A kit and method for detecting CCL1

Abstract

This invention relates to the field of biodetection technology, specifically to a kit and method for detecting CCL1. The kit comprises magnetic beads coated with capture antibodies, a CCL1 detection antibody conjugated with a first label, a β-galactosidase conjugated with a second label, an enzyme-catalyzed luminescent substrate, and CCL1 standards. The capture antibody in the magnetic beads is used to specifically bind to the CCL1 antigen. The first label in the CCL1 detection antibody conjugated with the first label is used to link with the second label in the β-galactosidase conjugated with the second label. The CCL1 detection antibody is used to specifically bind to the CCL1 antigen. The kit provided by this invention has advantages such as small sample volume, accurate detection results, high detection sensitivity, good specificity, wide linear range, good repeatability, short detection time, and convenient use in fully automated detection instruments.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a kit and method for detecting CCL1. Background Technology

[0002] Chemokine (CC motif) ligand 1 (CCL1), also known as I-309, is a small glycoprotein secreted by activated human T lymphocytes, monocytes, and endothelial cells. It is present in a large cluster of CC chemokines on human chromosome 17. CCL1 interacts with cell surface CC chemokine receptor 8 (CCR8) and plays a central role in immune regulation and inflammation.

[0003] CCL1, a potent chemotactic attractant, primarily participates in the recruitment and activation of macrophages and lymphocytes in inflammatory diseases, including atopic dermatitis, allergic asthma, type 1 diabetes, pulmonary fibrosis, and experimental autoimmune encephalomyelitis. For example, in the pathogenesis of allergic asthma, CCL1 can chemotactically attract and recruit CCR8+ inflammatory cells, thereby dominating the inflammatory response of type 2 T helper 2 (Th2) cells in the asthmatic airways. Increased CCL1 concentration in bronchoalveolar lavage fluid of asthmatic patients is associated with an increased number of lymphocytes. CCL1 levels reflect the severity of airway inflammation in asthma and can serve as a starting point for therapeutic drug research. During the lesion phase of atopic dermatitis, CCL1 levels are significantly elevated, recruiting T cells and Langerhans dendritic cells to the site of skin inflammation, thus amplifying the immune response. Monitoring CCL1 levels in patient tissue fluid and serum helps to explore its significance in the pathogenesis of atopic dermatitis. In the development of pulmonary fibrosis, chronic lung injury leads to the production of large amounts of CCL1 by alveolar macrophages and T cells. CCL1 binds to the autocrine motility factor receptor (AMFR) on the fibroblast membrane, causing AMFR phosphorylation, which promotes the synthesis of profibrotic proteins within fibroblasts, resulting in pulmonary fibrosis. As a specific biomarker for the development of pulmonary fibrosis, CCL1 provides a new perspective for the treatment and drug development of pulmonary fibrosis.

[0004] Furthermore, CCL1 is involved in tumor development, including gastric cancer, breast cancer, colorectal cancer, metastatic brain cancer, and metastatic liver cancer. In the tumor microenvironment, tumor stem cells, carcinoma-associated fibroblasts (CAFs), and tumor-associated macrophages (TAMs) secrete CCL1, which binds to CCR8 on the surface of cancer cells, inducing cancer cell proliferation and migration. It also induces angiogenesis by activating the CCR8 receptor on endothelial cells. Another important function of CCL1 is to convert CD4+ T cells into regulatory T cells (Tregs) and recruit them to the tumor niche. The CCL1 / CCR8 signaling axis has attracted worldwide attention as a potential tumor immune target.

[0005] Currently, the main clinical methods for detecting CCL1 include chemiluminescence analysis (CLIA) and enzyme-linked immunosorbent assay (ELISA). CLIA has a short luminescence process and a high background fluorescence, resulting in low detection accuracy. ELISA requires manual sample addition and plate washing, which is time-consuming and can be affected by different operator techniques, leading to inconsistent results. Furthermore, both CLIA and ELISA methods only achieve picogram-level detection, exhibiting relatively low sensitivity.

[0006] Therefore, developing a fully automated CCL1 detection kit with higher analytical sensitivity, less background signal interference, and a wider linear range has broad application prospects. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a kit for detecting CCL1, which has the advantages of high sensitivity, less background signal interference and a wide linear range.

[0008] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0009] A kit for detecting CCL1 is disclosed, comprising magnetic beads coated with a capture antibody, a CCL1 detection antibody conjugated with a first label, a β-galactosidase conjugated with a second label, an enzyme-catalyzed luminescent substrate, and a CCL1 standard. The capture antibody in the magnetic beads is used to specifically bind to the CCL1 antigen. The first label in the CCL1 detection antibody conjugated with the first label is used to link with the second label in the β-galactosidase conjugated with the second label. The CCL1 detection antibody is used to specifically bind to the CCL1 antigen. The invention also provides a method for preparing the kit for detecting CCL1.

[0010] The present invention also provides the use of the kit in the preparation of disease detection products.

[0011] The present invention also provides a method for detecting CCL1 using the kit, wherein the method involves using the kit to detect CCL1 via a two-step immunofluorescence assay; the method is a non-disease diagnostic method.

[0012] Furthermore, this invention utilizes the principle of the double-antibody sandwich method in enzyme-linked immunosorbent assay (ELISA) and combines it with SIMOA (Single Molecular Array) single-molecule immunoassay technology. This allows for the capture of single molecules in microwells the size of a flyby bead, enabling the digital reading of individual magnetic bead signals and converting the digital signals into the concentration of the analyte molecule, thus greatly improving the detection sensitivity.

[0013] In summary, this invention discloses a kit and method for detecting CCL1, and achieves the following beneficial effects:

[0014] The reagent kit provided by this invention has advantages such as small sample volume, accurate detection results, high detection sensitivity, good specificity, wide linear range, good repeatability, short detection time, and convenient use in fully automated detection instruments, overcoming many shortcomings of existing immunoassay techniques. It is easy to widely promote and apply, and has broad market prospects.

[0015] The beneficial effects of the present invention also include:

[0016] 1. This invention optimizes the working concentration of β-galactosidase coupled with a second label to 50-250 pM, which shows superior performance in parameters such as the limit of detection, lower limit of quantitation, upper limit of quantitation, and detection dynamic range; in particular, 150 pM is preferred for better results.

[0017] 2. This invention optimizes the working concentration of the CCL1 detection antibody conjugated with the first marker to 0.2-1.2 μg / mL, which shows superior performance in parameters such as signal-to-noise ratio, limit of detection (LOD) of the kit, and dynamic range of detection; in particular, 0.5 μg / mL is preferred for even better results.

[0018] 3. This invention optimizes the mixing ratio of magnetic beads and auxiliary magnetic beads for conjugating capture antibodies to 7:3-3:7, which shows superior results in parameters such as the lowest detection limit. In particular, the preferred ratio of 3:7 is even better.

[0019] 4. This invention optimizes the incubation sequence and incubation time of the capture antibody, analyte, detection antibody, and β-galactosidase coupled with a second label during the detection process.

[0020] 5. The reaction chamber volume of this invention is 50 fL. The extremely small reaction system can reduce background noise and signal scattering, making the detection sensitivity of this invention more than 1000 times higher on average than the existing conventional ELISA methods. It can detect very low concentrations of CCL1, requires less sample, saves precious samples, and reduces matrix effects.

[0021] 6. The detection and analysis instruments corresponding to this invention automatically complete the dilution, mixing, washing, incubation, and result reading and analysis, realizing one-stop detection from sample to result, without relying on staff, and ensuring the repeatability and accuracy of the results.

[0022] 7. The SIMOA detection technology corresponding to this invention can use two different analysis methods, digital detection and analog detection, for low-concentration and high-concentration samples, thereby improving the detection dynamic range. Attached Figure Description

[0023] Figure 1 This is a standard curve for CCL1. The horizontal axis represents the concentration of the standard; the vertical axis represents the signal value; and AEB (Average Number Enzymes per Bead) represents the number of β-galactosidases bound to each magnetic bead. Detailed Implementation

[0024] This invention provides a kit for detecting CCL1, comprising: magnetic beads coated with a capture antibody, a CCL1 detection antibody conjugated with a first label, a β-galactosidase conjugated with a second label, an enzyme reaction luminescent substrate, and a CCL1 standard; the capture antibody in the magnetic beads coated with the capture antibody is used to specifically bind to the CCL1 antigen; the first label in the CCL1 detection antibody conjugated with the first label is used to link with the second label in the β-galactosidase conjugated with the second label, and the CCL1 detection antibody is used to specifically bind to the CCL1 antigen.

[0025] The magnetic beads coated with capture antibodies comprise capture antibodies and magnetic beads. The capture antibodies are used to specifically bind to the CCL1 antigen. The magnetic beads are used to immobilize the capture antibodies.

[0026] Furthermore, the capturing antibody is either a monoclonal antibody or a polyclonal antibody. Preferably, the capturing antibody is a monoclonal antibody that specifically binds to the CCL1 antigen in the human body. Preferably, the CCL capturing antibody is purchased from R&D Company, catalog number MAB272.

[0027] Furthermore, the magnetic beads are chemically modified magnetic beads. The chemical groups are any one of succinimide ester, sulfonyl, hydroxyl, amino, carboxyl, and derivatives of the above chemical groups. Preferably, the magnetic beads are carboxyl-modified magnetic beads. The carboxyl-modified magnetic beads are simply referred to as carboxyl magnetic beads. In one embodiment, the carboxyl magnetic beads are purchased from Quanterix, product number 103612.

[0028] In one specific implementation, each 1 microgram of capture antibody is coated with 7 × 10⁻⁶ 6 On a carboxyl magnetic bead. More specifically, the amount of capture antibody used is 55-65 micrograms.

[0029] In one specific embodiment, the kit further includes auxiliary magnetic beads. In another specific embodiment, the magnetic beads coated with capture antibodies and the auxiliary magnetic beads are a mixed reagent. The ratio of the magnetic beads coated with capture antibodies to the auxiliary magnetic beads in the mixed reagent is 7:3 to 3:7. The ratio of the magnetic beads coated with capture antibodies to the auxiliary magnetic beads in the mixed reagent is selected from any of the following ranges: 7:3-6:4, 6:4-5:5, 5:5-4:6, or 4:6-3:7. More specifically, the ratio of the magnetic beads coated with capture antibodies to the auxiliary magnetic beads is 7:3, 5:5, or 3:7; preferably, the ratio is 3:7. In one embodiment, the auxiliary magnetic beads are purchased from Quanterix, catalog number 103208.

[0030] The first marker in the CCL1 detection antibody conjugated with the first marker is biotin or a biotin derivative; and / or, the second marker in the β-galactosidase conjugated with the second marker is avidin.

[0031] The biotin is used to bind to a kinase that catalyzes a luminescent reaction. Further, the kinase that catalyzes the luminescent reaction is a β-galactosidase coupled with a second label.

[0032] Further, the biotin is a chemical molecule comprising an imidazolium ring and a thiophene ring. The imidazolium ring is the site for binding with avidin. The thiophene ring is the site for binding with CCL1. The biotin can combine with other chemical groups to form biotin derivatives. The chemical groups comprise one or more types. The chemical groups are selected from one or more of amino (NH2), active ester (NHS), diphenylcyclooctyne (DBCO), azide, alkyne, or polyethylene glycol (PEG). Preferably, the biotin derivative is NHS-PEG4-Biotin.

[0033] Furthermore, the CCL1 detection antibody is either a monoclonal antibody or a polyclonal antibody. Preferably, the CCL1 detection antibody is a monoclonal antibody that specifically binds to the CCL1 antigen. The CCL detection antibody was purchased from R&D Company, catalog number BAF272.

[0034] In one specific implementation, each detection antibody molecule is linked to four NHS-PEG4-Biotin molecules.

[0035] In one specific embodiment, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.2-1.2 μg / mL. The working concentration of the CCL1 detection antibody conjugated with the first label is, for example, 0.2-0.4 μg / mL, 0.4-0.6 μg / mL, 0.6-0.8 μg / mL, 0.8-1.0 μg / mL, or 1.04-1.2 μg / mL. Preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.2-0.3 μg / mL, 0.3-0.5 μg / mL, 0.5-0.7 μg / mL, 0.7-0.9 μg / mL, or 0.9-1.2 μg / mL; more preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.4-0.7 μg / mL; even more preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.5 μg / mL.

[0036] In addition to specifically binding to the CCL1 antigen, the capture antibody and the CCL1 detection antibody conjugated with the first marker can form a "sandwich" structure with the antigen when used in pairs.

[0037] The β-galactosidase conjugated with the second label is used to bind the CCL1 detection antibody conjugated with the first label. The β-galactosidase is used to catalyze an enzymatic luminescent substrate reaction.

[0038] Furthermore, the avidin is selected from egg avidin, streptavidin, yolk avidin, and avidin-like substances. Preferably, the avidin is streptavidin.

[0039] In one specific embodiment, the working concentration of the β-galactosidase coupled with the second marker is 50-250 pM. More specifically, the working concentration of the β-galactosidase coupled with the second label is 50-70 pM, 70-90 pM, 90-110 pM, 110-130 pM, 130-150 pM, 150-170 pM, 170-190 pM, 190-210 pM, 210-230 pM, or 230-250 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 70-230 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 90-210 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 130-170 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 150 pM.

[0040] Furthermore, the luminescent substrate for the enzyme reaction is one or more. The luminescent substrate includes o-nitrophenyl-β-D-galactopyranoside (ONPG), chlorophenol red-β-D-galactopyranoside (CPRG), fluorescein digalactopyranoside (FDG), and phenoxazinone-β-D-galactopyranoside (RGP). Preferably, the luminescent substrate is phenoxazinone-β-D-galactopyranoside (RGP). The luminescent substrate is either self-produced or uses commercially available reagents. Preferably, the luminescent substrate is a commercially available reagent.

[0041] Furthermore, the CCL standard is a dry powder or a solution of known concentration. The CCL standard is either self-produced or uses commercially available reagents. In one specific embodiment, the CCL standard is purchased from an R&D company, catalog number 272-1.

[0042] Furthermore, the sample diluent comprises phosphate buffer, surfactant, and preservative. The phosphate buffer provides stable conditions for the enzymatic reaction. The surfactant solubilizes and disperses the solute to promote the enzymatic reaction. The preservative controls the growth of microorganisms in the sample diluent.

[0043] The phosphate in the phosphate buffer solution is selected from potassium phosphate or sodium phosphate. The potassium phosphate and sodium phosphate are selected from one or more of potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0044] The surfactant is a substance that contains both hydrophilic and hydrophobic groups, resulting in a significant decrease in the surface tension of the target solution. The surfactant is selected from one or more of stearic acid, sodium dodecylbenzenesulfonate, quaternary ammonium compounds, lecithin, amino acid-type surfactants, betaine-type surfactants, alkyl glucosides, fatty acid glycerides, fatty acid sorbitan, or polysorbates.

[0045] The preservative is either self-produced or uses commercially available reagents. Preferably, the preservative is any one or more of the commercially available ProClin series preservatives ProClin 150, 200, 300, or 5000. More preferably, the preservative is ProClin 300.

[0046] In some specific embodiments, the sample diluent is either self-prepared or a commercially available diluent. Preferably, the sample diluent is a commercially available diluent. More specifically, the commercially available diluent is purchased from Quanterix, product number 101359.

[0047] The CCL1 standard is a solution or a dry powder. The solvent for the standard solution is the sample diluent. The standard solution is one or more solutions with a certain concentration gradient. Preferably, the standard solution is at least five solutions with a certain concentration gradient, used to prepare a standard curve. In one specific embodiment, the concentrations of the standard solutions are 1000 pg / mL, 200 pg / mL, 40 pg / mL, 8 pg / mL, 1.6 pg / mL, and 0 pg / mL, respectively.

[0048] In one specific embodiment, the quality control sample is a solution or a dry powder. The solute in the quality control sample solution is the CCL1 standard, and the solvent is the sample diluent. The quality control sample solution is one or more solutions with a certain concentration. The concentration of the quality control sample is 50-900 pg / mL. The quality control sample concentration is 50-100 pg / mL, 100-300 pg / mL, 300-500 pg / mL, 500-700 pg / mL, or 700-900 pg / mL. Preferably, the quality control sample solution is 50-100 pg / mL or 700-900 pg / mL. In one specific embodiment, the quality control sample solution has two concentrations. In one specific embodiment, the concentrations of the quality control sample solutions are 80 pg / mL and 800 pg / mL, respectively.

[0049] The kit is used to prepare disease detection products. In some specific embodiments, the tumor includes one or more of gastric cancer, breast cancer, colorectal cancer, metastatic brain cancer, or metastatic liver cancer; the inflammatory disease is selected from one or more of atopic dermatitis, allergic asthma, type 1 diabetes, pulmonary fibrosis, or experimental autoimmune encephalomyelitis.

[0050] This invention also provides a method for detecting CCL1, wherein the method involves using the aforementioned kit to detect CCL1 via a two-step immunofluorescence assay. This method is not a disease diagnostic method.

[0051] The method includes the following steps:

[0052] 1) Mix magnetic beads coated with capture antibodies and auxiliary magnetic beads, CCL1 detection antibody coupled with the first label and the analyte to obtain a first mixture and incubate them together;

[0053] 2) The first mixture and β-galactosidase coupled with the second label are mixed to obtain a second mixture and then incubated together;

[0054] 3) After mixing the second mixture with the enzyme reaction substrate, perform the detection on the instrument.

[0055] The incubation time in step 1) is 30–40 minutes;

[0056] In step 1), the total amount of magnetic beads coated with capture antibodies and helper magnetic beads used is 3.0 × 10⁻⁶. 8 -5.0×10 8 Preferably, the total amount of magnetic beads coated with capture antibodies and auxiliary magnetic beads is 3.0 × 10⁻⁶. 8 -3.5×10 8 3.5-4.0×10 8 4.0-4.5×10 8 Or 4.5-5.0×10 8More preferably, the total amount of magnetic beads coated with capture antibodies and auxiliary magnetic beads is 3.5-4.5 × 10⁻⁶. 8 In one specific embodiment, the total amount of magnetic beads coated with capture antibodies and auxiliary magnetic beads is 4.2 × 10⁻⁶. 8 ;

[0057] In step 1), based on the total volume of the first mixture, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.2-1.2 μg / mL; preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.2-0.3 μg / mL, 0.3-0.5 μg / mL, 0.5-0.7 μg / mL, 0.7-0.9 μg / mL, or 0.9-1.2 μg / mL; more preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.4-0.7 μg / mL; even more preferably, the working concentration of the CCL1 detection antibody conjugated with the first label is 0.5 μg / mL.

[0058] In step 2), based on the total volume of the second mixture, the working concentration of the β-galactosidase coupled with the second label is 50-250 pM; more specifically, the working concentration of the β-galactosidase coupled with the second label is 50-70 pM, 70-90 pM, 90-110 pM, 110-130 pM, 130-150 pM, 150-170 pM, 170-190 pM, 190-210 pM, 210-230 pM. M or 230-250 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 70-230 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 90-210 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 130-170 pM; more preferably, the working concentration of the β-galactosidase coupled with the second label is 150 pM;

[0059] The incubation time in step 2) is 2–8 minutes;

[0060] The on-machine detection process described in step 3) uses SIMOA single-molecule immune array analysis technology.

[0061] All reagent components not mentioned in detail in the kit of this invention (e.g., washing solution, buffer solution, etc.), the outer packaging of the kit, and the individual packaging containers for each component were purchased from Quanterix, Inc., USA, and were operated in accordance with Quanterix's operating instructions. Operating steps not mentioned in detail in the method of this invention can also be performed by referring to conventional procedures in the relevant field, such as allowing each reagent to stand at room temperature (18-25°C) for half an hour before detection and mixing thoroughly before sample addition; the use of the detection instruments and equipment should be performed according to the instruction manual; in this invention, unless otherwise specified, the proportions and contents of units are defined as follows: solid components are defined by mass proportion and content, and liquid components by volume proportion and content.

[0062] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0063] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0065] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0066] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature; see Sambrook et al., *MOLECULAR CLONING: A LABORATORY MANUAL*, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, John Wiley & Sons, New York, 1987 and periodic updates; these series *METHODS IN ENZYMOLOGY*, Academic Press, San Diego; Wolffe, *CHROMATINSTRUCTURE AND FUNCTION*, Third edition, Academic Press, San Diego, 1998; *METHODS IN ENZYMOLOGY*, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and *METHODS IN MOLECULAR*. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0067] Example 1: Preparation of Standards and Quality Control Samples

[0068] The CCL1 standard was diluted with sample diluent to prepare CCL1 standard solutions and quality control solutions of specific concentrations. The concentrations of the CCL1 standard solutions were 1000 pg / mL, 200 pg / mL, 40 pg / mL, 8 pg / mL, 1.6 pg / mL, and 0 pg / mL, as shown in Table 1; the concentrations of the CCL1 quality control solutions were 80 pg / mL and 800 pg / mL, as shown in Table 2. The CCL1 standard used in this embodiment was purchased from the manufacturer, R&D Company.

[0069] Table 1 Preparation of Standards

[0070]

[0071]

[0072] Table 2 Preparation of quality control materials

[0073]

[0074] Example 2: Preparation of magnetic bead reagent

[0075] Preparation of a mixed reagent of magnetic beads coated with capture antibodies and auxiliary magnetic beads:

[0076] 1) Wash the capture antibody and replace the buffer with magnetic bead conjugation buffer. The magnetic bead conjugation buffer was purchased from Quanterix (catalog number 101357), and the CCL1 capture antibody was collected again (adjusted to a concentration of 0.2 mg / mL).

[0077] 2) Mix the thoroughly homogenized carboxyl magnetic bead concentrate (containing 4.2 × 10⁻⁶ mg / L) 8 Transfer 1 carboxyl magnetic bead to a 1.7 mL centrifuge tube, place the centrifuge tube on a magnetic rack for 1 min, aspirate the supernatant, add magnetic bead washing buffer to the centrifuge tube, and vortex to mix; place the centrifuge tube on a magnetic rack for another 1 min and aspirate the supernatant again, repeating this washing process 3 times; then wash the carboxyl magnetic beads 3 times with magnetic bead coupling buffer using the above method; finally, add magnetic bead coupling buffer to the carboxyl magnetic beads, vortex to mix, and place on ice for later use.

[0078] 3) Activate carboxyl magnetic beads: Add EDC (1-Ethyl-3dimethylaminopropyl carbodiimide, final concentration: 0.3 mg / mL) to the magnetic bead reagent in the magnetic bead coupling buffer, mix thoroughly, and then place the centrifuge tube on a mixer at 2-8℃ for 30 min.

[0079] 4) Couple CCL1 capture antibody to carboxyl magnetic beads: Place the carboxyl magnetic beads mixed with EDC on a magnetic rack for 1 min, remove the supernatant, add magnetic bead coupling buffer and mix well, place on a magnetic rack for another 1 min and remove the supernatant, add the capture antibody prepared in step 1), vortex to mix thoroughly, place the centrifuge tube on a mixer at 2-8℃ and mix for 2 hours.

[0080] 5) Blocking the carboxyl magnetic beads after coupling: Place the magnetic beads coupled with the capture antibody on a magnetic rack for 1 min, remove the supernatant, add magnetic bead washing buffer and vortex to mix; then place the centrifuge tube on the magnetic rack, remove the supernatant, and wash the coupled magnetic beads twice in this way; finally, remove the magnetic bead washing buffer on the magnetic rack, add magnetic bead blocking solution, mix thoroughly, and place on a mixer to block for 45 min.

[0081] 6) Washing: Place the centrifuge tube containing the sealed magnetic beads on a magnetic rack for 1 minute, aspirate the supernatant, add magnetic bead washing buffer, and mix thoroughly; then place it on a magnetic rack for 1 minute, aspirate the supernatant, add magnetic bead diluent and mix thoroughly; repeat the above method, aspirate the magnetic bead diluent, add new magnetic bead diluent, and store in a 4°C refrigerator for later use.

[0082] Example 3: Preparation method of biotinylated CCL1 detection antibody, i.e., NHS-PEG4-Biotin-detection antibody conjugate

[0083] The preparation method of the NHS-PEG4-Biotin-detection antibody conjugate is as follows:

[0084] 1) Replace the buffer solution with biotinylation reaction buffer. The biotinylation reaction buffer was purchased from Quanterix, catalog number 101358, and the detection antibody (1 mg / mL) was collected again.

[0085] 2) Biotin-labeled CCL1 detection antibody: Add biotin to the CCL1 detection antibody at a mass ratio of 4:1, mix thoroughly, and incubate at room temperature for 30 minutes.

[0086] 3) Purify biotinylated CCL1 detection antibody: Wash the detection antibody three times with biotinylated reaction buffer using a centrifuge filter tube, collect the detection antibody again, and store it in a 4°C refrigerator for later use.

[0087] Example 4: Kit for detecting CCL1 and method for detecting CCL1

[0088] This invention also provides a method for detecting CCL1 using this CCL1 detection kit:

[0089] 1) Preparation of standard solutions: CCL1 standard was diluted to 1000 pg / mL, 200 pg / mL, 40 pg / mL, 8 pg / mL, 1.6 pg / mL and 0 pg / mL using sample diluent, and 230 μL of each of the 6 concentrations was added to a 96-well plate.

[0090] 2) Preparation of quality control solution: Dilute CCL1 standard to 80 pg / mL and 800 pg / mL using sample diluent, add 80 μL of each concentration to a 96-well plate.

[0091] 3) Add the sample to the 96-well plate and load it into the detection instrument.

[0092] 4) Load the prepared or diluted magnetic bead reagent (including magnetic beads coated with capture antibody and auxiliary magnetic beads), CCL1 detection antibody coupled with NHS-PEG4-Biotin, β-galactosidase coupled with avidin, standard solution, quality control solution and sample dilution into the detection instrument.

[0093] 5) The reaction steps in the detection instrument are as follows:

[0094] A. 25 μL of a mixture of magnetic beads and auxiliary magnetic beads containing CCL1 capture antibody was incubated with 100 μL of the analyte and 100 μL of biotinylated CCL1 detection antibody for 35 min.

[0095] B. Add 100 μL of 150 pM streptavidin-β-galactosidase (SBG) coupled with a second label to the above mixed solution and incubate for 5 min. After the above two steps are completed, a "sandwich" structure of "capture antibody-analyte-detection antibody" is formed.

[0096] C. Wash the magnetic beads to remove non-specifically bound proteins.

[0097] D. Add 100 μL of enzyme reaction substrate RGP. The RGP-containing magnetic bead immune complex is transferred to the wells on the Simoa optical disc and generates a fluorescent signal.

[0098] E. The oil seal confines the fluorescent signal within the small hole.

[0099] F. Fluorescence is digitally interpreted by a CCD imaging system.

[0100] G. The instrument automatically outputs the CCL1 concentration in the sample.

[0101] The samples to be tested in the above methods are selected from plasma, serum, cell culture supernatant or cell lysate.

[0102] Example 5: Comparison of CCL1 detection kits and methods for CCL1 detection

[0103] According to the CCL1 detection kit described in Example 4, the incubation sequence and time among the following four components were optimized: magnetic beads coated with capture antibody, biotinylated CCL1 detection antibody conjugated with a first label, the analyte, and streptavidin-β-galactosidase conjugated with a second label. This invention compared two methods: a two-step method and a three-step method, with specific implementation methods shown in Tables 3 and 4. The results are shown in Table 5. By comparing parameters such as the limit of detection, lower limit of quantitation, upper limit of quantitation, and detection dynamic range, the two-step method was determined to be superior to the three-step method.

[0104] Table 3 Two-step method

[0105]

[0106] Table 4 Three-Step Method

[0107]

[0108] Table 5 Comparison of results between the two-step and three-step methods

[0109] Minimum detection limit 0.2478 pg / mL 0.5395 pg / mL Lower limit of quantitation 0.605 pg / mL 0.902 pg / mL upper limit of quantification 12100pg / mL 3098pg / mL Detection dynamic range 3.25 logs 2.82 logs

[0110] The detection dynamic range is expressed as a logarithm to base 10, representing the range between the highest and lowest detection limits. Taking the data in Table 5 as an example, 3.25 logs means that the highest detection limit of the detection dynamic range is "10 to the power of 3.25 times" the lowest detection limit.

[0111] Example 6: Optimization of SBG working concentration

[0112] The working concentration of SBG was optimized according to the CCL1 detection kit method described in Example 4. This invention compared three working concentrations of SBG: 250 pM, 150 pM, and 100 pM. The results are shown in Table 6. By comparing parameters such as the limit of detection, lower limit of quantitation, upper limit of quantitation, and detection dynamic range, it was determined that a 150 pM SBG concentration was superior to 250 pM and 100 pM.

[0113] Table 6. Kit test results for three SBG concentrations (250 pM, 150 pM, 100 pM)

[0114] Minimum detection limit 1.7862 pg / mL 0.2478 pg / mL 0.5185 pg / mL Lower limit of quantitation 1.98 pg / mL 0.605 pg / mL 1.14 pg / mL upper limit of quantification 645pg / mL 12100pg / mL 8821 pg / mL Detection dynamic range 2.23logs 3.25 logs 3.19logs

[0115] Example 7

[0116] The working concentration of the biotinylated detection antibody was optimized according to the method for detecting CCL1 using the kit described in Example 4. This invention compared the working concentrations of two detection antibodies: 0.3 μg / mL and 0.5 μg / mL.

[0117] Table 7 compares the test results using kits with two working concentrations of CCL1 detection antibodies (0.3 μg / mL and 0.5 μg / mL).

[0118] Table 7. Test results of working concentrations of CCL1 detection antibodies with different biotinylation levels.

[0119] Minimum detection limit 0.6340 pg / mL 0.2478 pg / mL Lower limit of quantitation 1.41 pg / mL 0.605 pg / mL upper limit of quantification 9230pg / mL 12100pg / mL Detection dynamic range 3.08logs 3.25 logs

[0120] After comparing the signal-to-noise ratio, the limit of detection (LOD) of the kit, and the dynamic range of the detection, it was finally determined that a two-step method should be used for antibody incubation and analyte incubation during the detection process using this kit; the working concentration of the detection antibody is 0.5 μg / mL; and the working concentration of SBG is 150 pM.

[0121] Example 8

[0122] The method for detecting CCL1 using the kit described in Example 4 was optimized by adjusting the ratio of carboxyl magnetic beads coated with capture antibodies to auxiliary magnetic beads. This invention compared three ratios of carboxyl magnetic beads coated with capture antibodies to auxiliary magnetic beads: 7:3, 5:5, and 3:7.

[0123] Comparison of kit results using three different ratios of carboxyl magnetic beads coated with capture antibodies to helper magnetic beads (7:3, 5:5, 3:7):

[0124] Table 8. Test results of different ratios of carboxyl magnetic beads and auxiliary magnetic beads used for three types of coated capture antibodies.

[0125] Minimum detection limit 0.2799 pg / mL 0.3330 pg / mL 0.1765 pg / mL Lower limit of quantitation 0.555 pg / mL 0.593 pg / mL 0.337 pg / mL upper limit of quantification 2772 pg / mL 1789 pg / mL 1210 pg / mL Detection dynamic range 3.06logs 2.96 logs 3.00logs

[0126] The optimization results are shown in Table 9. The four-parameter equation for the standard curve was obtained by nonlinearly fitting the concentration and signal values ​​of the standard samples: Y = 44.6666 + (0.0065 - 44.6666) / (1 + (X / 588.3610)^1.2373); R 2 =0.9999. The standard curve is as follows: Figure 1 As shown in Table 9, the horizontal axis represents the concentration of the standard, and the vertical axis represents the signal value. Table 9 shows that the limit of detection is below 0.176 pg / mL; the limit of quantitation is approximately 0.337 pg / mL; the upper limit of quantitation is approximately 1210 pg / mL; and the dynamic range is 3.0 logs. This indicates that the kit has high sensitivity, a wide dynamic range, and can be used with fully automated instruments.

[0127] Table 9. Test results of the optimal ratio of carboxyl magnetic beads to auxiliary magnetic beads for coating and capturing antibodies.

[0128]

[0129] In summary, the above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A kit for detecting CCL1, characterized in that, The kit comprises magnetic beads coated with capture antibodies, a CCL1 detection antibody conjugated with a first label, a β-galactosidase conjugated with a second label, an enzyme-catalyzed luminescent substrate, and a CCL1 standard. The capture antibody in the magnetic beads is used to specifically bind to the CCL1 antigen. The first label in the CCL1 detection antibody conjugated with the first label is linked to the second label in the β-galactosidase conjugated with the second label, and the CCL1 detection antibody is used to specifically bind to the CCL1 antigen. The working concentration of the CCL1 detection antibody conjugated with the first label is 0.4-0.7 μg / mL. The working concentration of the β-galactosidase conjugated with the second label is 130-170 pM. The first label in the CCL1 detection antibody conjugated with the first label is biotin or a biotin derivative, wherein the biotin derivative is NHS-PEG4-Biotin. The second label in the β-galactosidase coupled with the second label is avidin; the kit further comprises auxiliary magnetic beads, the magnetic beads coated with the capture antibody and the auxiliary magnetic beads are a mixed reagent, the ratio of the magnetic beads coated with the capture antibody to the auxiliary magnetic beads in the mixed reagent is 3:7, and the mixed amount of the magnetic beads coated with the capture antibody and the auxiliary magnetic beads is 3.0×10 8 -5.0×10 8 individual beads; The kit uses a two-step immunofluorescence assay to detect CCL1.

2. The reagent kit according to claim 1, characterized in that, The capture antibody is an antibody that can specifically bind to the human CCL1 antigen.

3. The reagent kit according to claim 2, characterized in that, The capture antibody and the CCL1 detection antibody conjugated with the first label are monoclonal antibodies; and / or, the affinity of the capture antibody and the CCL1 detection antibody conjugated with the first label to the CCL1 antigen respectively satisfies the dissociation constant KD < 10. -9 M.

4. The reagent kit according to claim 1, characterized in that, The kit contains sample diluent and quality control materials.

5. The reagent kit according to claim 1, characterized in that, The magnetic beads coated with the capture antibody are magnetic beads modified with chemical groups; the chemical groups are any one of succinimide ester group, sulfonyl group, hydroxyl group, amino group, and carboxyl group.

6. The reagent kit according to claim 5, characterized in that, The magnetic beads are carboxyl-modified magnetic beads.

7. Use of the kit according to any one of claims 1-6 in the preparation of a disease detection product, wherein the disease is a tumor and / or an inflammatory disease, wherein the tumor is one or more of gastric cancer, breast cancer, colorectal cancer, metastatic brain cancer or metastatic liver cancer, and the inflammatory disease is selected from one or more of atopic dermatitis, allergic asthma, type 1 diabetes, pulmonary fibrosis or experimental autoimmune encephalomyelitis.

8. A method for detecting CCL1, characterized in that, The method is to detect CCL1 using a two-step immunofluorescence assay with the kit described in any one of claims 1-6; the method is a non-disease diagnostic method.

9. The method as described in claim 8, characterized in that, The method includes the following steps: 1) Mix magnetic beads coated with capture antibody and auxiliary magnetic beads, CCL1 detection antibody coupled with the first label and the analyte to obtain a first mixture and incubate them together; 2) The first mixture and β-galactosidase coupled with the second label are mixed to obtain a second mixture, and then incubated together; 3) Mix the second mixture with the enzyme reaction substrate and then perform the detection.

10. The method as described in claim 9, characterized in that, It also includes one or more of the following features: The incubation time in step 1) is 30-40 minutes; In step 1), the mixed amount of magnetic beads coated with capture antibodies and helper magnetic beads is 3.0 × 10⁻⁶. 8 -5.0×10 8 indivual; In step 1), the working concentration of the CCL1 detection antibody conjugated with the first marker is 0.4-0.7 μg / mL; In step 2), the working concentration of β-galactosidase coupled with the second marker is 130~170 pM, based on the total volume of the first mixture. In step 2), the incubation time is 2-8 min, based on the total volume of the second mixture.

11. The method as described in claim 9, characterized in that, Step 3) The on-machine detection is performed using SIMOA single-molecule immune array analysis technology.

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