A kit for rapid detection of adenosine deaminase 2 and a preparation method thereof

Abstract

The present invention relates to the field of ADA2 detection, and particularly relates to a kit for rapidly detecting adenosine deaminase 2 and a preparation method thereof. The present invention provides a single-chain antibody against adenosine deaminase 2, wherein the amino acid sequence of the light chain variable fragment of the single-chain antibody is as shown in SEQ ID NO:1, and the amino acid sequence of the heavy chain variable fragment of the single-chain antibody is as shown in SEQ ID NO:2. The present invention also provides a kit for rapidly detecting adenosine deaminase 2 deficiency. The present invention can early identify and screen DADA2 patients in the population by detecting the saliva of the subject.

Description

Technical Field

[0001] The present invention relates to the field of ADA2 detection, and in particular to a kit for rapidly detecting adenosine deaminase 2 and a preparation method thereof. Background Art

[0002] The role of adenosine deaminase (ADA) is to regulate purine metabolism and irreversibly convert adenosine in cells into inosine. Currently, two types of adenosine deaminases are known: ADA1 and ADA2. Deficiency of adenosine deaminase 2 (DADA2) is a monogenic autosomal recessive autoinflammatory disease. Most patients develop the disease in childhood and can cause multi-system and multi-organ involvement. Some patients may experience ischemic or hemorrhagic stroke, other vascular disease-related manifestations (hypertension, gastrointestinal symptoms), hepatosplenomegaly, peripheral neuropathy, blood system symptoms, mild immunodeficiency, nodular arteritis, etc. However, the clinical phenotypic spectrum of DADA2 has been significantly expanded, and different patients vary greatly in clinical manifestations, age of onset, and severity of the disease. Current studies have shown that patients with ADA2 deficiency are more common than expected.

[0003] It is not clear what causes DADA2, so the early symptoms of the disease may be severe, disabling, or even life-threatening. Therefore, early diagnosis and treatment of DADA2 are necessary to reduce organ damage and morbidity. However, the diagnosis of DADA2 is mainly carried out by ADA2 gene sequencing and ELISA to measure ADA2 enzyme activity. The above methods have disadvantages such as high cost and slow detection speed, which makes it difficult to popularize them for early diagnosis and screening of DADA2. Summary of the invention

[0004] In the first aspect, the present invention provides a single-chain antibody against adenosine deaminase 2, characterized in that 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. The single-chain antibody against adenosine deaminase 2 provided by the present invention has the advantages of high affinity and strong specificity for adenosine deaminase 2 (ADA2), and is particularly effective in detecting ADA2.

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

[0006] In a second aspect, the present invention provides a kit for rapid diagnosis of adenosine deaminase 2 deficiency, characterized in that the kit comprises a test strip, the test strip comprises a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, the sample pad, the conjugation pad, the nitrocellulose membrane and the water absorbent pad are pasted on the base plate in a chromatographic direction, the conjugation pad is coated with an anti-ADA2 antibody labeled with colloidal gold, and the anti-ADA2 antibody is the above-mentioned single-chain antibody against adenosine deaminase 2.

[0007] The present invention provides a kit for rapid detection of adenosine deaminase 2 deficiency based on the above-mentioned single-chain antibody against adenosine deaminase 2. The sensitivity of the kit is just above 0.5ng / ml ADA2, that is, it allows early identification and screening of DADA2 patients in the population by testing the saliva of the subject (the test results of DADA2 patients are negative, and the test results of healthy people are positive). The kit provided by the present invention is non-invasive, rapid and convenient, and is particularly suitable for large-scale screening of DADA2 patients and immediate detection in the home.

[0008] In some embodiments, the method for preparing the conjugate pad comprises the following steps:

[0009] S101: mixing the colloidal gold solution and the anti-ADA2 antibody at room temperature to react, adding BSA for blocking after the reaction is completed, and collecting the precipitate of the anti-ADA2 antibody labeled with colloidal gold;

[0010] S102 resuspending the precipitate of the colloidal gold-labeled anti-ADA2 antibody in a first solution to obtain a colloidal gold-labeled anti-ADA2 antibody solution, wherein the first solution includes 0.1% PEG6000, 0.2% BSA and 0.01M Tris-HCl;

[0011] S103: impregnating the glass fiber membrane with the colloidal gold-labeled anti-ADA2 antibody solution to obtain the conjugate pad.

[0012] In some embodiments, the nitrocellulose membrane is coated with a detection line and a quality control line.

[0013] In some embodiments, the method for preparing the test line comprises printing the second solution comprising the anti-ADA2 antibody on the nitrocellulose membrane, and the concentration of the anti-ADA2 antibody comprises 0.8-1 mg / mL.

[0014] In some embodiments, the method for preparing the quality control line comprises printing a second solution comprising anti-rabbit IgG on the nitrocellulose membrane, and the concentration of the anti-rabbit IgG comprises 0.8-1 mg / mL.

[0015] In some embodiments, the concentration of the colloidal gold-labeled anti-ADA2 antibody solution comprises 1-1.5 mg / mL.

[0016] In some embodiments, the second solution comprises 2 wt % sucrose in PBS.

[0017] In some embodiments, the sample pad is saturated with a third solution prior to drying, the third solution comprising 0.5% Tween 20 and 0.1% (wt / vol) BSA in PBS.

[0018] In some embodiments, the kit is for saliva testing.

[0019] In some embodiments, the kit is used to screen patients for DADA2.

[0020] In some embodiments, the DADA2 patient carries one or more of the following mutations: Y453C, G47R, R169Q, R131Sfs*52, T33Nfs*29, G358R, Arg169Gly, G47W, R49Gfs*4, exon 7 deletion.

[0021] In a third aspect, the present invention provides a use of a single-chain antibody against adenosine deaminase 2 in the preparation of a kit for rapid diagnosis of adenosine deaminase 2 deficiency.

[0022] In some embodiments, 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.

[0023] In some embodiments, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO:3.

[0024] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0025] The lack of enzyme activity is a common feature of DADA2 patients. Carriers of ADA2 gene mutations usually appear healthy and do not have the hallmark manifestations of DADA2. These individuals are usually discovered through targeted screening after a family member is diagnosed with DADA2. In other words, the existing technology mainly screens DADA2 patients through genetic testing. However, genetic testing has disadvantages such as high cost and slow detection speed, making it difficult to popularize in the early diagnosis and screening of DADA2.

[0026] The present invention provides a single-chain antibody against adenosine deaminase 2, which has the advantages of high affinity and high specificity for ADA2, and demonstrates its advantages in biological detection in the present invention.

[0027] The present invention provides a kit for rapid detection of adenosine deaminase 2 deficiency based on the above-mentioned single-chain antibody against adenosine deaminase 2. The sensitivity of the kit is just above 0.5ng / ml ADA2, which allows early identification and screening of DADA2 patients in the population by testing the saliva of the subject. The actual results of the present invention show that for DADA2 patients with different types of gene mutations, the present invention can accurately and sensitively detect them, which is non-invasive, rapid and convenient, and is particularly suitable for large-scale screening of DADA2 patients and instant detection in the family. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0029] Figure 1 It is a lateral flow test strip for rapid detection of ADA2 in saliva;

[0030] Figure 2 It is a schematic diagram of the composition of the kit of the present invention;

[0031] Figure 3 This is a typical test result diagram of the kit of the present invention;

[0032] Figure 4 This is a graph showing the calibration test results using ADA2. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0035] In this document, unless otherwise clearly specified and limited, the terms "installed", "provided with", "connected", etc. should be understood in a broad sense. For example, "connected" 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, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0036] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0037] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0038] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0039] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0040] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges 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., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0041] Example 1: Preparation of anti-ADA2 single-chain antibody

[0042] 1.1 Coating magnetic beads with ADA2 protein

[0043] According to the manufacturer's instructions, Tosylactivated M-280 Dynabeads (Invitrogen, Norway) were coated with ADA2 protein. Specifically, the coating process was to incubate 60 μg ADA2 protein and 80 μl (2.4 mg) magnetic beads in 200 μl 75 mM borate buffer (pH 9.5) containing 75 mM ammonium sulfate at 37°C overnight. The coated magnetic beads were then blocked with PBS (pH 7.4) containing 0.5% (w / v) BSA at 37°C for 1 hour and then washed once with PBS (pH 7.4) containing 0.1% (w / v) BSA. Finally, the magnetic beads were dissolved in the washing buffer to a concentration of 20 mg / ml.

[0044] 1.2 Screening of ADA2-specific scFv antibodies by phage display

[0045] Antibodies against ADA2 were enriched from a synthetic human antibody library by phage display technology. For screening, two libraries were mixed: scFvP (size 1x10 10 ) and scFvM (size 6x10 9), both libraries used the same single scFv gene as the framework, but differed in the design of the binding sites (Huovinen, Syrjanpaa et al. 2013). The diversity design of the scFvP library was similar to that described by Brockmann et al. (Brockmann, Akter et al. 2011). The design of the ScFvM library was similar to that described by Huovinen et al. (Huovinen, Syrjanpaa et al. 2013). Both libraries used the phage vector pEB32x, which uses a truncated p3 coat protein to display scFv on the surface of the filamentous phage M13. In the first round of panning, 2.4x10 12 Library phage (mixture of scFvP and scFvM libraries) was mixed with 50 μl (1 mg) ADA2-coated magnetic beads in 2.5 ml 50 mM Tris-PBS pH 7.0, 150 mM NaCl, 1% (w / v) BSA, 0.05% (v / v) Tween20 solution and incubated with rotation at room temperature for 2 hours. The magnetic beads were then collected with a magnet, washed twice with 1 ml 50 mM Tris-PBS pH 7.0, 1% (w / v) BSA, 0.1% (v / v) Tween20, and then washed once with 1 ml 50 mM Tris-PBS pH 7.0, 0.1% (v / v) Tween20. To elute the bound phages, the 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 TBS. Escherichia coli XL1-Blue cells were infected with the eluted phages and amplified with the help of VCS M13 helper phage (Stratagene, USA). The subsequent two rounds of panning were performed in a similar manner, but with a smaller number of phages (10 in 1 ml for the second and third rounds, respectively). 11 and 10 10 phages) and the binding time was 1 hour. After each round of panning, the enriched phage library was tested for its ability to bind to ADA2. Phage particles bound to ADA2 were absorbed on a 96-well plate, and then anti-phage Eu-labeled antibodies were detected by time-resolved spectroscopy. A plate coated with BSA was used to detect background signals.

[0046] 1.3 96-well plate phage culture screening

[0047] Infect Escherichia coli XL1-Blue cells with the third round of phage. The gradually diluted infected cells were added with 25 μg / ml chloramphenicol, 12.5 μg / ml tetracycline and 0.5% glucose at 37°C LA plates (tryptone 10g / l, yeast extract 5g / l, sodium chloride 5g / l, agar 15g / l) to obtain isolated bacterium colonies. Then, a single bacterium colony (95 bacterium colonies were selected in this embodiment) was inoculated in each hole of 96-well V-bottom plate SB culture medium, 10 μg / ml tetracycline, 25 μg / ml chloramphenicol and 0.05% glucose. VCS M13 auxiliary phage (Stratagene, the United States) was used to produce phage particles. In order to release phage from cells, the plate was frozen to -70°C and thawed 2 times. Screening was performed on the Maxisorp colony count plate (Nunc, Denmark) coated with ADA2. Coating was performed with TSA (50 mM Tris-HCl, 150 mM NaCl, 0.02% NaN3) containing 1 μg / ml ADA2, 100 μl / well, overnight at room temperature. The coated plate was blocked with TSA containing 1% BSA. Binders were examined using Eu-N1 labeled anti-phage Mab (Brockmann, Akter et al. 2011).

[0048] 1.4 Expression and purification of antibody variable domain single-chain fragment (scFv)

[0049] 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 a single phage clone 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 is ampicillin-resistant but not chloramphenicol-resistant, there is no need to purify the digested fragments. The digested phage DNA was then ligated to the digested gel-purified pAK600H using T4 DNA ligase (Fermentas). pAK600 E. coli XL1-Blue cells were then transformed with the ligation mixture using electroporation and cultured overnight on LA plates (added with 100 μg / ml ampicillin and 12.5 μg / ml tetracycline) at 37°C. 5 ml of SB / Amp-Tet-Glu medium (tryptone 30 g / L, yeast extract 20 g / L, MOPS 10 g / L, ampicillin 100 μg / mL, tetracycline 12.5 μg / mL, glucose 0.5%) was inoculated into a single colony and cultured overnight at 37°C. The next morning, dilute 100-200 times with fresh SB / Amp-Tet medium (without glucose) until OD600 reaches 0.6. Then 200 μg / l IPTG was used to induce the production of scFv. The induced culture was cultured overnight at 20°C. The bacterial cells were centrifuged the next day, resuspended in IMAC30 (20 mM phosphate buffer, 150 mM NaCl, 30 mM imidazole, pH 7.4) and sonicated. Purification was performed using a nickel agarose HiTrap column (GE Healthcare). IMAC30 was used as 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 and the buffer was changed to TSA.

[0050] This example focuses on testing the binding ability of phage clones to ADA2, and found that 15 phage clones can selectively bind to ADA2. Part of the phage DNA corresponding to the anti-ADA2 single-chain antibody was cut out 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 of the anti-ADA2 single-chain antibodies has the highest apparent dissociation constant (Kd <10nM).

[0051] The amino acid sequence of the light chain variable (VL) fragment of the anti-ADA2 single-chain antibody is: AEIVLTQSPGTLSLSPGERATLSCRASQSVSS SYLA WYQQKPGQAP RLLIYGASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQQ SSYPW TFGQGTKVEIKRT (SEQ ID NO: 1, wherein SYLA and QQSSSYPW are the antigen recognition sites of the VL fragment).

[0052] The amino acid sequence of the heavy chain variable (VH) fragment of the anti-ADA2 single-chain antibody is: EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYSMH WVRQAPGKG LEWVS SIWGVNGETD YADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYC AADWSLDY WGQGTLVTVSSASGAEF (SEQ ID NO: 2, wherein SYSMH, SIWGVNGETD and AADWSLDY are the antigen recognition sites of the VL fragment and the VH fragment).

[0053] The amino acid sequence of the anti-ADA2 single-chain antibody is: AEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQSSSYPWTFGQGTKVEIKRTGGGGSGAGGSGGGGTGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYSMHWVRQAPGKGLEWVSSIWGVNGETDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAADWSLDYWGQGTLVTVSSASGAEF (SEQ ID NO: 3, wherein GGGGSGAGGSGGGGTGGGGS is a linker).

[0054] Example 2: Preparation of DADA2 detection kit

[0055] 2.1 Preparation of colloidal gold anti-ADA2 antibody conjugate

[0056] In a clean glass beaker (250 ml), add 1% HAuCl 2100 ml of colloidal gold solution was obtained by stirring with a magnetic stirrer. The mixture was heated to 100°C. Then, 0.7 ml of 1% sodium citrate (Na 3 C 6 H 5 O 7 ·2H 2 O) Slowly add HAuCl 2 The solution was stirred continuously for 20 minutes. Afterwards, the solution was cooled to room temperature. 150 μg of antigen-purified anti-ADA2 antibody was added to 10 ml of colloidal gold solution at pH 7.8 and stirred at room temperature for 30 minutes. Aqueous bovine serum albumin (BSA) (5% wt / vol; 2.5 ml) was used to block any endogenous colloidal gold reaction. The resulting mixture was centrifuged at 12000 rpm and 4°C for 30 minutes to remove unbound antibodies. Finally, the precipitate was resuspended in a 0.01 M Tris-HCl (pH 8.0) suspension containing 0.1% PEG6000 and 0.2% BSA to obtain a colloidal gold anti-ADA2 antibody conjugate.

[0057] 2.2 Preparation of immunochromatographic test strips

[0058] The sample pad was saturated with a PBS solution (pH 8.5) containing 0.5% Tween 20 and 0.1% (wt / vol) bovine serum albumin. Subsequently, the glass fiber paper was impregnated with a colloidal gold anti-ADA2 antibody conjugate solution. The sample pad was then dried at 37°C for one hour and stored in a desiccator for future use. Then, the diluted anti-ADA2 antibody and anti-rabbit IgG (1 mg / ml) were blotted on a nitrocellulose (NC) membrane using PBS containing 2% sucrose as a medium. After drying at 37°C for 2 hours, the NC membrane was also stored in a desiccator. The absorbent pad, nitrocellulose membrane, gold conjugated pad (conjugation pad) and sample pad were combined onto a plastic base plate (the sample pad, conjugation pad, nitrocellulose membrane and absorbent pad were pasted onto the base plate in the chromatography direction). Then, these components were cut into test strips, assembled into a plastic box and sealed in an aluminum foil bag.

[0059] The anti-ADA2 antibody used in this example is the anti-ADA2 single-chain antibody prepared in Example 1.

[0060] Example 3: Detection using the DADA2 detection kit

[0061] 3.1 Clinical samples

[0062] This example includes saliva samples from healthy subjects (n=59) and 6 patients diagnosed with DADA2. The DADA2 detection kit of the present invention is used to detect ADA2 in the saliva samples.

[0063] 3.2 Sample preparation and test procedure

[0064] To collect saliva for the test, a sterile cotton swab is rolled in the mouth for 90 seconds to increase absorption. There are two options for reading the results. The first method is to insert the cotton swab containing saliva directly into the plastic cavity, squeezing the saliva inside and into the test strip ( Figure 1 A). Alternatively, you can squeeze saliva into a plastic tube and place 3 drops of collected saliva onto the sample pad ( Figure 1 B) In both cases, the results were read within 15 minutes to confirm the result.

[0065] 55 μl of buffer containing different concentrations of ADA2 was applied to the lateral flow test strip and the results were obtained after 15 minutes. Figure 1 As shown in A, when a solution containing 0.5 ng / mL ADA2 is applied to the sample pad on the test strip, a faint band can still be seen. In contrast, when the ADA2 concentration is 0.1 ng / mL, no ADA2 is detected. The above results show that the kit of the present invention has a high sensitivity to ADA2.

[0066] Figure 4 It is further shown that the DADA2 detection kit of the present invention can distinguish between 0.8 ng / mL and 0.4 ng / mL of ADA2, which lead to positive and negative results, respectively. 100 μl of buffer containing different concentrations of ADA2 (ng / mL) was applied to the kit and the results were obtained after 15 min.

[0067] It is reported that the average concentration of ADA2 in normal human saliva is about 0.5 ng / mL. The above results show that the sensitivity of the DADA2 detection kit of the present invention is just at 0.5 ng / mL ADA2, so the results displayed can be used to quickly distinguish DADA2 patients from healthy people.

[0068] Since the concentration of ADA2 in the saliva of DADA2 patients is at least 10 times lower than that of healthy subjects, the test results of saliva samples from DADA2 patients should be negative. Testing of saliva samples from healthy subjects (55 μl of saliva from healthy subjects was applied to the lateral flow test strip and the results were obtained after 15 minutes) showed that ADA2 was detected in the saliva of all healthy subjects except one ( Figure 1 B, Table 1), with a calculated specificity of 98%.

[0069] Table 1 Summary of test results of healthy subjects

[0070] quantity ADA2 positive ADA2 negative Specificity Healthy subjects 59 58 1 98.3％

[0071] The present invention designs two types of test kits for further testing. The first test kit includes a swab for collecting saliva from the mouth, a saliva collection tube and a plastic box ( Figure 2 A). The saliva collection tube also has a chamber for applying 3 drops of saliva sample from the saliva collection tube onto the sample pad. This kit also works well when saliva is collected directly into the saliva collection tube. The second kit includes a swab with a hole in the top and a plastic box containing a lateral flow test strip for collecting saliva from the mouth. The saliva can be inserted into the plastic cavity and the sample can be applied directly to the lateral flow test strip ( Figure 2 B).

[0072] Both designs showed similar results when tested with saliva samples from healthy subjects or with 1x PBS buffer as a control ( Figure 3 ).in Figure 3 A Results obtained after 15 min of placing three drops of undiluted saliva sample, saliva sample diluted with 1xPBS or 1xPBS (100 μl) on the sample pad. Figure 3 B is the result obtained after 15 minutes when the swab containing undiluted saliva sample, saliva sample diluted with 1xPBS or 1xPBS was inserted into the plastic chamber.

[0073] Example 3: Using the DADA2 detection kit to detect saliva samples of DADA2 patients

[0074] In order to test the effect of the DADA2 detection kit provided by the present invention in diagnosing DADA2, this example collected saliva samples from 9 DADA2 patients with different mutations or deletions in the ADA2 gene.

[0075] The results showed (Table 2) that ADA2 was not detected in the saliva of 9 DADA2 patients, indicating that the kit provided by the present invention can be used as a screening tool for patients with DADA2 symptoms.

[0076] Table 2 Summary of test results of DADA2 patients

[0077] Number age gender mutation ADA2 negative 1 29 female Y453C and Y453C yes 2 36 male Y453C and Y453C yes 3 18 male G47R and G47R yes 4 13 male R169Q and R131Sfs*52 yes 5 35 male Exon 7 deletion and exon 7 deletion yes 6 10 female T33Nfs*29 and G358R yes 7 17 male Arg169Gly and Arg169Gly yes 8 20 male Arg169Gly and Arg169Gly yes 9 1 female G47W and R49Gfs*4 yes

[0078] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A single-chain antibody against adenosine deaminase 2, characterized in that: The VL sequence of the single-chain antibody is shown in SEQ ID NO: 1, and the VH sequence of the single-chain antibody is shown in SEQ ID NO:

2.

2. The single-chain antibody according to claim 1, wherein The amino acid sequence of the single-chain antibody is shown in SEQ ID NO:

3.

3. A kit for rapid diagnosis of adenosine deaminase 2 deficiency, characterized in that: The kit comprises a test strip, which comprises a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water absorbent pad are attached to the base plate in a chromatography direction, the conjugation pad is coated with an anti-ADA2 antibody labeled with colloidal gold, and the anti-ADA2 antibody is a single-chain antibody against adenosine deaminase 2 as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that The preparation method of the conjugate pad comprises the following steps: S101: mixing the colloidal gold solution and the anti-ADA2 antibody at room temperature to react, adding BSA for blocking after the reaction is completed, and collecting the precipitate of the anti-ADA2 antibody labeled with colloidal gold; S102 resuspending the precipitate of the colloidal gold-labeled anti-ADA2 antibody in a first solution to obtain a colloidal gold-labeled anti-ADA2 antibody solution, wherein the first solution comprises 0.1% PEG6000, 0.2% BSA and 0.01 M Tris-HCl; S103: impregnating the glass fiber membrane with the colloidal gold-labeled anti-ADA2 antibody solution to obtain the conjugate pad.

5. The kit according to claim 3, wherein The nitrocellulose membrane is coated with a detection line and a quality control line. The preparation method of the detection line comprises printing a second solution containing the anti-ADA2 antibody on the nitrocellulose membrane, and the concentration of the anti-ADA2 antibody is 0.8-1 mg / mL.

6. The kit according to claim 5, characterized in that The method for preparing the quality control line comprises printing a second solution containing anti-rabbit IgG on the nitrocellulose membrane, wherein the concentration of the anti-rabbit IgG is 0.8-1 mg / mL.

7. The kit according to claim 4, characterized in that The concentration of the colloidal gold-labeled anti-ADA2 antibody solution is 1 – 1.5 mg / mL.

8. The kit according to claim 5 or 6, characterized in that The second solution contained 2 wt % sucrose in PBS.

9. The kit according to claim 3, characterized in that The sample pad was saturated with a third solution containing 0.5% Tween 20 and 0.1% (wt / vol) BSA in PBS before drying.

10. The kit according to claim 3, characterized in that The kit is used for saliva detection.

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