A kit for detecting alpha-interferon and related applications

Abstract

This invention discloses a kit for detecting α-interferon and related applications, relating to the biological field. The kit includes a capture protein for capturing α-interferon and a detection protein for detecting α-interferon; the capture protein includes an α-interferon receptor protein; the detection protein includes an antibody against α-interferon. This kit is applied to IFN-α detection, covering multiple IFN-α subtypes, including natural and recombinant interferon, and exhibits no cross-reactivity with INF-β and INF-ε. It has high detection accuracy and good repeatability, enabling rapid and effective detection of IFN-α levels in human serum, thereby more accurately reflecting the overall level and activity status of IFN-α in the body. This is of great significance for understanding human immune status, diagnosing and monitoring disease development, and evaluating the effectiveness of treatment.

Description

Technical Field

[0001] This invention relates to the field of biology, and more specifically, to a kit for detecting α-interferon and related applications. Background Technology

[0002] Alpha-interferon (IFN-α) is a class of cytokines with antiviral and immunomodulatory functions, primarily produced by macrophages, dendritic cells, and epithelial cells under the induction of viruses and nucleic acids. IFN-α has direct antiviral effects and further exerts indirect antiviral effects by regulating primary and secondary immune responses. Studies have found that IFN-α levels are significantly higher in patients with chronic hepatitis B than in healthy individuals. Furthermore, IFN-α levels are elevated in the blood of some autoimmune diseases (such as systemic lupus erythematosus). IFN-α has potential anti-inflammatory and tissue-protective effects in diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. In vitro quantitative detection of IFN-α concentrations in human serum or plasma is mainly used clinically to monitor the body's immune status and inflammatory responses.

[0003] Currently, 15 IFN-α subtypes are widely recognized after detailed research: IFNA-1α, IFNA-1β, IFNA-2, IFNA-2β, IFNA-4A, IFNA-4B, IFNA-5, IFNA-6, IFNA-7, IFNA-8, IFNA-10, IFNA-14, IFNA-16, IFNA-17, and IFNA-21. Each subtype has a molecular weight of 19-26 kDa and is composed of 149-172 amino acids. They share similar sequences and structures, with homology approaching 70%. However, clinical observations have revealed significant differences in the types of IFN-α subtypes induced by the same virus in different cells or by different viruses infecting the same type of cells. Existing IFN-α chemiluminescence detection kits generally only detect IFN-α1 or IFN-α2 subunits, while flow cytometry platforms commonly suffer from low sensitivity, poor specificity, and poor reproducibility. Therefore, clinical testing often results in missed detections or poor reproducibility.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a kit for detecting α-interferon and related applications. Addressing the problem that existing technologies cannot detect all IFN-α subtypes, this application provides a novel composition that covers all IFN-α subtypes (including IFNA-1α, IFNA-1β, IFNA-2, IFNA-2β, IFNA-4A, IFNA-4B, IFNA-5, IFNA-6, IFNA-7, IFNA-8, IFNA-10, IFNA-14, IFNA-16, IFNA-17, and IFNA-21), offering advantages such as high detection sensitivity and good repeatability.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a composition for IFN-α detection, comprising: a capture protein for capturing α-interferon and a detection protein for detecting α-interferon; wherein the capture protein comprises: an α-interferon receptor protein, the α-interferon receptor protein comprising any one or both of IFNAR1 and IFNAR2; and the detection protein comprises: an antibody against α-interferon or an antigen-binding fragment thereof.

[0008] In some embodiments, the amino acid sequence of the IFNAR1 has at least 80% identity with the sequence shown in SEQ ID NO:1.

[0009] In some embodiments, having at least 80% identity means having a range of any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%. Identity means that the two sequences are completely identical at the same site using nucleotide or amino acid residues.

[0010] In some embodiments, the amino acid sequence of IFNAR1 is shown in SEQ ID NO:1.

[0011] The amino acid sequence of the IFNAR2 has at least 80% identity with the sequence shown in SEQ ID NO:2.

[0012] In some embodiments, the amino acid sequence of IFNAR2 is shown in SEQ ID NO:2.

[0013] In some embodiments, when the receptor protein of α-interferon comprises a combination of IFNAR1 and IFNAR2, the mass ratio of IFNAR1 to IFNAR2 is 1 to 4:2. Specifically, the mass ratio of IFNAR1 to IFNAR2 can be any one or a range between any two of the following: 1:2, 1.5:2, 2:2, 2.5:2, 3:2, 3.5:2, and 4:2.

[0014] In some embodiments, the mass ratio of IFNAR1 to IFNAR2 is 1 to 2:1.

[0015] In some embodiments, the composition further includes a solid support.

[0016] In some embodiments, the capture protein is coated on the solid support.

[0017] In some embodiments, the solid support includes any one or more of magnetic beads, plates, and membranes.

[0018] In some embodiments, the ratio of the capture protein to the solid-phase carrier is 10–50 μg / mg, specifically any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg / mg.

[0019] In some embodiments, the detected protein includes any one or more of Santa Cruz Biotechnology's anti-IFN-α antibody and Mabtech's anti-IFN-α antibody.

[0020] In some embodiments, the composition further includes a marker.

[0021] In some embodiments, the marker is coupled to the detection protein.

[0022] In some embodiments, the markers include any one or more of the following: fluorescent dyes / proteins, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle markers.

[0023] In some embodiments, the enzyme includes any one or more of horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

[0024] In some embodiments, the working concentration of the detection protein coupled with the marker is 0.1 to 0.6 μg / mL, specifically any one or any two of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 μg / mL.

[0025] In some embodiments, the working concentration of the detection protein coupled with the marker is 0.3 to 0.5 μg / mL, specifically any one or any two of 0.3, 0.4, and 0.5 μg / mL.

[0026] Secondly, the present invention provides the use of the compositions described in any of the foregoing embodiments in the preparation of α-interferon detection kits.

[0027] Thirdly, the present invention provides an α-interferon detection kit, which includes the composition described in any of the foregoing embodiments.

[0028] In some embodiments, the compositions are stored in respective reservoirs;

[0029] After capturing proteins and coating them with magnetic beads, they are stored in a magnetic bead preservation solution, which typically contains components such as buffer, protective proteins, surfactants, and preservatives.

[0030] If a protein is labeled for testing, it is stored in a labeling preservation solution. Taking alkaline phosphatase labeling as an example, the preservation solution usually contains components such as buffer, salt, reducing sugar, protective protein, and preservative.

[0031] In some embodiments, the kit further includes any one or more of the following: standards, dilution buffer, coating buffer, blocking solution, and washing solution.

[0032] The kit of the present invention can be used to detect multiple α-interferon subtypes; the α-interferon subtypes include, but are not limited to, any one or more combinations of IFNA-1α, IFNA-1β, IFNA-2, IFNA-2β, IFNA-4A, IFNA-4B, IFNA-5, IFNA-6, IFNA-7, IFNA-8, IFNA-10, IFNA-14, IFNA-16, IFNA-17 and IFNA-21.

[0033] The present invention has the following beneficial effects:

[0034] The composition provided by this invention is used for IFN-α detection, covering multiple IFN-α subtypes, including natural interferon and recombinant interferon, and has no cross-reactivity with INF-β and INF-ε. The prepared IFN-α detection kit has high accuracy and good repeatability, and can rapidly and effectively detect the IFN-α content in human serum, thereby more accurately reflecting the overall level and activity status of IFN-α in the body. This is of great significance for understanding the human immune status, diagnosing and monitoring the development of diseases, and evaluating the effectiveness of treatment. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0037] Example 1, Material Preparation

[0038] Anti-IFN-α antibody 1 (purchased from Santa Cruz Biotechnology, catalog number sc-80996), anti-IFN-α antibody 2 (purchased from Mabtech, catalog number 3425-3-1000), alkaline phosphatase (purchased from BBISolutions), interferon receptor protein α (IFNAR1) (purchased from R&D Systems), and interferon receptor protein β (IFNAR2) (purchased from Sinobiological) were all commercially available raw materials.

[0039] The sequence of the interferon receptor protein α (IFNAR1) is as follows (SEQ ID NO:1):

[0040] GGKNLKSPQKVEVDIIDDNFILRWNRSDESVGNVTFSFDYQKTGMDNWIKLSGCQNITSTKCNFSSLKLNVYEEIKLRIRAEKENTSSWYEVDSFTPFRKAQI GPPEVHLEAEDKAIVIHISPGTKDSVMWALDGLSFTYSLVIWKNSSGVEERIENIYSRHKIYKLSPETTYCLKVKAALLTSWKIGVYSPVHCIKTTVENELPP PENIEVSVQNQNYVLKWDYTYANMTFQVQWLHAFLKRNPGNHLYKWKQIPDCENVKTTQCVFPQNVFQKGIYLLRVQASDGNNTSFWSEEIKFDTEIQAFLLP PVFNIRSLSDSFHIYIGAPKQSGNTPVIQDYPLIYEIIFWENTSNAERKIIEKKTDVTVPNLKPLTVYCVKARAHTMDEKLNKSSVFSDAVCEKTKPGNTSK.

[0041] The sequence of the interferon receptor protein β (IFNAR2) is as follows (SEQ ID NO:2):

[0042] MLLSQNAFIFRSLNLVLMVYISLVFGISYDSPDYTDESCTFKISLRNFRSILSWELKNHSIVPTHYTLLYTIMSKPEDLKVVKNCANTTRSFCDLTDEWRSTHEAYVTVLEGFSGNTTLFSC SHNFWLAIDMSFEPPFEIVGFTNHINVMVKFPSIVEEELQFDLSLVIEEQSEGIVKKHKPEIKGNMSGNFTYIIDKLIPNTNYCVSVYLEHSDEQAVIKSPLKCTLLPPGQESESAESAK.

[0043] (1) Preparation of sulfonated magnetic microparticle working solution

[0044] 1. The supernatant of sulfonated magnetic microparticles was removed by magnetic separation, and the microparticles were washed with PBS at pH 7.0 and a concentration of 20 mM. After washing, the washing solution was aspirated by a magnetic rack.

[0045] 2. Mix the antibody / protein, magnetic beads, and ammonium sulfate in a certain proportion, and place them on a multi-purpose rotary shaker at 4°C for 24 hours at a speed of 30 times / minute;

[0046] 3. Place the centrifuge tube from step 2 on a magnetic rack for 5 minutes, discard the supernatant, wash with PBS at pH 7.0 and a concentration of 20 mM, and aspirate the washing solution;

[0047] 4. Resuspend the washed magnetic beads from step 3 in a blocking solution of PBS (pH 7.0, 20 mM) and 5% BSA, mix well, and then react at 37°C for 5 hours on a multipurpose rotary shaker.

[0048] 5. Repeat step 4;

[0049] 6. Resuspend the magnetic beads in a working solution to obtain a magnetic bead working solution with a concentration of 0.3 mg / mL. The working solution contains 0.05 mg / mL bovine serum albumin, 0.005 mg / mL Triton X-100 and 0.005 mg / mL Proclin 300, and PBS buffer with a pH of 7.0 and a concentration of 0.1 mol / L.

[0050] (2) Preparation of alkaline phosphatase-labeled antibody / protein working solution

[0051] sulfo-SMCC, also known as sodium 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester, is a water-soluble amine-thiol crosslinking agent.

[0052] Antibody / protein intermediates and alkaline phosphatase intermediates were prepared separately according to a sulfo-SMCC:antibody / protein molar ratio of 20:1 and a reducing agent:alkaline phosphatase molar ratio of 20:1. Subsequently, the mixtures were incubated at 30°C for 2 hours at a mass ratio of antibody / protein intermediate to alkaline phosphatase intermediate of 2:1. After further desalting, alkaline phosphatase-labeled antibody / protein stock solutions were prepared.

[0053] The antibody / protein working solution was further diluted with enzyme-labeled diluent to prepare a 0.25 μg / mL alkaline phosphatase-labeled solution. The enzyme-labeled diluent contained 0.1 mol / L phosphate buffer (pH 6.5), 0.2 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, and 0.005 mg / mL proclin 300.

[0054] (3) Preparation of working solution for test samples

[0055] The test samples used are as follows:

[0056] S: Multi-subtype natural antigen, a mixture of multi-subtype natural interferons produced by human leukocytes—Interferonalpha (Human, leukocyte derived) WHO International Standard (NIBSC, 94 / 784);

[0057] R: Multi-subtype recombinant antigen, multi-subtype recombinant interferon mixture (15 recombinant IFN-α subtype proteins) — HumanIFN-α All Subtype Standard (R&D Systems, PART#899172);

[0058] Cross-reactive sample 1: IFN-β (purchased from Genscript);

[0059] Cross-reactive sample 2: IFN-ε (purchased from MedChemExpress);

[0060] Dilute each test sample to the working concentration using the sample diluent:

[0061] Multi-subtype natural antigens S1 and S2 (10 pg / mL, 10000 pg / mL);

[0062] Multi-subtype recombinant antigens R1 and R2 (10 pg / mL, 10000 pg / mL);

[0063] Cross-reactive samples were diluted to 10000 pg / mL.

[0064] The sample diluent consisted of: phosphate buffer at pH 7.0 with a concentration of 0.01 mol / L, bovine serum albumin at 0.05 mg / mL, sodium chloride at 0.05 mg / mL, and Tween 20 at 0.01 mg / mL.

[0065] Example 2: The effect of different combinations of capture and detection proteins on detection results

[0066] This embodiment provides multiple combinations of capture and detection proteins formed from different proteins, and tests their detection results for different antigens.

[0067] Following the preparation method in Example 1, working solutions of anti-IFN-α antibody 1, anti-IFN-α antibody 2, IFNAR1, and IFNAR2 with a concentration of 0.3 mg / mL were prepared. Four alkaline phosphatase-labeled antibody / protein working solutions with a concentration of 0.25 μg / mL were also prepared: alkaline phosphatase-labeled anti-IFN-α antibody 1, alkaline phosphatase-labeled anti-IFN-α antibody 2, alkaline phosphatase-labeled IFNAR1, and alkaline phosphatase-labeled IFNAR2.

[0068] Different combinations of magnetic bead working solutions and alkaline phosphatase-labeled protein working solutions were used to detect the test samples. The combinations are shown in the table below.

[0069] Table 1 Combinations

[0070]

[0071]

[0072] Using the combinations in Table 1, the following sample dilutions were tested: S0 (0 pg / mL, no antigen), S1 and S2 (10 pg / mL and 10,000 pg / mL) diluted with a mixture of multi-subtype natural interferon, and R1 and R2 (10 pg / mL and 10,000 pg / mL) diluted with a mixture of multi-subtype recombinant interferon, as well as cross-reactive sample 1 (10,000 pg / mL IFN-β) and cross-reactive sample 2 (10,000 pg / mL IFN-ε). The test results are shown in Table 2 below.

[0073] Table 2 Test Results

[0074]

[0075]

[0076] Table 2 shows that Group 1 and Group 2 (when anti-IFN-α antibody 1 and anti-IFN-α antibody 2 are paired) have poor repeatability (CV>3%), but there is no cross-interference for IFN-β and IFN-ε. This indicates that anti-IFN-α antibody 1 and anti-IFN-α antibody 2 can recognize one or more IFN-α subtypes. However, during the antibody coating or labeling process, the antibody activity is affected or some sites are masked, resulting in randomness in the recognition of natural or recombinant multi-subtype antigens, thus leading to poor repeatability.

[0077] Groups 3 and 4 (IFNRA1 and IFNRA2 are receptor proteins for INF-α, and the two receptors pair up with each other) showed good repeatability (CV < 3%), indicating that the combination can recognize multiple subtype antigens; however, the overall signal-to-noise ratio was low, and there was cross-interference between IFN-β and IFN-ε.

[0078] Groups 5-8 (receptor protein and antibody combination) showed good reproducibility (CV < 3%), excellent signal-to-noise ratio, and no cross-interference between IFN-β and IFN-ε. Among them, group 5 (IFNAR1 combined with anti-IFN-α antibody 1) had the best signal-to-noise ratio, with a CV of less than 2%, and no cross-reactivity.

[0079] Example 3: The effect of different capture proteins on detection results

[0080] IFNAR1 and IFNAR2 were mixed according to the ratios shown in Table 3 (mass ratio, the total protein:magnetic bead mass = 20 μg / mg after coating the magnetic beads). The corresponding magnetic bead working solutions were prepared according to the preparation method of the sulfonated magnetic microparticle working solution in Example 1. These solutions were then combined with alkaline phosphatase-labeled anti-IFN-α antibody 1 working solution to detect the results on different samples. The combinations of capture and detection proteins are shown in Table 3 below.

[0081] Table 3 Combinations of capture and detection proteins

[0082]

[0083] Using the combinations in Table 3, the following sample dilutions were tested: S0 (0 pg / mL, no antigen), S1 and S2 (10 pg / mL and 10,000 pg / mL) diluted with a mixture of multi-subtype natural interferon, and R1 and R2 (10 pg / mL and 10,000 pg / mL) diluted with a mixture of multi-subtype recombinant interferon, as well as cross-reactive sample 1 (10,000 pg / mL of IFN-β) and cross-reactive sample 2 (10,000 pg / mL of IFN-ε). The test results are shown in Table 4 below.

[0084] Table 4 Test Results

[0085]

[0086] As shown in Table 4, groups 9-13 showed good repeatability (CV < 3%), good signal-to-noise ratio, and no IFN-β or IFN-ε cross-interference. Group 13 was not significantly different from group 5, while groups 9-12 showed improvement compared to group 5 in Example 1. Among them, group 10 (when IFNAR1:IFNAR2 = 1.5:1 in total protein) had the best CV and specificity, and a significantly improved signal-to-noise ratio.

[0087] Example 4: The effect of enzyme-labeled antibody concentration on detection results

[0088] Using the capture protein and detection protein combination of Group 10 in Example 3, with the magnetic bead working solution unchanged, alkaline phosphatase-labeled anti-IFN-α antibody 1 working solutions of different concentrations were prepared according to the preparation method of alkaline phosphatase-labeled antibody / protein working solution in Example 1, and their detection results on different samples were examined, as shown in Table 5 below.

[0089] Table 5 Combinations

[0090]

[0091] The antibody combinations in Table 5 were used to test gradient reference standards P1-P5, with corresponding concentrations of 0 pg / mL, 10 pg / mL, 100 pg / mL, 1000 pg / mL, and 10000 pg / mL (obtained by diluting the multi-subtype natural antigen S with antigen diluent). The test results are shown in Table 6 below.

[0092] Table 6 Test Results

[0093]

[0094] The results of groups 14 to 18 showed that the repeatability and signal-to-noise ratio of each group were good; when the enzyme concentration was 0.3 to 0.5 μg / mL, the CV and signal-to-noise ratio were good; among them, group 16 (enzyme concentration of 0.4 μg / mL) was the best.

[0095] Example 5, Clinical Application

[0096] Using the magnetic bead-labeled working solution and enzyme-labeled working solution prepared in Group 16 of Example 4, along with the washing solution, substrate solution, and sample diluent produced by our company, we formulated the α-interferon detection kit of this application. Simultaneously, we used two commercial kits, α-interferon detection kit 1 (single subtype IFN-α2) and α-interferon detection kit 2 (all subtypes, referring to a mixture of multiple subtypes of natural interferon produced by human leukocytes, mainly IFNA-1α, IFNA-1β, IFNA-2, IFNA-2β, IFNA-4A, IFNA-4B, IFNA-5, IFNA-6, IFNA-7, IFNA-8, IFNA-10, IFNA-14, IFNA-16, IFNA-17, and IFNA-21), as controls. We simultaneously tested 500 healthy human samples, 300 systemic lupus erythematosus (SLE) patient samples, and 100 novel coronavirus positive patient samples. The test results are shown in Table 7 below.

[0097] Table 7 Test Results

[0098]

[0099] Negative detection rate = Number of samples with negative test results / Total number of samples;

[0100] Positive detection rate = Number of samples with positive test results / Total number of samples.

[0101] As shown in Table 7, the α-interferon detection kit prepared in this application exhibits sensitivity comparable to the commercially available α-interferon detection kit 2 (multi-subtype) in SLE patient samples and SARS-CoV-2 positive patient samples, reaching 34.33% and 36%, respectively, significantly higher than the commercially available α-interferon detection kit 1 (single subtype). Results in healthy individuals show that the α-interferon detection kit prepared in this application has better specificity, exceeding the commercially available α-interferon detection kit 1 (single subtype) and commercially available α-interferon detection kit 2 (multi-subtype) by 0.6% and 1.6%, respectively.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition for detecting α-interferon, characterized in that, It includes: The invention comprises a capture protein for capturing α-interferon and a detection protein for detecting α-interferon; wherein the capture protein includes: an α-interferon receptor protein, wherein the α-interferon receptor protein includes any one or both of IFNAR1 and IFNAR2; and the detection protein includes: an antibody against α-interferon or an antigen-binding fragment thereof. The amino acid sequence of the IFNAR1 is shown in SEQ ID NO:1; The amino acid sequence of IFNAR2 is shown in SEQ ID NO:

2.

2. The composition according to claim 1, characterized in that, The receptor proteins for α-interferon include IFNAR1 and IFNAR2, with a mass ratio of IFNAR1 to IFNAR2 of 1 to 4:

2.

3. The composition according to claim 2, characterized in that, The mass ratio is 1~2:

1.

4. The composition according to any one of claims 1 to 3, characterized in that, The composition further includes a solid support.

5. The composition according to claim 4, characterized in that, The capture protein is coated on the solid support.

6. The composition according to claim 4, characterized in that, The solid support includes any one or more of magnetic beads, plates, and membranes.

7. The composition according to claim 1, characterized in that, The detected proteins include any one or more of Santa Cruz Biotechnology's anti-IFN-α antibody and Mabtech's anti-IFN-α antibody.

8. The composition according to claim 7, characterized in that, The composition also includes a marker.

9. The composition according to claim 8, characterized in that, The marker is coupled to the detection protein.

10. The composition according to claim 8, characterized in that, The markers include any one or more of the following: fluorescent dyes, fluorescent proteins, enzymes, radioactive isotopes, chemiluminescent reagents, and nanoparticle markers.

11. The composition according to claim 10, characterized in that, The enzymes include any one or more of horseradish peroxidase, alkaline phosphatase, and β-galactosidase.

12. The composition according to claim 8, characterized in that, The working concentration of the detection protein conjugated with the marker is 0.1~0.6 μg / mL.

13. The composition according to claim 12, characterized in that, The working concentration of the detection protein conjugated with the aforementioned label is 0.3–0.5 μg / mL.

14. Use of the composition according to any one of claims 1 to 13 in the preparation of an α-interferon detection kit.

15. An α-interferon detection kit, characterized in that, It includes: The composition according to any one of claims 1 to 13.

16. The reagent kit according to claim 15, characterized in that, The kit also includes any one or more of the following: standards, dilution buffer, coating buffer, blocking solution, and washing solution.

17. The kit according to claim 15, characterized in that, The kit can detect multiple α-interferon subtypes.

18. The kit according to claim 17, characterized in that, The α-interferon subtypes include any one or more combinations of IFNA-1α, IFNA-1β, IFNA-2, IFNA-2β, IFNA-4A, IFNA-4B, IFNA-5, IFNA-6, IFNA-7, IFNA-8, IFNA-10, IFNA-14, IFNA-16, IFNA-17, and IFNA-21.

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