserted between, and the His tag fragment (CATCACCATC ATCATCAT) was connected to the tail end of PD-L1, and EcoR1 and Xhol double restriction sites were introduced, and a pair of forward and reverse primers (Table 1) were designed to carry out PCR amplification. The CD63-PD-L1 gene fragment is obtained by increasing, and its nucleic acid sequence is shown in SEQ ID No: 1, and its amino acid sequence is shown in SEQ ID No: 4.

Table 1. CD63-PD-L1 PCR primers

2. Connect the CD63-PD-L1 gene fragment with the Pet28a plasmid that has been double digested by Ecorl and Xhol by Exnase recombinase (Novizon, product number C113-01), and obtain the recombinant plasmid CD63-PD after transformation and screening -L1.

3. The recombinant plasmid CD63-PD-L1 was transformed into E.coli DH5α competent bacteria, and the plasmid was extracted after extensive amplification in Kanamycin-resistant agarose medium (K ⁺ LB).

4. Transfer the extracted recombinant plasmid CD63-PD-L1 into Rosetta (DE3) competent bacteria, and evenly spread it on K ⁺ LB solid medium to obtain the target cloned strain.

5. Pick a single colony to 50mL K ⁺ LB and culture to OD ₆₀₀ =0.6-0.8, add IPTG to a final concentration of 0.5-1mmol/L to stimulate protein expression, culture continuously for 4-8 hours, centrifuge at 12000r/min for 15 minutes to collect separately The supernatant and pellet were diluted or resuspended in PBS, and then 20 μL of 6× protein loading buffer was added to each 100 μL, and SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were carried out for analysis and identification.

6. Add the collected bacterial pellet to 8 mL bacterial lysate (TRIS-HCl 50mmol/L, NaCl 300mmol/L, TritonX-100 0.5%, pH=8.0), mix on ice for 30 minutes, and centrifuge at 4°C at 12000g For 10 minutes, resuspend the pellet with 15 times the volume of inclusion body lysis buffer (TRIS-HCl 50mmol/L, NaCl 100mmol/L, EDTA 1mmol/L, TritonX-100 0.5%, urea 8mol/L, pH=8.0).

7. The above re-suspension is added after adding 10mmol/L imidazole, and the chromatographic column is added, and the flow rate is controlled 0.5-1mL/min, and the washing buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole) is 1mL/min with the flow rate. 10mmol/L; pH=8.0) to wash the column to remove impurity proteins, repeat the washing 6 times and then use elution buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole 250mmol/L; pH=8.0) to wash 4 times Remove and collect the eluate.

8. Dialyze the collected eluate into PBS using a semi-permeable membrane, dialyze overnight at 4°C, collect the solution in the membrane, and obtain the CD63-PD-L1 fusion protein

Example 2: Construction of CD9-PD-L1 fusion protein

1. According to the Pubmed Gene query, the human CD9 gene sequence (Gene ID: 928) was selected, and the Ser112 to Ile195 translation fragment was selected; the PD-L1 gene sequence (Gene ID: 29126), the Phe19 to Thr239 translation fragment was selected. A segment of connexin sequence (GGTGGTGGTGGTAGCGGTGGTGGCGGT AGTGGTGGTGGTGGTAGC) was inserted between, and a His tag fragment (CATCACCATC ATCATCAT) was connected to the tail end of PD-L1, and EcoR1 and Xhol double restriction sites were introduced, and a pair of forward and reverse primers (Table 2) were designed to carry out PCR amplification. The CD9-PD-L1 gene fragment was obtained by increasing, and its nucleic acid sequence was shown in SEQ ID No: 2, and its amino acid sequence was shown in SEQ ID No: 5.

Table 2 CD9-PD-L1 PCR primers

2. Connect the CD9-PD-L1 gene fragment with the Pet28a plasmid that has been double digested by Ecorl and Xhol by Exnase recombinase, and obtain the recombinant plasmid CD9-PD-L1 after transformation and screening.

3. The recombinant plasmid CD63-PD-L1 was transferred into E.coli DH5α competent bacteria, and the plasmid was extracted after a large amount of amplification in K ⁺ LB medium.

4. Transfer the extracted recombinant plasmid CD9-PD-L1 into Rosetta (DE3) competent bacteria, and evenly spread it on K ⁺ LB solid medium to obtain the target cloned strain.

5. Pick a single colony to 50mL K ⁺ LB and culture to OD ₆₀₀ =0.6-0.8, add IPTG to a final concentration of 0.5-1mmol/L to stimulate protein expression, culture continuously for 4-8 hours, centrifuge at 12000r/min for 15 minutes to collect separately The supernatant and pellet were diluted or resuspended in PBS, and then 20 μL of 6× protein loading buffer was added to each 100 μL, and SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were carried out for analysis and identification.

6. Add the collected bacterial precipitate to 8 mL bacterial lysate (TRIS-HCl 50mmol/L, NaCl 300mmol/L, TritonX-100 0.5%, pH=8.0), mix on ice for 30 minutes, under the condition of 12,000g at 4°C Centrifuge for 10 minutes, and resuspend the pellet with 15 times the volume of inclusion body lysis buffer (TRIS-HCl 50mmol/L, NaCl 100mmol/L, EDTA 1mmol/L, TritonX-100 0.5%, urea 8mol/L, pH=8.0).

7. The above re-suspension is added after adding 10mmol/L imidazole, and the chromatographic column is added, and the flow rate is controlled 0.5-1mL/min, and the washing buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole) is 1mL/min with the flow rate. 10mmol/L; pH=8.0) to wash the column to remove impurity proteins, repeat the washing 6 times and then use elution buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole 250mmol/L; pH=8.0) to wash 4 times Remove and collect the eluate.

8. The collected eluate was dialyzed into PBS using a semi-permeable membrane, dialyzed overnight at 4°C, and the solution in the membrane was collected to obtain the CD9-PD-L1 fusion protein.

Example 3: Construction of CD81-PD-L1 fusion protein

1. According to Pubmed Gene, the human CD81 gene sequence (Gene ID: 975) was selected, and the translation fragment from Phe113 to Lys201 was selected; the PD-L1 gene sequence (Gene ID: 29126), the translation fragment from Phe19 to Thr239 was selected; A segment of connexin sequence (GGTGGTGGTGGTAGCGGTGGTGGCGGT AGTGGTGGTGGTGGTAGC) was inserted between, and a His tag fragment (CATCACCATC ATCATCAT) was connected to the tail end of PD-L1, and EcoR1 and Xhol double restriction sites were introduced, and a pair of forward and reverse primers (Table 3) were designed to carry out PCR amplification. The CD81-PD-L1 gene fragment was obtained by increasing, and its nucleic acid sequence was shown in SEQ ID No: 3, and its amino acid sequence was shown in SEQ ID No: 6.

Table 3. CD81-PD-L1 PCR primers

2. Connect the CD81-PD-L1 gene fragment with the Pet28a plasmid that has been double digested by Ecorl and Xhol by Exnase recombinase, and obtain the recombinant plasmid CD81-PD-L1 after transformation and screening.

3. The recombinant plasmid CD81-PD-L1 was transferred into E.coli DH5α competent bacteria, and the plasmid was extracted after a large amount of amplification in K ⁺ LB medium.

4. Transfer the extracted recombinant plasmid CD81-PD-L1 into Rosetta (DE3) competent bacteria, and evenly spread it on K ⁺ LB solid medium to obtain the target cloned strain.

5. Pick a single colony to 50mL K ⁺ LB and culture to OD ₆₀₀ =0.6-0.8, add IPTG to a final concentration of 0.5-1mmol/L to stimulate protein expression, culture continuously for 4-8 hours, centrifuge at 12000r/min for 15 minutes to collect separately The supernatant and pellet were diluted or resuspended in PBS, and then 20 μL of 6× protein loading buffer was added to each 100 μL, and SDS-polyacrylamide gel electrophoresis and Coomassie brilliant blue staining were carried out for analysis and identification.

6. Add the collected bacterial pellet to 8 mL bacterial lysate (TRIS-HCl 50mmol/L, NaCl 300mmol/L, TritonX-100 0.5%, pH=8.0), mix on ice for 30 minutes, and centrifuge at 4°C at 12000g For 10 minutes, resuspend the pellet with 15 times the volume of inclusion body lysis buffer (TRIS-HCl 50mmol/L, NaCl 100mmol/L, EDTA 1mmol/L, TritonX-100 0.5%, urea 8mol/L, pH=8.0).

7. The above re-suspension is added after adding 10mmol/L imidazole, and the chromatographic column is added, and the flow rate is controlled 0.5-1mL/min, and the washing buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole) is 1mL/min with the flow rate. 10mmol/L; pH=8.0) to wash the column to remove impurity proteins, repeat the washing 6 times and then use elution buffer (TRIS-HCl 50mmol/L; NaCl 300mmol/L; imidazole 250mmol/L; pH=8.0) to wash 4 times Remove and collect the eluate.

8. The collected eluate was dialyzed into PBS using a semi-permeable membrane, dialyzed at 4°C overnight, and the solution in the membrane was collected to obtain the CD81-PD-L1 fusion protein.

Example 4: ELISA detection of PD-L1 levels in extracellular vesicles derived from oral cancer and lung cancer cell lines

1. Human normal cell line HUVEC, human oral cancer cell line Cal27 and human lung cancer H1975 cell line cultured with exosome-free fetal bovine serum (100,000g, centrifuged overnight at 4°C) were used to collect low-concentration fetal bovine serum (5% ) starved cultured cell supernatant.

2. Centrifuge 10 mL of the collected cell supernatant at 4°C and 2000g for 30 minutes, retain the supernatant to remove cells and cell debris, take 1mL of the supernatant for testing, and centrifuge the remaining 9mL at 4°C and 100,000g for 70 minutes. After 10 minutes, the centrifugation supernatant was collected and the pellet was resuspended in 20 μL of PBS to obtain extracellular vesicles.

3. The resuspended extracellular vesicles were detected by Nanoparticle tracking analysis (NTA), and it was found that the average diameter of extracellular vesicles derived from HUVEC, Cal27 and H1975 was about 100 nm (Figure 1, a-c).

4. Take 100 μL of the supernatant collected from the above HUVEC, Cal27 and H1975 cell lines, extracellular vesicles (diluted 20 times), and the supernatant obtained by ultracentrifugation into the sample well of the ELISA plate that has been plated with the CD63 capture antibody. The CD63-PD-L1 standard protein obtained in Example 1 was set to eight concentrations according to 16ng/mL, 12ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, and 0ng/mL Gradient, add 100 μL to each standard well, seal the well plate with parafilm, and incubate in a 37°C incubator for 1 hour.

5. While operating step 5, mix the enzyme-labeled antibody A and B solutions at a ratio of 1:1, and place them in a 37°C incubator for 1 hour.

6. Discard the liquid in the wells of the ELISA plate, inject 300 μL of PBST washing solution (0.05% Tween 20 in PBS), wash 4 times, invert on absorbent paper and pat dry, add 100 μL of the enzyme-labeled antibody mixture from step 6, Incubate in a 37°C incubator for 1 hour.

7. Discard the liquid in the wells of the ELISA plate, inject 300 μL of PBST washing solution, wash 5 times repeatedly, put it on absorbent paper and pat dry; mix the chromogenic solutions A and B at 1:1, add 100 μL to each well, avoid light color.

8. After the color of the standard well changes linearly (10-30 minutes), add 50 μL of stop solution to each well, shake gently until the liquid in the well is completely terminated, and measure the OD value at the wavelengths of 450nm and 570nm with a microplate reader within 15 minutes. (The OD value at 450nm minus the OD value at 570nm is the final OD value of the sample).

9. Draw a standard curve according to the results of the standard product (Figure 2), and calculate the PD-L1 concentration of each cell line sample (Figure 3). The results show that: the three samples of the normal cell line HUVEC can hardly detect PD-L1, and tumor cells can hardly detect PD-L1. The results of extracellular vesicles obtained by supernatant and centrifugation of Cal27 and H1975 cells were similar, while the supernatant obtained by ultracentrifugation almost did not contain PD-L1, suggesting that this ELISA kit can directly quantitatively detect PD on EVs in culture supernatant -L1.

Example 5: Application of an ELISA kit for efficient quantitative detection of PD-L1 levels in extracellular vesicles in monitoring tumor progression

1. Take log-phase WM164 cells and inoculate them subcutaneously on the back of 8-week-old nude mice, and inject 5×10 ⁶ cells (100 μL) into each mouse, inoculating 20 mice in total; culture for three weeks, take pictures and measure the tumor volume, and calculate the tumor volume. Calculated as width ² × length/2.

2. Euthanize nude mice, immediately collect 500 μL of ocular arterial blood, and centrifuge at 1550g at 25°C for 10 minutes to collect upper plasma; centrifuge for 10 minutes under the same conditions to collect upper plasma.

3. Take out the ELISA plate pre-laid with CD63 capture antibody, and set the CD63-PD-L1 recombinant protein prepared in Example 1 to 16ng/mL, 12ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng A total of 8 concentration gradients of /mL, 0.5ng/mL, and 0ng/mL were added to the standard wells; 100 μL of nude mouse plasma was added to the sample wells; the wells were sealed with parafilm, and incubated in a 37°C incubator for 1 hour.

4. While operating step 3, mix the enzyme-labeled antibody A and B solutions at a ratio of 1:1, and place them in a 37°C incubator for 1 hour.

5. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes and then absorb the liquid in the well, repeat the washing 4 times, the last time need to absorb the washing solution in the well, and put it on absorbent paper and pat dry; add 100 μL of the enzyme-labeled antibody mixture from step 3 was placed in a 37°C incubator for 1 hour.

6. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes, and then absorb the liquid in the well, and repeat the washing for 5 times. For the last time, the washing solution in the well needs to be aspirated, and then placed on absorbent paper and patted dry; Chromogenic solutions A and B were mixed at a ratio of 1:1, and 100 μL was added to each well, and the color was developed in the dark.

7. After observing the color gradient change in the standard wells within 10-30 minutes, add 50 μL of stop solution to stop the color reaction, shake gently until the end of the well is complete, and measure each color at the 450nm and 570nm wavelengths of the enzyme-linked immunosorbent assay within 15 minutes. The OD value of the hole, calculate the OD value at 450nm minus the OD value at 570nm to obtain the final OD value of the sample, and calculate the concentration of the sample to be tested.

8. Combined with tumor measurement results, the correlation between tumor volume and PD-L1 concentration on blood extracellular vesicles was analyzed (Fig. 4). The results showed that the concentration of PD-L1 on extracellular vesicles in nude mice increased with the increase of tumor volume (P=0.0002), suggesting that PD-L1 on extracellular vesicles in body fluids could predict tumor progression.

Example 6: Application of an ELISA kit for efficient quantitative detection of PD-L1 levels on human fluid extracellular vesicles in tumor diagnosis and treatment

1. Collect 2 mL of blood from five normal subjects (Healthy donor, HD), five non-small cell lung cancer (NSCLC) patients, and five malignant melanoma (MP) patients, centrifuge at 25°C and 1550g for 10 centrifuged for 10 minutes under the same conditions to collect the upper plasma; reserved part of the plasma for testing, the remaining part was centrifuged at 16000g at 4°C for 10 minutes, and the supernatant was collected; centrifuged at 120000g at 4°C for 70 minutes, the supernatant was collected And resuspend the pellet with 20 μL PBS to obtain extracellular vesicles.

2. Take 100 μL of the collected plasma, extracellular vesicles (diluted 25 times), and the supernatant obtained by ultracentrifugation into the sample well of the ELISA plate that has been pre-laid with CD63 capture antibody; PD-L1 recombinant protein was set to 8 concentration gradients of 16ng/mL, 12ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, and 0ng/mL, and standard wells were added; Membrane-sealed the well plate and placed in a 37°C incubator for 1 hour.

3. While operating step 3, mix the enzyme-labeled antibody A and B solutions at a ratio of 1:1, and incubate them in a 37°C incubator for 1 hour.

4. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes, and then absorb the liquid in the well, repeat the washing for 4 times, and absorb the washing solution in the well for the last time, and place it on absorbent paper and pat dry; add 100 μL of the enzyme-labeled antibody mixture from step 3 was placed in a 37°C incubator for 1 hour.

5. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake it gently for 2 minutes, blot dry the liquid in the well, repeat the washing 5 times, the last time it is necessary to aspirate the washing solution in the well, and put it on absorbent paper and pat dry; Chromogenic solutions A and B were mixed at a ratio of 1:1, and 100 μL was added to each well, and the color was developed in the dark.

6. Observe the color gradient change in the standard wells within 10-30 minutes, then add 50 μL of stop solution to stop the color reaction, shake gently until the wells are completely terminated, and measure each at 450nm and 570nm wavelengths of the enzyme-linked immunosorbent assay within 15 minutes. The OD value of the hole, calculate the OD value at 450nm minus the OD value at 570nm to obtain the final OD value of the sample, and calculate the concentration of the sample to be tested.

7. Comparison of PD-L1 concentrations in blood samples from normal subjects, non-small cell lung cancer patients, and malignant melanoma patients (Figure 5). The results showed that the results of plasma and extracellular vesicles obtained by centrifugation in the three groups of samples were similar, but PD-L1 could not be detected in the supernatant obtained after ultracentrifugation, which further suggested that this ELISA kit can exclude free PD-L1 protein. Interference, quantitative detection of PD-L1 on extracellular vesicles in the culture supernatant; the results of the two groups of patients with non-small cell lung cancer and malignant melanoma were significantly higher than those in the HD group, indicating that this kit can be used for the diagnosis of patients with malignant tumors .

Example 7: Application of an ELISA kit for efficient quantitative detection of PD-L1 levels on extracellular vesicles in the detection of oral cancer salivary extracellular vesicles PD-L1 levels

1. Collect 5 mL of saliva from five patients with oral cancer (OSCC), centrifuge at 2600g at 4°C for 15 minutes, collect the supernatant, centrifuge twice in total, and store at 4°C for future use.

2. Take anti-CD63 antibody (Thermo Fisher, Catalog No. TJ26539346), anti-CD9 antibody (ThermoFisher, Catalog No. TL2686763), and anti-CD81 antibody (Novus, Catalog No. 531413) at a concentration of 5 μg/mL and dilute them in carbonic acid coating buffer (8.4 g NaHCO3 and 3.56g Na2CO3 were dissolved in 1L of double distilled water, adjusted to pH 9.5), and anti-CD63, CD9, and CD81 antibodies were mixed and diluted at a concentration of 5μg/mL as the capture antibody of total extracellular vesicles in body fluids.

3. Add 50 μL of the above-mentioned anti-CD63, CD9, CD81 and CD63/9/81 mixed antibodies to each well of the ELISA plate, 5 wells in each group, 20 wells in total; the well plate is sealed with parafilm and kept at 4°C overnight.

4. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes and then absorb the liquid in the well, repeat the washing for 4 times, the last time need to aspirate the washing solution in the well, and put it on absorbent paper and pat dry.

5. Add 200 μL blocking solution (10% FBS, 2 mL fetal bovine serum plus 18 mL PBS) to each well, and block at room temperature for 1 hour.

6. Take 100 μL of the supernatant collected in step 1 and add it to the above sample wells. Each patient has a well that has not been incubated with the capture antibody as a blank control; the well plate is sealed with parafilm and placed in a 37°C incubator for 1 hour.

7. While operating step 6, mix the enzyme-labeled antibody A and B solutions at a ratio of 1:1 and incubate in a 37°C incubator for 1 hour.

8. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes, and then absorb the liquid in the well; repeat the washing 4 times, the last time you need to absorb the washing solution in the well, and place it on absorbent paper and pat dry; add 100 μL of the enzyme-labeled antibody mixture from step 3 was placed in a 37°C incubator for 1 hour.

9. Aspirate the reaction solution in the well, inject 300 μL of washing solution, shake gently for 2 minutes, and then absorb the liquid in the well; repeat the washing 5 times, the last time you need to absorb the washing solution in the well, and put it on absorbent paper and pat dry; Chromogenic solutions A and B were mixed at a ratio of 1:1, and 100 μL was added to each well, and the color was developed in the dark.

10. Observe the color change of the sample wells within 10-30 minutes, add 50 μL of stop solution to stop the color reaction, shake gently until the end of the well is complete, and measure the OD of each well at the wavelengths of 450nm and 570nm in an enzyme-linked immunosorbent assay within 15 minutes. Calculate the OD value at 450nm minus the OD value at 570nm to obtain the final OD value of the sample.

11. Compare the detection results obtained after the anti-CD63, CD9, CD81, CD63/9/81 mixed antibody capture (Figure 6), with CD63/9/81 as the control, it can be seen that the CD9 capture antibody is more effective than the CD63 and CD81 capture antibodies. The PD-L1 content was slightly higher, even though more extracellular vesicles were captured with the anti-CD9 antibody, and the anti-CD63/9/63 mixed capture antibody captured more extracellular vesicles and More PD-L1 was detected, indicating that the surface protein expression of extracellular vesicles in different body fluids is different, and the use of anti-CD63/9/81 mixed capture antibodies can avoid errors caused by differences in the expression of surface protein molecules in different body fluids.

The above descriptions are only the preferred embodiments of the present invention, of course, it cannot limit the scope of rights of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, Several improvements and changes are made, and these improvements and changes are also regarded as the protection scope of the present invention.

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Claims (10)

1. A method for efficiently and quantitatively detecting the level of PD-L1 in extracellular vesicles is characterized by comprising the following steps: the specificity of an anti-CD 63 antibody, an anti-CD 9 antibody or an anti-CD 81 antibody is used for capturing extracellular vesicles, the level of PD-L1 protein in the extracellular vesicles is detected by using an anti-PD-L1 antibody, and the constructed corresponding CD63-PD-L1 fusion protein, CD9-PD-L1 fusion protein or CD81-PD-L1 fusion protein is used as a standard substance to realize the quantitative detection of PD-L1 in the extracellular vesicles.

2. The method for efficiently and quantitatively detecting the level of PD-L1 in extracellular vesicles according to claim 1, wherein: the nucleic acid sequence of the CD63-PD-L1 fusion protein is shown as SEQ ID No: 1, the nucleic acid sequence of the CD9-PD-L1 fusion protein is shown as SEQ ID No: 2, the nucleic acid sequence of the CD81-PD-L1 fusion protein is shown as SEQ ID No: 3, respectively.

3. The method for efficiently and quantitatively detecting the level of PD-L1 in extracellular vesicles according to claim 1, wherein: the extracellular vesicle is derived from any one of blood, saliva, urine, cerebrospinal fluid, abdominal cavity hydrops, pleural effusion, sweat, semen, lymph fluid and cell culture supernatant.

4. An ELISA kit for efficiently and quantitatively detecting the PD-L1 level in extracellular vesicles is characterized in that: the kit comprises an ELISA plate, a sealing plate membrane, standard protein diluent, sample diluent, concentrated washing liquid, an ELISA antibody A, an ELISA antibody B, a color developing agent A liquid, a color developing agent B liquid and stop solution;

wherein, the ELISA plate is coated with anti-CD 63 antibody, anti-CD 9 antibody or anti-CD 81 antibody in advance according to the concentration of 1-5 mug/mL;

the standard protein corresponds to the constructed CD63-PD-L1, CD9-PD-L1 or CD81-PD-L1 fusion protein.

5. The ELISA kit for high-efficiency quantitative detection of the PD-L1 level in extracellular vesicles according to claim 4, characterized in that: the anti-CD 63 antibody is an anti-CD 63 antibody from Thermo Fisher, cat # TJ 26539346; the anti-CD 9 antibody is an anti-CD 9 antibody from Thermo Fisher, cat # TL 2686763; the anti-CD 81 antibody is an anti-CD 81 antibody from Novus, cat No. 531413.

6. The ELISA kit for high-efficiency quantitative detection of the PD-L1 level in extracellular vesicles according to claim 4, characterized in that: the nucleic acid sequence of the CD63-PD-L1 fusion protein is shown as SEQ ID No: 1, the nucleic acid sequence of the CD9-PD-L1 fusion protein is shown as SEQ ID No: 2, the nucleic acid sequence of the CD81-PD-L1 fusion protein is shown as SEQ ID No: 3, respectively.

7. The ELISA kit for high-efficiency quantitative detection of the PD-L1 level in extracellular vesicles according to claim 4, characterized in that: the concentrated washing solution is an aqueous solution containing 0.01-0.05mol/L PBS and 1-5 per mill of Tween-20 in mass percentage; the enzyme-labeled antibody A is a1 mu g/mL PD-L1 detection antibody, and the enzyme-labeled antibody B is a1 mu g/mL horseradish peroxidase-coupled chromogenic antibody.

8. The ELISA kit for high-efficiency quantitative detection of the PD-L1 level in extracellular vesicles according to claim 4, characterized in that: the color developing agent A liquid comprises 2.72% of sodium acetate, 0.32% of citric acid and 0.02% of hydrogen peroxide by mass percent, and is configured in distilled water; the color developing agent B liquid comprises 0.04% of disodium ethylene diamine tetraacetate, 0.19% of citric acid, 9% of glycerol and 0.03% of 3,3 ', 5, 5' -tetramethyl benzidine in percentage by mass, and is configured in distilled water; the stop solution is 2mol/L dilute sulfuric acid.

9. The use method of the ELISA kit for the high-efficiency quantitative detection of the PD-L1 level in the extracellular vesicles according to any one of claims 4 to 8, is characterized by comprising the following steps:

(1) after balancing at room temperature, taking out the ELISA plate, arranging a standard substance hole and a sample hole on the ELISA plate, adding standard proteins with different concentration gradients into the standard substance hole, adding a body fluid sample to be detected into the sample hole, and culturing for 1 hour at 37 ℃;

(2) discarding liquid in the hole, injecting washing liquid 300 mu L, washing, repeating for many times, discarding the washing liquid for the last time, and then inversely placing on absorbent paper for drying;

(3) adding 100 mu L of enzyme-labeled antibody A liquid and enzyme-labeled antibody B liquid which are mixed in advance into an ELISA plate hole, sealing the ELISA plate hole by using a sealing plate film, and incubating for 1 hour at 37 ℃;

(4) discarding liquid in the hole, injecting washing liquid 300 mu L, washing, repeating for many times, discarding the washing liquid for the last time, and then inversely placing on absorbent paper for drying;

(5) mixing the color developing agent A solution and the color developing agent B solution according to the volume ratio of 1:1, adding 100 mu L of the mixture into each hole, and developing at room temperature in a dark place;

(6) and observing the standard sample hole, after the color gradient changes within 10-30 minutes, adding 50 mu L of stop solution to stop color development, shaking until the color in the hole is yellow, measuring the OD value of each hole at the wavelength of 450nm and 570nm within 15 minutes, calculating the final OD value of the sample by subtracting the OD value at the wavelength of 570nm from the OD value at the wavelength of 450nm, and calculating the concentration of the sample to be measured.

10. The use method of the ELISA kit for high-efficiency quantitative detection of the PD-L1 level in the extracellular vesicles according to claim 9, characterized in that: in the step (1), the concentration gradient of the standard substance is eight concentration gradients of 16ng/mL, 12ng/mL, 8ng/mL, 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL and 0ng/mL, and 100 mu.L of the standard substance is added into each standard hole.

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