Method for detecting adenosine deaminase 2 in sample

Abstract

The invention relates to the field of ADA2 detection, in particular to a method for detecting adenosine deaminase 2 in a sample. The invention provides a fusion protein, the fusion protein comprises an anti-ADA2 single-chain antibody and alkaline phosphatase, the amino acid sequence of a light chain variable fragment of the single-chain antibody is as shown in SEQ ID NO: 1, and the amino acid sequence of a heavy chain variable fragment of the single-chain antibody is as shown in SEQ ID NO: 2. The invention also provides a method for detecting adenosine deaminase 2 in a sample. According to the present invention, the concentration of ADA2 in the biological sample can be quantitatively detected, the specificity and the sensitivity are high, the AUC is more than 99%, and the method is suitable for various types of samples.

Description

Technical Field

[0001] This invention relates to the field of ADA2 detection, and more specifically to a method for detecting adenosine deaminase 2 in a sample. Background Technology

[0002] Tuberculosis (TB) is a global threat to public health, with an estimated 8 million new cases each year. Early diagnosis and effective treatment are crucial for reducing TB morbidity and mortality. Pleural tuberculosis accounts for as much as 25% of all TB cases.

[0003] Tuberculous pleurisy (or pleural tuberculosis) is an inflammation of the pleura caused by a high-intensity allergic reaction resulting from Mycobacterium tuberculosis invading the pleural cavity. If left undiagnosed and untreated, tuberculous pleurisy can lead to severe ventilation dysfunction and lung function impairment. The diagnostic criteria for tuberculous pleurisy published in the "Expert Consensus on Ultrasound Diagnosis, Classification, and Interventional Treatment of Tuberculous Pleurisy (2022 Edition)" include: imaging examination showing pleural effusion, the effusion being exudate, elevated adenosine deaminase, and any one of the following: ① moderately or strongly positive tuberculin purified protein derivative (PPD) skin test; ② positive interferon-gamma release test; ③ positive Mycobacterium tuberculosis antibody. In other words, current technology usually requires a combination of methods to diagnose tuberculous pleurisy, making it difficult to diagnose using conventional methods. Summary of the Invention

[0004] In a first aspect, the present invention provides a fusion protein, characterized in that the fusion protein comprises an anti-ADA2 single-chain antibody and an alkaline phosphatase, wherein the amino acid sequence of the light chain variable fragment of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable fragment of the single-chain antibody is shown in SEQ ID NO:2.

[0005] In some embodiments, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO:3.

[0006] In some embodiments, the alkaline phosphatase is Escherichia coli alkaline phosphatase.

[0007] In some embodiments, the amino acid sequence of the alkaline phosphatase is shown in SEQ ID NO:6.

[0008] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0009] In a second aspect, the present invention provides a kit for detecting adenosine deaminase 2 in a sample, characterized in that the kit comprises the above-mentioned fusion protein, an enzyme-labeled plate coated with a capture antibody, adenosine deaminase 2 standard, blocking solution, and enzyme-labeled plate washing solution.

[0010] In some embodiments, the kit further includes a sample diluent and a coating buffer.

[0011] In some embodiments, the sample includes one or more of pleural effusion, saliva, serum, and cell culture medium.

[0012] Thirdly, the present invention provides a kit for preparing a fusion protein to detect adenosine deaminase 2 in a sample, characterized in that the fusion protein comprises an anti-ADA2 single-chain antibody and alkaline phosphatase, wherein the amino acid sequence of the light chain variable fragment of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable fragment of the single-chain antibody is shown in SEQ ID NO:2.

[0013] In some embodiments, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO:3.

[0014] In some embodiments, the alkaline phosphatase is Escherichia coli alkaline phosphatase.

[0015] In some embodiments, the amino acid sequence of the alkaline phosphatase is shown in SEQ ID NO:6.

[0016] In some embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0017] In some embodiments, the kit is used to detect exudative pleural effusion.

[0018] In some embodiments, the kit is used to distinguish between tuberculous exudative pleural effusion samples and non-tuberculous exudative pleural effusion samples.

[0019] In some embodiments, when the concentration of ADA2 in the sample is ≤300 ng / mL, the sample is assessed as a non-tuberculous exudative pleural effusion sample.

[0020] In some embodiments, the kit is used to detect lymphoma.

[0021] Fourthly, the present invention provides a method for detecting adenosine deaminase 2 in a sample, characterized in that the method comprises:

[0022] S101 coats an ELISA plate with anti-ADA2 rabbit polyclonal antibody to obtain an ELISA plate coated with capture antibody;

[0023] S102 Add adenosine deaminase 2 standard dilution solution and the sample to the wells of the ELISA plate coated with capture antibody, and then incubate.

[0024] S103 Add the above fusion protein to the corresponding wells of the ELISA plate obtained in step S102;

[0025] S104 Add p-nitrophenyl phosphate solution to the corresponding wells of the ELISA plate obtained in step S103.

[0026] S105 uses an ELISA reader to read the absorbance of the corresponding wells of the ELISA plate obtained in step S104 at a wavelength of 405 nm, thereby obtaining the concentration of adenosine deaminase 2 in the sample.

[0027] In some embodiments, the sample includes one or more of pleural effusion, saliva, serum, and cell culture medium.

[0028] In some embodiments, the pleural effusion includes tuberculous exudative pleural effusion and non-tuberculous exudative pleural effusion.

[0029] In some embodiments, the concentration of the fusion protein includes 1-1.5 μg / mL.

[0030] In some embodiments, the concentration of the p-nitrophenyl phosphate solution includes 5-10 mM.

[0031] In some embodiments, the concentration of the anti-ADA2 rabbit polyclonal antibody includes 5-10 μg / mL.

[0032] In some embodiments, after steps S101, S102, S103, and S104, the step of washing the ELISA plate with ELISA plate washing solution is further included.

[0033] In some embodiments, the ELISA plate washing solution comprises PBS containing 0.05% Tween 20.

[0034] In some embodiments, step S101 is followed by a step of sealing the ELISA plate with a blocking solution.

[0035] In some embodiments, the blocking solution comprises PBS containing 2% BSA.

[0036] In some embodiments, the amount of adenosine deaminase 2 standard diluent, the sample, and the p-nitrophenyl phosphate solution added per well comprises 100 μL.

[0037] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0038] Existing techniques typically measure total ADA activity by detecting ammonia (a result of adenosine deamination) or peroxide ions (formed during the degradation of inosine to uric acid by purine nucleotidase and xanthine oxidase). Furthermore, ADA2 activity can be determined by adding EHNA (racemic-9-(2-hydroxy-3-nonyl)adenine hydrochloride, a potent ADA1 inhibitor). Therefore, the difference between the total ADA activity and the remaining ADA2 activity measured in the presence of EHNA is considered ADA1 activity. The inventors of this invention have found that the ADA1 and ADA2 activities, as well as the total ADA activity, measured by existing methods are significantly affected by the pH of the buffer solution and the concentration of adenosine used in the assay. This may be attributed to the approximately 100-fold difference in the Km values ​​of ADA1 and ADA2 relative to adenosine, and the different optimal pH values ​​for their respective ADA activities. Therefore, the accuracy of ADA1, ADA2, and total ADA activities measured by existing techniques lacks a clear standard, making it difficult to compare data obtained from different clinical laboratories. Moreover, sample quality is also crucial for determining ADA activity. Contamination of biological samples with lysed red blood cells or bacteria (the source of ADA1) may lead to an erroneous increase in total ADA and ADA1 activity.

[0039] This invention provides an anti-ADA2 single-chain antibody fused to alkaline phosphatase (hereinafter referred to as a fusion protein), which exhibits high affinity and specificity. When applied to sandwich enzyme-linked immunosorbent assay (ELISA), compared to existing methods for detecting ADA2 (e.g., "ELISA-based assays to measure adenosine deaminases concentration in serum and saliva for the diagnosis of ADA2 deficiency and cancer"), the detection method provided by this invention not only has a low limit of detection (1 ng / mL) but also a wide detection range, capable of detecting ADA2 concentrations above 300 ng / mL (even 600 ng / mL). Compared to methods for detecting total ADA activity, the detection method provided by this invention can quantitatively detect the concentration of ADA2 in biological samples while being less affected by buffer pH, adenosine concentration used in the assay, and the sample itself, and is also less affected by ADA1. It has higher specificity and sensitivity, with an AUC greater than 99%, and is suitable for various sample types. The detection method provided by this invention can be used to differentiate between tuberculous and non-tuberculous exudative pleural effusions, thereby helping to rule out tuberculosis as a cause in clinical practice.

[0040] Furthermore, the detection method provided by this invention can also be used for monitoring diseases related to changes in ADA2 concentration and for evaluating subsequent treatments, such as HIV, ADA2 deficiency, autoimmune diseases, and cancer. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0042] Figure 1 This is a schematic diagram of the anti-ADA2 scFv-AP structure provided by the present invention;

[0043] Figure 2A This is a schematic diagram of a detection method based on anti-ADA2 scFv-AP;

[0044] Figure 2B The standard curve is shown for the detection method based on anti-ADA2 scFv-AP.

[0045] Figure 3 The results are shown in the figure for ADA2 isoenzyme concentration (A) and total ADA activity (B) in different pleural effusions;

[0046] Figure 4 The graph shows the correlation between total ADA and ADA2 isoenzymes.

[0047] Figure 5 ROC curves for total ADA activity (A) and ADA2 concentration (B);

[0048] Figure 6 The standard curve was obtained after culturing anti-ADA2 scFv-AP with alkaline phosphatase substrate pNPP for 5 hours. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] In this document, the terms "upper," "lower," "inner," "outer," "front," "rear," "one end," and "the other end," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the present invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0051] In this document, unless otherwise explicitly specified and limited, the terms "installed," "equipped with," "connected," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, a direct connection, or an indirect connection through an intermediate medium; it can be a connection within two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0052] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0053] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0054] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0055] As used in this specification, the term "about" typically means + / - 5% of the value, more typically + / - 4%, more typically + / - 3%, more typically + / - 2%, even more typically + / - 1%, even more typically + / - 0.5% of the value.

[0056] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0057] Example 1: Preparation of anti-ADA2 single-chain antibody

[0058] 1.1 Coating magnetic beads with ADA2 protein

[0059] Following the manufacturer's instructions, Tosylactivated M-280 Dynabeads (Invitrogen, Norway) were coated with ADA2 protein. Specifically, the coating process involved incubating 60 μg of ADA2 protein and 80 μl (2.4 mg) of magnetic beads overnight at 37°C in 200 μl of 75 mM borate buffer (pH 9.5) containing 75 mM ammonium sulfate. The coated magnetic beads were then blocked for 1 hour at 37°C with PBS (pH 7.4) containing 0.5% (w / v) BSA, followed by a wash with PBS (pH 7.4) containing 0.1% (w / v) BSA. Finally, the magnetic beads were dissolved in wash buffer to achieve a concentration of 20 mg / ml.

[0060] 1.2 Screening for ADA2-specific scFv antibodies via phage display

[0061] Antibodies against ADA2 were enriched from a synthetic human antibody library using phage display technology. For screening, two libraries were mixed: scFvP (1x10⁻¹⁰). 10 ) and scFvM (size 6x10) 9Both libraries use the same single scFv gene as a framework, but differ in their binding site design (Huovinen, Syrjanpaa et al. 2013). The diversity design of scFvP in the libraries is similar to that described by Brockmann et al. (Brockmann, Akter et al. 2011). The design of the scFvM library follows the description of Huovinen et al. (Huovinen, Syrjanpaa et al. 2013). Both libraries use the phage vector pEB32x, which displays scFv on the surface of filamentous phage M13 using a truncated p3 capsid protein. In the first round of panning, 2.4 x 10⁻⁶ scFv genes were used. 12 Library phages (a mixture of scFvP and scFvM libraries) were mixed with 50 μl (1 mg) of ADA2-coated magnetic beads in 2.5 mL of 50 mM Tris-PBS pH 7.0, 150 mM NaCl, 1% (w / v) BSA, and 0.05% (v / v) Tween 20 solution and incubated by rotation at room temperature for 2 h. The magnetic beads were then collected using a magnet, washed twice with 1 mL of 50 mM Tris-PBS pH 7.0, 1% (w / v) BSA, and 0.1% (v / v) Tween 20, and then once with 1 mL of 50 mM Tris-PBS pH 7.0 and 0.1% (v / v) Tween 20. To elute the bound phage, magnetic beads were resuspended in 100 μl of TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) and 100 μg / ml trypsin (Sigma, USA) and incubated at room temperature for 30 minutes. Then, 100 μl of 100 μg / ml soybean trypsin inhibitor (Sigma, USA) was added to the TBS. *E. coli* XL1-Blue cells were infected with the eluted phage, and the phage was amplified with the aid of VCS M13 helper phage (Stratagene, USA). Subsequent two rounds of washing were performed in a similar manner, but with a smaller number of phages used (10-1 phages per ml in the second and third rounds, respectively). 11 and 10 10 (1 phage), with a binding time of 1 hour. After each round of washing, the binding ability of the enriched phage library to ADA2 was tested. Phage particles bound to ADA2 were absorbed onto 96-well plates, and the anti-phage Eu-labeled antibody was detected by time-resolved spectroscopy. Background signal was detected using BSA-coated plates.

[0062] 1.3 96-well plate phage culture screening

[0063] E. coli XL1-Blue cells were infected with a third round of phage. Gradually diluted infected cells were plated on LA plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar) at 37°C, with 25 μg / ml chloramphenicol, 12.5 μg / ml tetracycline, and 0.5% glucose added to obtain isolated colonies. Single colonies were then inoculated into each well of a 96-well V-type plate with SB medium, 10 μg / ml tetracycline, 25 μg / ml chloramphenicol, and 0.05% glucose (95 colonies were selected in this example). Phage particles were produced using VCS M13 helper phage (Stratagene, USA). To release the phage from the cells, the plates were frozen to -70°C and thawed twice. Screening was performed on Maxisorp colony count plates (Nunc, Denmark) coated with ADA2. The plates were coated with TSA containing 1 μg / ml ADA2 (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3), 100 μl / well, and incubated overnight at room temperature. The coated plates were blocked with TSA containing 1% BSA. Eu-N1-labeled antiphage Mab binding reagent (Brockmann, Akter et al. 2011) was used for detection.

[0064] 1.4 Expression and purification of antibody variable domain single-chain fragments (scFv)

[0065] The most promising phage clones were cloned into the pAK600H vector. pAK600H was digested with SfiI and EcoO109I and purified from a 1% agarose gel. *E. coli* XL1-Blue cells were infected with single phage clones and grown in SB medium containing 25 μg / ml chloramphenicol. Phage DNA was extracted from infected *E. coli* XL1-Blue cells using a plasmid mini-kit (TermoFisher). The DNA was then digested with SfiI. Since the pAK600H vector was ampicillin-resistant rather than chloramphenicol-resistant, purification of the digested fragments was not required. The digested phage DNA was then ligated to the gel-purified pAK600H using T4 DNA ligase (Fermentas). *E. coli* XL1-Blue cells were then transformed with the pAK600H ligation mixture using electroporation and cultured overnight in LA plates (supplemented with 100 μg / ml ampicillin and 12.5 μg / ml tetracycline) at 37°C. Inoculate a single colony with 5 mL of SB / Amp-Tet-Glu medium (30 g / L tryptone, 20 g / L yeast extract, 10 g / L MOPS, 100 μg / mL ampicillin, 12.5 μg / mL tetracycline, 0.5% glucose) and incubate overnight at 37°C. The next morning, dilute 100-200 times with fresh SB / Amp-Tet medium (without glucose) until the OD600 reaches 0.6. Then induce scFv production with 200 μg / L IPTG. Incubate the induced culture overnight at 20°C. The next day, centrifuge the bacterial cells, resuspend them in IMAC30 (20 mM phosphate buffer, 150 mM NaCl, 30 mM imidazole, pH 7.4) and sonicate. Purify using a nickel agarose HiTrap column (GE Healthcare). IMAC30 was used as the binding / washing buffer, and IMAC300 (20 mM phosphate buffer, 150 mM NaCl, 300 mM imidazole, pH 7.4) was used for elution. A GE desalting column (Sigma-Aldrich) was used, with the buffer changed to TSA.

[0066] This embodiment focuses on testing the binding ability of phage clones to ADA2, finding that 15 phage clones can selectively bind to ADA2. A portion of the phage DNA corresponding to the anti-ADA2 single-chain antibody was cleaved from the phage plasmid and inserted into the pAK600H expression plasmid. The binding ability of all expressed antibodies to ADA2 was detected using the BLITZ protein interaction system. One anti-ADA2 single-chain antibody exhibited the highest apparent dissociation constant (Kd < 10 nM).

[0067] The amino acid sequence of the light chain variable (VL) fragment of this anti-ADA2 single-chain antibody is: AEIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLA WYQQKPGQAP RLLIYGASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQS SSYPW TFGQGTKVEIKRT (SEQ ID NO:1, where SYLA and QQSSSYPW are antigen recognition sites of the VL fragment).

[0068] The amino acid sequence of the heavy chain variable (VH) fragment of this anti-ADA2 single-chain antibody is: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYSMH WVRQAPGKG LEWVS SIWGVNGETD YADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYC AADWSLDY WGQGTLVTVSSASGAEF (SEQ ID NO:2, where SYSMH, SIWGVNGETD and AADWSLDY are the antigen recognition sites of the VL fragment and the VL fragment, respectively).

[0069] The amino acid sequence of the anti-ADA2 single-chain antibody is AEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQS SSYPWTFGQGTKVEIKRTGGGGSGAGGSGGGGTGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMHWVRQAPGKGLEWVSSIWGVNGETDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADWSLDYWGQGTLVTVSSASGAEF (SEQ ID NO:3, where GGGGSGAGGSGGGGTGGGGS is the linker).

[0070] Example 2: Anti-ADA2 single-chain antibody-alkaline phosphatase fusion protein (anti-ADA2scFv-AP)

[0071] Based on the anti-ADA2 single-chain antibody obtained in Example 1, this example designs an anti-ADA2 single-chain antibody-alkaline phosphatase fusion protein, the structural schematic of which is shown in Figure 1.

[0072] The amino acid sequence of the anti-ADA2 single-chain antibody-alkaline phosphatase fusion protein is as follows: MKYLLPTAAAGLLLLAAQPAMAEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSSYPWTFGQGTKVEIKRTGGGGSGAGGSGGGGTGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMHWVRQAPGKGLEWVSSIWGVNGETDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADWSLDYWGQGTLVTVSSASGAEFRTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQDATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDAASLNSVTEANQQKPLLGLFAD GNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIGETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKSGHHHHHH(SEQ ID NO:4)

[0073] Among them, MKYLLPTAAAGLLLLAAQPAM(SEQ ID NO:5) is the pelB boot sequence, RTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTD SAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQDATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGE WQGKTLREQAQARGYQLVSDAASLNSVTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIGETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKSG (SEQ ID NO: 6) is the sequence of Escherichia coli alkaline phosphatase, and HHHHHH is the 6xHis tag sequence.

[0074] Example 3: A method for ADA2 detection using an anti-ADA2 single-chain antibody-alkaline phosphatase fusion protein.

[0075] 3.1 Cloning, expression, and purification of ADA2

[0076] To ensure stable expression of the human ADA2 gene in cells transfected with lentiviral vectors, specific primers (f) 5'-ATCTCGAGCCACCATGTTGGTGGATGGCCCATCTG-3' were used. The open reading frame of the ADA2 gene was amplified by PCR using (SEQ ID NO:7) and (r)5'-TAGGATCCTCACTTTGTAGCCACATCTGC-3' (SEQ ID NO:8) (hADA2). The PCR product was subcloned into the pCR2.1-TOPO plasmid, digested with XhoI and BamHI, and finally ligated into the self-inactivation (SIN) transfer plasmid (phr-cppt-hb7-SIN) digested with XhoI / BamHI.

[0077] HEK 293T cells were transfected with the lentiviral transfer plasmid pCMV-VSV-G containing the ADA gene or mutant and the packaging plasmid pCMVΔR8.2. Transfection was performed using an optimized calcium phosphate method. The lentiviral vector was then concentrated from the conditioned medium of the transfected HEK-293T cells by ultracentrifugation. The concentrated lentiviral vector was used to infect new 293T cells. Finally, recombinant ADA2 was purified from the conditioned medium of the lentivirally transduced 293T cells. The cell culture medium was diluted three times with water, and 1M Tris HCl pH 6.8 was added to obtain a final concentration of 20mM Tris HCl pH 6.8, 0.02% NaN3. Heparinized HiTrap columns (Cytivia) were equilibrated with buffer A (20mM Tris HCl pH 6.8, 50mM NaCl, 0.02% NaN3) and cultured with diluted cell culture medium. The column was washed with buffer A until a stable UV baseline was reached. A HiTrap protein G column equilibrated in buffer A was ligated to a heparin column to remove traces of IgG. ADA2 was eluted with 20 mM Tris HCl pH 6.8, 0.5 M NaCl, and 0.02% NaN3, and the fraction containing ADA2 activity was collected. The enzyme was concentrated using a 30 kDa Amicon Ultra centrifugal filter. The concentrate was diluted 10 times with 50 mM Tris HCl pH 8.0, 10 μM ZnCl2, and 0.02% NaN3 to obtain a final concentration of 50 mM NaCl. Next, the DEAE HiTrap Sepharose column was washed with 20 mM Tris HCl pH 8.0, 10 μM ZnCl2, and 0.02% NaN3, and a fraction of the purified ADA2 was applied to the column. The flow-through was collected and concentrated using a 30 kDa Amicon Ultra centrifugal filter. The concentrate was applied to a Superdex 200 column (Cytivia) and equilibrated with 1xPBS, 10 μM ZnCl2, and 0.02% NaN3. After gel filtration, fractions containing ADA2 activity were combined and rabbits were immunized with the recombinant protein.

[0078] 3.2 Production and purification of polyclonal antibodies

[0079] Polyclonal antibodies against recombinant ADA2 were prepared by administering 1 mg of recombinant ADA2 to rabbits twice. The antibody was purified from rabbit serum using a 5 mL HiTrap protein G antibody purification column. The column was equilibrated with 1xPBS buffer and serum was loaded onto it. The column was washed with 1xPBS buffer until a stable UV baseline was reached. The antibody was then eluted with 0.1 M glycine buffer at pH 2.7 and immediately neutralized with a 1:10 volume of 1 M Tris HCl buffer. The buffer was then replaced with 1xPBS buffer containing 0.02% NaN3 using a 30 kDa Amicon Ultra Centrifugal filter. To isolate ADA2-specific antibodies, recombinant ADA2 was chemically biotinylated with EZ-Link™ NHS-PEG4-Biotin (ThermoFisher) and purified on a 1xPBS-equilibrated HiTrap desalted column. The biotinylated ADA2 was then bound to a HiTrap Streptavidin column in 1xPBS. The anti-ADA2 antibody was then purified on a column loaded with biotinylated ADA2. The binding buffer was 1xPBS, and the elution buffer was 0.1M glycine buffer containing 200Mm NaCl at pH 2.7. To achieve pH neutrality, 1M Tris HCl buffer was used, and the buffer was replaced with 1xPBS buffer containing 0.02% NaN3 at a ratio of 1:10 v / v, using a 30kD Amicon Ultra Centrifugal filter.

[0080] To establish a sandwich enzyme-linked immunosorbent assay (ELISA), Maxisorp plates (Nunc, Denmark) were first coated with 5 μg / ml anti-hADA2 rabbit polyclonal antibody (in PBS) and incubated overnight at 4°C. The next day, the plates were washed three times with PBS containing 0.05% Tween 20 and blocked for 2 hours at room temperature with 2% BSA (in PBS). After removing the blocking solution, the plates could be stored at 4°C for 3 months. Next, 100 μl of sample and hADA2 dilution buffer (0.5% BSA, PBS) were added to the wells and incubated for 2 hours at room temperature. Then, the plates were washed three times with PBS containing 0.05% Tween 20, and 100 μl of 1 μg / ml H11 scFv-AP was added, followed by incubation at room temperature for 1 hour. Then, the plate was washed four times with PBS containing 0.05% Tween 20, and 100 μl of 5 mM para-nitrophenylphosphate (TerrmoFisher) (50 mM Tris pH 9, 200 mM NaCl, 1 mM MgCl2) was added to each well. The plate was incubated at 37°C for 5 hours. The optical density at 405 nm was then measured using a Synergy H4 microplate reader (BioTek).

[0081] The method is summarized as follows:

[0082] First, anti-ADA2 rabbit polyclonal antibody was coated onto 96-well high-binding plates. Figure 2A (Step S101). Add standard dilutions of recombinant ADA2 and samples with unknown ADA2 concentrations ( Figure 2A (Step S102). After incubation and washing of unbound proteins, anti-ADA2 scFv-AP was added to the plate. Figure 2A (Step S103). The concentration of anti-ADA2 scFv-AP bound to ADA2 was detected using the alkaline phosphatase substrate pNPP. Figure 2A (Step S104). After incubation, the increase in absorbance at 405 nm was measured using a microplate reader. Figure 2A (Step S105). A typical standard curve is as follows: Figure 2B As shown.

[0083] The results showed that the detection limit of the method for ADA2 concentration in clinical samples was 1 ng / mL. Furthermore, anti-ADA2scFv-AP was very stable, still providing reliable and reproducible results after 5 years of storage. Figure 6 ).

[0084] Example 4: Detection of ADA2 in clinical samples

[0085] This embodiment examined pleural effusions in patients with TB (tuberculosis), UPPE (uncomplicated pneumonia-like pleural effusion), CPPE (complication-related pneumonia-like pleural effusion), MPE (malignant pleural effusion), and other miscellaneous effusions. The results are shown in Tables 1 and 2.

[0086] This study included 41 patients with pleural tuberculosis and 48 patients with non-tuberculous effusion. A prerequisite was that at least 20% of the non-tuberculous pleural effusion samples had elevated ADA levels >35 U / L (the diagnostic threshold for pulmonary tuberculosis).

[0087] Diagnostic criteria: Tuberculous effusion can be diagnosed if auramine staining or culture (solid or liquid) of pleural effusion, sputum, or pleural biopsy specimens is positive, or if the latter shows granulomas in the parietal pleura. Tuberculosis is suspected if the patient's first or subsequent thoracentesis shows lymphocytic exudate, high ADA levels in the pleural effusion (>35 U / L), negative cytology, and effusion clearance after anti-tuberculosis treatment. If malignant tumor cells are detected in the pleural effusion cytology or biopsy sample, the effusion is classified as malignant effusion. Pneumonoid pleural effusion refers to any effusion associated with bacterial pneumonia that either heals with antibiotics alone (without complications) or requires chest tube drainage (complications). Other causes of pleural effusion are determined according to established clinical criteria.

[0088] ADA Measurement: Pleural effusion samples obtained by thoracentesis were collected in 5 mL sterile heparinized tubes for immediate routine biochemical analysis, including ADA analysis. Total ADA activity was determined using an automated spectrophotometric method (Roche Diagnostics, Barcelona, ​​Spain).

[0089] Statistical analysis: Continuous and categorical variables are expressed as medians (25th and 75th percentiles) and percentages. Kruskal-Wallis tests or Fisher tests were used for comparisons between groups. Spearman correlation coefficients were used to assess correlation. Receiver operating characteristic (ROC) curves were constructed to illustrate the diagnostic accuracy of ADA and isoenzymes. Measures of effectiveness included sensitivity, specificity, and likelihood ratio. The significance level was set at p < 0.05 (two-tailed). Data were analyzed using SPSS 18.0.

[0090] The analytical results were compared with data obtained from the total ADA activity assay (Porcel, Esquerda et al. 2010).

[0091] Table 1: Thoracic ADA and ADA2 Remark: 1 According to the Kruskal-Wallis test, it was significantly higher than that of other groups (p<0.01).

[0092] Table 2: Patient Baseline Information Remark: 1 According to the Kruskall-Wallis test, the value was significantly lower than the corresponding value in other groups (P<0.01). 2Fisher's test showed no significant difference between the groups (P = 0.34). 3 Seven cases of lung cancer (three adenocarcinomas, two squamous cell carcinomas, and two small cell carcinomas); five cases of unknown origin; four cases of lymphoma; one case of breast cancer; one case of ovarian cancer; one case of mesothelioma; and one case of sarcoma. 4 Two cases of acute pericarditis, one case of heart failure, one case of Dresler syndrome, and one case of idiopathic pericarditis.

[0093] like Figure 3 As shown in Figure A, this embodiment found that the ADA2 concentration in tuberculous pleural effusion (TB) was significantly higher than that in non-tuberculous exudate. Based on the calculation that 1 ng / mL of ADA2 represents 0.114 U / L, the critical value of 300 ng / mL ADA2 in pleural effusion is equivalent to 34.2 U / L, which is close to the diagnostic critical value of 35 U / L for pulmonary tuberculosis obtained using the total ADA detection method. Figure 3 B). ADA2 levels in non-tuberculous exudative pleural effusions rarely exceed the diagnostic threshold for pulmonary tuberculosis mentioned above. The above results demonstrate that the detection method provided by this invention is not only simple and has a low detection limit, but also a wide detection range, capable of detecting ADA2 levels above 300 ng / mL (even up to 600 ng / mL).

[0094] Furthermore, as shown in Table 3, both total ADA activity and ADA2 concentration were higher in pleural effusions from patients with lymphoma and small cell lung cancer. However, total ADA activity was higher, but ADA2 concentration was lower, in patients with adenocarcinoma of unknown origin and breast cancer. This difference may be due to their higher ADA1 levels. In other words, total ADA activity is affected by both ADA1 and ADA2 activities. Therefore, current techniques for detecting total ADA activity are prone to falsely elevated ADA values ​​in pleural effusions, making it difficult to simply interpret them as ADA2 activity. The correlation between total ADA activity and ADA2 isoenzymes is shown in [reference needed]. Figure 4 (Pearson: 0.727, Spearman: 0.788).

[0095] Table 3: Falsely elevated ADA values ​​in pleural effusion Note: *Two cases of lymphoma and one case of small cell lung cancer; Two cases of lymphoma, one case of adenocarcinoma of unknown origin, one case of breast cancer, and one case of small cell lung cancer.

[0096] As shown in Table 4 and Figure 5 The results showed that, compared with the detection of total ADA activity, the method for detecting ADA2 provided by this invention has higher specificity and sensitivity, and the AUC can reach over 99%.

[0097] In summary, if the ADA2 concentration in an exudative pleural effusion sample is below 300 ng / mL, the method for detecting ADA2 provided by this invention can be used to rule out tuberculosis as a cause, thereby helping to differentiate between tuberculosis and other exudative pleural effusions.

[0098] Table 4: Accuracy of ADA and ADA2 scFv-AP in diagnosing tuberculous pleural effusion Sensitivity Specificity Positive likelihood ratio Negative likelihood ratio ADA2 > 300 ng / mL 98(87-100) 91(80-97) 11.5(4.5-29.3) 0.03(0.004-0.19) ADA2 > 400 ng / mL 90(77-96) 98(89-100) 42.4(6.1–295.6) 0.1(0.04-0.25) ADA2 > 500 ng / mL 90(77-96) 98(89-100) 42.4(6.1-295.6) 0.1(0.04-0.25) ADA2 > 600 ng / mL 78(63-88) 98(89-100) 36.7(5.2-257) 0,22(0.13-0.4) Total ADA > 35 U / L 99(90-100) 76(62-86) 4.1(2.5-6.8) 0.02(0.001-0.25) Total ADA > 40 U / L 93(81-97) 83(70-91) 5.4(2.9-10.3) 0.09(0.03-0.26)

[0099] The above results also indicate that the method for detecting ADA2 provided by this invention can be used to distinguish certain types of cancer patients (e.g., lymphoma patients and small cell lung cancer patients) from other cancer patients (Table 5).

[0100] Table 5: ADA-2 and total ADA levels in malignant pleural effusion (MPE)

[0101] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises an anti-ADA2 single-chain antibody and alkaline phosphatase, wherein the amino acid sequence of the light chain variable fragment of the single-chain antibody is shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable fragment of the single-chain antibody is shown in SEQ ID NO:

2.

2. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the single-chain antibody is shown in SEQ ID NO:

3.

3. The fusion protein as described in claim 1, characterized in that, The alkaline phosphatase is Escherichia coli alkaline phosphatase.

4. The fusion protein as described in claim 3, characterized in that, The amino acid sequence of the alkaline phosphatase is shown in SEQ ID NO:

6.

5. A kit for detecting adenosine deaminase 2 in a sample, characterized in that, The kit includes the fusion protein as described in any one of claims 1-4, an ELISA plate coated with capture antibody, adenosine deaminase 2 standard, blocking buffer, and ELISA plate washing buffer.

6. A method for detecting adenosine deaminase 2 in a sample, characterized in that, The method includes: S101 coats an ELISA plate with anti-ADA2 rabbit polyclonal antibody to obtain an ELISA plate coated with capture antibody; S102 Add adenosine deaminase 2 standard dilution solution and the sample to the wells of the ELISA plate coated with capture antibody, and then incubate. S103 Add the fusion protein as described in any one of claims 1-4 to the corresponding wells of the ELISA plate obtained in step S102; S104 Add p-nitrophenyl phosphate solution to the corresponding wells of the ELISA plate obtained in step S103. S105 uses an ELISA reader to read the absorbance of the corresponding wells of the ELISA plate obtained in step S104 at a wavelength of 405 nm, thereby obtaining the concentration of adenosine deaminase 2 in the sample.

7. The method as described in claim 6, characterized in that, The samples include one or more of pleural effusion, saliva, serum, and cell culture medium.

8. The method as described in claim 6, characterized in that, The concentration of the fusion protein ranges from 1 to 1.5 μg / mL.

9. The method as described in claim 6, characterized in that, The concentration of the p-nitrophenyl phosphate solution ranges from 5 to 10 mM.

10. The method as described in claim 6, characterized in that, The concentration of the anti-ADA2 rabbit polyclonal antibody includes 5-10 μg / mL.

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