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US-20260126448-A1 - KIT FOR DETECTING SOLUBLE GROWTH STIMULATION EXPRESSED GENE 2 PROTEIN

US20260126448A1US 20260126448 A1US20260126448 A1US 20260126448A1US-20260126448-A1

Abstract

The present disclosure relates to a kit for detecting a soluble growth stimulation expressed gene 2 protein. In particular, the present disclosure relates to a latex-enhanced turbidimetric immunoassay kit for detecting the concentration and/or content of the sST2 in human samples. The kit can be used in transmission immunoturbidimetry and scattering immunoturbidimetry. The kit comprises a buffer system, an anti-interference component, latex microspheres, an anti-sST2 antibody, etc. The latex-enhanced immunoturbidimetric agent of the present disclosure can detect sST2 proteins within a range of <400 ng/ml in a sample, with a sensitivity of up to 0.1 ng/ml and a high specificity, accuracy and precision. The kit is suitable for a fully automatic biochemical analyzer and a scattering analyzer, and has the advantages of convenient and fast use and low cost, and can be used clinically to detect the sST2 protein.

Inventors

  • Yao Liu
  • Xi Liu

Assignees

  • BEIJING STRONG BIOTECHNOLOGIES, INC.

Dates

Publication Date
20260507
Application Date
20251219
Priority Date
20190328

Claims (10)

  1. 1 - 9 . (canceled)
  2. 10 . A method for fixing polypeptides to latex microspheres, comprising: providing a polypeptide in a buffer; providing latex microspheres in the buffer; mixing the polypeptide and the latex microspheres at a temperature of 10° C. to 35° C. for 6 to 10 hours to obtain a first mixture by shaking and mixing; providing a crosslinking agent; contacting the crosslinking agent with the first mixture at a temperature of 30° C. to 60° C. for 2 to 5 hours to obtain latex microspheres cross-linked with the polypeptide; wherein the polypeptide carries at least one functional group selected from the group consisting of amino, carboxyl, hydroxyl, and sulfhydryl; wherein the polypeptide is selected from the group consisting of an antibody, an antigen-binding fragment, an antigen, an enzyme, and a recombinant protein; wherein a surface functional group of the latex microsphere is selected from the group consisting of carboxyl, sulfhydryl, chloromethyl, and no surface functional group; wherein the latex microspheres have an average particle size of 350 nm to 450 nm; wherein the buffer is selected from the group consisting of HEPES buffer, glycine buffer, Tris buffer, PBS buffer, MOPS buffer, and boric acid buffer; wherein the buffer provides a pH of 7.0 to 9.0; and wherein a concentration of the buffer is 10 mmol/L to 1000 mmol/L.
  3. 11 . The method of claim 10 , wherein: the mixing is performed at a temperature of 20° C. to 25° C. for 7 to 9 hours, the crosslinking agent comprises carbodiimide; the contacting is performed at a temperature of 45° C. to 55° C. for 2 to 4 hours; the buffer comprises boric acid buffer at pH 9.0; and the concentration of the buffer is 10 mmol/L to 1000 mmol/L.
  4. 12 . The method of claim 10 , further comprising blocking the latex microspheres cross-linked with the polypeptide by: contacting the latex microspheres cross-linked with the polypeptide with a blocking system at a temperature of 15° C. to 28° C. for 1 hour to 24 hours; wherein the blocking system comprises a blocking agent, a buffer having a pH of 5.0 to 8.0, and a surfactant; wherein the blocking agent is selected from the group consisting of: 0.1% to 5.0% w/v polyethylene glycol having a molecular weight of 1000 to 5000, 0.1% to 5.0% w/v polyethylene glycol polyamine having a molecular weight of 1000 to 5000, 2.0% to 10.0% w/v bovine serum albumin, 2.0% to 10.0% w/v dextran gel, and 2.0% to 10.0% w/v casein; wherein the buffer of the blocking system is selected from the group consisting of: phosphate buffer, glycine buffer, and HEPES buffer; and wherein the surfactant is selected from the group consisting of: 2.0% to 8.0% w/v Triton X, 2.0% to 8.0% w/v Tween, and 2.0% to 8.0% w/v AEO.
  5. 13 . The method of claim 12 , further comprising one or more steps selected from the group consisting of: rinsing the latex microspheres cross-linked with the polypeptide; centrifuging and collecting the latex microspheres cross-linked with the polypeptide; and packaging the latex microspheres cross-linked with the polypeptide.
  6. 14 . A method of performing immunoturbidimetric detection, comprising: providing an immunoturbidimetric detection kit comprising an anti-interference composition; and performing immunoturbidimetric detection using the kit; wherein the anti-interference composition comprises 0.1% to 10% w/v surfactant and 0.5 KU/L to 10 KU/L lipid digestive enzyme; wherein the surfactant is selected from the group consisting of: Triton X, Tween, AEO, Thesit, Brij, and NP; and wherein the lipid digestive enzyme is selected from the group consisting of: lipase and triglyceride oxidase.
  7. 15 . The method of claim 14 , wherein the immunoturbidimetric detection kit is selected from the group consisting of: (a) a first kit comprising: a first reagent comprising: 200 mmol/L sodium chloride, 1% w/v surfactant, 0.1% w/v sodium azide, and 100 mmol/L HEPES buffer, pH 8.0; a second reagent comprising: 0.1% w/v 400 nm latex microspheres and 30 g/mL anti-sST2 monoclonal antibody; (b) a second kit comprising: a first reagent comprising: 150 mmol/L sodium chloride, 1% w/v surfactant, 0.1% w/v sodium azide, 5 KU/L lipase, 0.5% methacryloyloxy phosphatidylcholine, and 100 mmol/L Tris buffer, pH 7.4; a second reagent comprising: 0.15% w/v 400 nm latex microspheres, 20 g/mL sST2 antibody Fab fragment, 100 mmol/L glycine buffer at pH 8.0, 0.1% w/v NaCl, 0.5% w/v methacryloyloxy phosphatidylcholine, and 0.1% w/v NaN 3 ; and (c) a third kit comprising: a first reagent comprising: 1% w/v surfactant, 0.1% w/v sodium azide, 5 KU/L lipase, 2% w/v sodium cholate, 5 mmol/L CaCl 2 , 2.0% w/v methacryloyloxy phosphatidylcholine, 3% w/v blocker HBR-8 TM , and 200 mmol/L Tris buffer, pH 7.4; and a second reagent comprising: 0.15% w/v 400 nm latex microspheres, 20 g/mL sST2 antibody Fab fragment, 100 mmol/L glycine buffer at pH 8.0, 0.1% w/v NaCl, 0.5% w/v methacryloyloxy phosphatidylcholine, and 0.1% w/v NaN 3 ; wherein the surfactant is selected from the group consisting of: Triton X, Tween, AEO, Thesit, Brij, and NP.
  8. 16 . The method of claim 15 , wherein the kit further comprises at least one of a calibrator and a control material.
  9. 17 . The method of claim 16 , wherein: the calibrator comprises sST2 protein at a concentration selected from the group consisting of: 25 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, and 400 ng/mL; the control material comprises sST2 protein at a concentration selected from the group consisting of: 30 ng/mL and 100 ng/mL; the calibrator or the control material comprises a buffer selected from the group consisting of: phosphate buffer, HEPES buffer, MOPS buffer, MES buffer, and PIPES buffer; the buffer has a concentration of 5 mmol/L to 250 mmol/L; the buffer has a pH of 4.0 to 8.0; the calibrator or the control material comprises 1.0% to 10% w/v protectant selected from the group consisting of: bovine serum albumin, saccharide, and alcohol; and the calibrator or the control material comprises 0.05% to 1.5% w/v preservative selected from the group consisting of: sodium azide, PC, and dithiothreitol.
  10. 18 . The method of claim 17 , wherein the calibrator or the control material comprises sST2, 100 mmol/L phosphate buffer at pH 5.5, 150 mmol/L NaCl, 5% w/v BSA, 10% w/v sorbitol, and 0.1% w/v NaN 3 .

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of, and claims priority to, U.S. patent application Ser. No. 17/440,930, which was filed on Sep. 20, 2021, which is a U.S. National Phase Entry of International PCT Application No. PCT/CN2020/073461, which was filed on Jan. 21, 2020, and which claims the benefit of Chinese Patent Application Serial No. 201910241518.7, which was filed on Mar. 28, 2019. The contents of each of those applications are incorporated herein by reference in their entireties. TECHNICAL FIELD OF THE INVENTION The present disclosure relates to a kit for detecting a soluble growth stimulation expressed gene2 protein and protein product (sST2) thereof inhuman blood. In particular; the present disclosure relates to a latex-enhanced turbidimetric immunoassay kit for detecting the concentration or content of the sST2. The kit can be used in transmission immunoturbidimetry and scattering immunoturbidimetry. BACKGROUND OF THE INVENTION The isolation of murine specific growth stimulation expressed genes from BALB/c-3T3 cells was described by Tominaga; they called one of these genes “St2” (for growth stimulation expressed gene 2). The St2 gene encodes two protein products: ST2, which is a soluble secreted form; and ST2L, a form of transmembrane receptor very similar to the interleukin-1 receptor. The HUGO Nomenclature Committee refers to the human homologue as interleukin 1 receptor-like 1 (IL1R-L1). ST2 is a member of the interleukin-1 receptor family and has two existing forms: transmembrane (ST2L) and soluble (sST2). Th (helper T cell) can secrete a variety of cytokines. Th cells express CD4, and the so-called CD4+ T cell refers to Th. HIV can specifically damage Th cells, causing destroyed immune system of patients. ST2L has immunomodulatory functions and plays an important role in T cell-mediated immune diseases such as asthma and rheumatoid arthritis. ST2L neutralizing antibody or sST2 will block the binding of ST2L to ligands, thereby down-regulating Th2 (Th2 cells mainly secrete IL-4, IL-5, IL-6 and IL-10, etc., the main function thereof is to stimulate B cell proliferation and produce antibodies of immunoglobulin G1 and immunoglobulin E, and are related to humoral immunity). Lymphocyte function suggests that sST2 has an inhibitory effect on inflammation. SUMMARY OF THE INVENTION According to some embodiments of the application, provided is a detection kit for soluble growth stimulation expressed gene 2 protein, which comprises: a first reagent,a second reagent,optionally, calibrator(s),optionally, control material(s). In some embodiments, the first reagent comprises: 10 mmol/L to 500 mmol/L buffer, with pH range of 5.0 to 8.0,10 mmol/L to 700 mmol/L dispersing agent,0.05% to 5% w/v coagulant,anti-interference composition, andoptionally, 0.05% to 0.5% w/v preservative. In some embodiments, the second reagent comprises: 10 mmol/L to 500 mmol/L buffer,0.05% to 0.25% w/v latex microspheres, andanti-sST2 antibody or antigen-binding fragment thereof. In some embodiments, the buffer in the first reagent and the second reagent is the one or combination thereof selected from the group consisting of: HEPES buffer, glycine buffer, Tris buffer, PBS buffer, MOPS buffer and boric acid buffer. In specific embodiments, the buffer of the second reagent is boric acid buffer, pH 9.0. In some embodiments, the dispersing agent is the one or combination thereof selected from the group consisting of: salt ion, thiocyanate, organic dispersant and surfactant. In some embodiments, the coagulant is the one or combination thereof selected from the group consisting of: polyethylene glycol, methacryloyloxy phosphatidylcholine and polyether diamine. In some embodiments, the preservative is the one or combination thereof selected from the group consisting of: azide and PC preservative. In some embodiments, the surface functional group of the latex microspheres is selected from one of the following: carboxyl, sulfhydryl, chloromethyl or without surface functional group. In some embodiments, the average particle size of the latex microspheres is 300 nm to 600 nm, preferably 350 nm to 450 nm. In some embodiments, the anti-sST2 antibody is the one or combination thereof selected from the group consisting of: non-affinity IgG antibody, affinity IgG antibody and IgY antibody. In some embodiments, the anti-sST2 antibody is derived from murine, rabbit, goat or avian. In some embodiments, the anti-sST2 antibody is a monoclonal antibody or a polyclonal antibody. In specific embodiments, the antigen-binding fragment is selected from: Fab, Fab′, (Fab′)2, Fv or scFv. In some embodiments, the anti-sST2 antibody or antigen-binding fragment thereof is covalently bound to the surface of the latex microspheres. In some embodiments, the anti-interference composition comprises: 0.1% to 10% w/v surfactant, and 0.5 KU/L to 10 KU/L lipid digesting enzyme. In some embodiments, the surfactant is the one or