A free mesothelin detection kit

Abstract

The present application provides a mesothelin binding molecule for detecting free mesothelin, which comprises a mesothelin single domain antibody targeting mesothelin I region or II region and a rabbit IgG Fc segment. The present application also provides a free mesothelin detection kit, which comprises a first binding molecule and a second binding molecule, and the first binding molecule and the second binding molecule respectively comprise a mesothelin single domain antibody targeting mesothelin I region and II region, or respectively comprise a mesothelin single domain antibody targeting mesothelin II region and I region, and the kit adopts a sandwich ELISA method. The single domain antibody used in the present application can significantly improve the detection sensitivity of soluble mesothelin in human plasma, and the free mesothelin detection kit has high detection specificity, high sensitivity and good stability.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, and more specifically, to a free mesothelin detection kit. Background Technology

[0002] Camels possess a naturally occurring heavy chain antibody (HCAbs) lacking the constant CH1 region of both the light and heavy chains. Its variable heavy chain region is a single-domain antibody or nanobody (VHH). The relative molecular weight of a single-domain antibody is approximately 15 kDa, only about one-tenth that of a traditional antibody. Its crystals are oval-shaped, approximately 2.5 nm in diameter and 4.2 nm in length, making it the smallest functional antigen-binding fragment found in nature.

[0003] Traditional antibody Fab fragments and single-chain antibody ScFvs typically form concave topologies on their antigen-binding surfaces, meaning they can usually only recognize sites located on the antigen surface. In contrast, single-domain antibodies have a CDR3 region of only 8–15 amino acids, and some single-domain antibodies have even longer CDR3 regions, forming a stable convex ring structure. Furthermore, the small size of single-domain antibodies helps reduce steric hindrance, allowing them to better penetrate the antigen and recognize and bind to epitopes that traditional antibodies cannot, thereby improving antigen specificity and affinity.

[0004] The mesothelin gene is located at chromosome 16p13.3, comprising 17 exons. The full-length cDNA is approximately 2138 base pairs, with an open reading frame of 1884 base pairs, encoding a 628-amino acid precursor protein (69 kDa). This precursor protein can be hydrolyzed by furin protease into a 31 kDa secretory fragment and a 40 kDa mesothelin fragment. Mesothelin is anchored to the cell surface via GPI. Various extracellular proteases cleave mesothelin in its proximal-membrane region III, with seven main cleavage sites, producing various forms of soluble mesothelin-associated peptides (SMRPs). These peptides are commonly used as biomarkers for detecting malignant mesothelioma and other tumors.

[0005] Early studies have shown that soluble mesothelin expression is elevated in pleural and peritoneal effusions of patients with malignant mesothelioma compared to benign effusions. Canessa et al. confirmed elevated mesothelin expression in effusions in 72% of epithelial mesothelioma cases (Canessa PA, Ferro P, Manta C, et al. Clinical value of mesothelin in pleural effusions versus histology by medical thoracoscopy: brief report. Med Oncol 2013; 30:649). Recent studies have further explored soluble mesothelin as a diagnostic indicator and confirmed that elevated soluble mesothelin expression in effusions is highly predictive of mesothelioma, even when cytological methods are insufficient for diagnosis.

[0006] Therefore, soluble mesothelin is an important biomarker for the early diagnosis of malignant tumors, especially malignant mesothelioma, and is of great significance for the early non-invasive detection of malignant tumors, particularly malignant mesothelioma. Thus, there is a need to develop more methods for detecting soluble mesothelin in human blood and effusions.

[0007] The only commercially available kit for detecting soluble mesothelin peptides is R&D Systems' Quantikine ELISAMSLN (catalog number DMLSN0). This kit uses a sandwich ELISA, but its detection accuracy is limited, with the lower limit of the standard curve being only 0.156 ng / mL.

[0008] In view of this, the present invention is hereby proposed. Summary of the Invention

[0009] A first aspect of the present invention provides a mesothelin-binding molecule for detecting free mesothelin, said mesothelin-binding molecule comprising a mesothelin single-domain antibody, wherein the complementarity-determining region (CDR) of said single-domain antibody comprises CDR1, CDR2, and CDR3, wherein...

[0010] The sequence of CDR1 is selected from any one of SEQ ID NO:1-6;

[0011] The sequence of CDR2 is selected from any one of SEQ ID NO:7-13;

[0012] The sequence of CDR3 is selected from any one of SEQ ID NO:14-19.

[0013] In one or more embodiments, CDR1, CDR2, and CDR3 are selected from any combination of the following:

[0014] The sequence of CDR1 is shown in SEQ ID NO:1, the sequence of CDR2 is shown in SEQ ID NO:7, and the sequence of CDR3 is shown in SEQ ID NO:14; or,

[0015] The sequence of CDR1 is shown in SEQ ID NO:2, the sequence of CDR2 is shown in SEQ ID NO:8, and the sequence of CDR3 is shown in SEQ ID NO:15; or,

[0016] The sequence of CDR1 is shown in SEQ ID NO:3, the sequence of CDR2 is shown in SEQ ID NO:9, and the sequence of CDR3 is shown in SEQ ID NO:16; or,

[0017] The sequence of CDR1 is shown in SEQ ID NO:4, the sequence of CDR2 is shown in SEQ ID NO:10, and the sequence of CDR3 is shown in SEQ ID NO:17; or,

[0018] The sequence of CDR1 is shown in SEQ ID NO:5, the sequence of CDR2 is shown in SEQ ID NO:11, and the sequence of CDR3 is shown in SEQ ID NO:18; or,

[0019] The sequence of CDR1 is shown in SEQ ID NO:6, the sequence of CDR2 is shown in SEQ ID NO:12, and the sequence of CDR3 is shown in SEQ ID NO:19; or,

[0020] The sequence of CDR1 is shown in SEQ ID NO:6, the sequence of CDR2 is shown in SEQ ID NO:13, and the sequence of CDR3 is shown in SEQ ID NO:19.

[0021] In one or more embodiments, the single-domain antibody further comprises four backbone regions (FRs), namely FR1, FR2, FR3, and FR4, wherein,

[0022] The sequence of FR1 is selected from any one of SEQ ID NO:20-26;

[0023] The sequence of FR2 is selected from any one of SEQ ID NO:27-33;

[0024] The sequence of FR3 is selected from any one of SEQ ID NO:34-40;

[0025] The sequence of FR4 is selected from any one of SEQ ID NO:41-45.

[0026] In one or more embodiments, FR1, FR2, FR3, and FR4 are selected from any combination of the following:

[0027] The sequence of FR1 is shown in SEQ ID NO:20, the sequence of FR2 is shown in SEQ ID NO:27, the sequence of FR3 is shown in SEQ ID NO:34, and the sequence of FR4 is shown in SEQ ID NO:41; or,

[0028] The sequence of FR1 is shown in SEQ ID NO:21, the sequence of FR2 is shown in SEQ ID NO:27, the sequence of FR3 is shown in SEQ ID NO:34, and the sequence of FR4 is shown in SEQ ID NO:41; or,

[0029] The sequence of FR1 is shown in SEQ ID NO:22, the sequence of FR2 is shown in SEQ ID NO:28, the sequence of FR3 is shown in SEQ ID NO:35, and the sequence of FR4 is shown in SEQ ID NO:42; or,

[0030] The sequence of FR1 is shown in SEQ ID NO:23, the sequence of FR2 is shown in SEQ ID NO:29, the sequence of FR3 is shown in SEQ ID NO:36, and the sequence of FR4 is shown in SEQ ID NO:41; or,

[0031] The sequence of FR1 is shown in SEQ ID NO:24, the sequence of FR2 is shown in SEQ ID NO:30, the sequence of FR3 is shown in SEQ ID NO:37, and the sequence of FR4 is shown in SEQ ID NO:41; or,

[0032] The sequence of FR1 is shown in SEQ ID NO:25, the sequence of FR2 is shown in SEQ ID NO:31, the sequence of FR3 is shown in SEQ ID NO:38, and the sequence of FR4 is shown in SEQ ID NO:43; or,

[0033] The sequence of FR1 is shown in SEQ ID NO:26, the sequence of FR2 is shown in SEQ ID NO:32, the sequence of FR3 is shown in SEQ ID NO:39, and the sequence of FR4 is shown in SEQ ID NO:44; or,

[0034] The sequence of FR1 is shown in SEQ ID NO:25, the sequence of FR2 is shown in SEQ ID NO:33, the sequence of FR3 is shown in SEQ ID NO:40, and the sequence of FR4 is shown in SEQ ID NO:45.

[0035] In one or more embodiments, the amino acid sequence of the mesothelin single-domain antibody is as shown in any of SEQ ID NO: 46-53.

[0036] In one or more embodiments, the mesothelin binding molecules are used in pairs, divided into binding molecules targeting mesothelin I region and mesothelin II region, wherein the binding molecule targeting mesothelin I region comprises a mesothelin single-domain antibody as shown in any of SEQ ID NO: 46-49, and the binding molecule targeting mesothelin II region comprises a mesothelin single-domain antibody as shown in any of SEQ ID NO: 50-53.

[0037] In one or more embodiments, the binding molecule targeting the mesothelin I region comprises a mesothelin single-domain antibody as shown in any one of SEQ ID NO: 46-49, and the binding molecule targeting the mesothelin II region comprises a mesothelin single-domain antibody as shown in SEQ ID NO: 51 or 53.

[0038] In one or more embodiments, the first binding molecule and the second binding molecule respectively comprise mesothelin single-domain antibodies of any of the following sets of amino acid sequences: SEQ ID NO: 46 and 53, SEQ ID NO: 47 and 51, SEQ ID NO: 48 and 51, SEQ ID NO: 49 and 53.

[0039] In one or more embodiments, the mesothelin-binding molecule further includes a constant region or a portion thereof, such as CH2 and CH3.

[0040] In one or more embodiments, the mesothelin-binding molecule further includes a rabbit IgG Fc fragment, for example, the amino acid sequence of which is shown in SEQ ID NO: 54. The Fc fragment can increase the stability of single-domain antibodies and facilitate secondary antibody design.

[0041] The present invention also provides a free mesothelin detection kit, comprising mesothelin-binding molecules according to any of the above embodiments. Preferably, the detection kit employs a sandwich ELISA method.

[0042] A second aspect of the present invention provides a free mesothelin detection kit, the kit comprising a first binding molecule and a second binding molecule, wherein the kit performs detection in the form of "support-first binding molecule-analyte-second binding molecule-label"; the first binding molecule and the second binding molecule respectively contain mesothelin single-domain antibodies targeting mesothelin regions I and II, or respectively contain mesothelin single-domain antibodies targeting mesothelin regions II and I. Free mesothelin is generated by cleavage of the MSLN protein, with the cleavage site being the juxtamembranous region III, and the cleavage position being diverse, leading to diversity of free mesothelin in plasma. Therefore, the present invention selects single-domain antibodies that bind to regions I and II at the distal membrane end.

[0043] In one or more embodiments, the amino acid sequence of the mesothelin single-domain antibody targeting the mesothelin I region is as shown in any of SEQ ID NO: 46-49; the amino acid sequence of the mesothelin single-domain antibody targeting the mesothelin II region is as shown in any of SEQ ID NO: 5-8.

[0044] In one or more embodiments, the binding molecule targeting the mesothelin I region comprises a mesothelin single-domain antibody as shown in any one of SEQ ID NO: 46-49, and the binding molecule targeting the mesothelin II region comprises a mesothelin single-domain antibody as shown in SEQ ID NO: 51 or 53.

[0045] In one or more embodiments, the first binding molecule and the second binding molecule respectively comprise mesothelin single-domain antibodies of any of the following sets of amino acid sequences: SEQ ID NO: 46 and 53, SEQ ID NO: 47 and 51, SEQ ID NO: 48 and 51, SEQ ID NO: 49 and 53.

[0046] Support materials include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) plates, microspheres, microparticles, glass slides, chips, plastics, and films made from materials such as polystyrene, polyethylene, cellulose, nitrocellulose, cellulose acetate, and silicon compounds.

[0047] The reagent kit provided by this invention completes the detection in the form of "support-first binding molecule-analyte-second binding molecule-label", and the main detection process is as follows:

[0048] Coating: The first binding molecule is coated onto the support and then washed.

[0049] Blocking: The vacant sites on the support that are not coated with the first binding molecule are blocked with an inert protein to prevent non-specific adsorption in subsequent reactions.

[0050] The first binding molecule captures the antigen: After adding the sample to be tested and incubating, the first binding molecule captures the antigen in the sample.

[0051] Antigen captures second binding molecule: After adding the second binding molecule and incubating, the antigen captures the second binding molecule.

[0052] Detection: Detection is performed by introducing markers in one or more steps.

[0053] Furthermore, the label is selected from any one of fluorescent substances, quantum dots, digoxigenin-labeled probes, biotin, radioactive isotopes, radioactive contrast agents, paramagnetic ion fluorescent microspheres, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers, or enzymes.

[0054] In some embodiments, the fluorescent material includes Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, and Cascade. Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitation compounds, Tribispyridyldiamine europium, europium cavitation compounds or chelates, Diamine, Dianthocyanin, La Jolla Blue dye, Allococyanin B. Phycocyanin C. Phycocyanin R. Thiamine, Phycoerythrin, Phycoerythrin R. REG. Rhodamine Green. Rhodamine Isothiocyanate. Rhodamine Red. ROX. TAMRA. TET. TRIT (Tetramethylrhodamine isothiol). Tetramethylrhodamine. Texas Red.

[0055] In some embodiments, the radioactive isotope includes 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc, 99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C 13 N、 15 O、 186 Re、 188 Re、 51 Mn, 52 mMn, 55 Co、 72 As、 75 Br、 76 Br、 82 mRb and 83 Any of the following in Sr.

[0056] In some embodiments, the fluorescent microspheres are polystyrene fluorescent microspheres internally encapsulated with rare-earth fluorescent ions europium.

[0057] Furthermore, the marker is followed by an identification signal, and the marker and the identification signal are color-developed using a biotin-avidin system.

[0058] This invention employs a biotin-avidin system for colorimetric development, thereby improving sensitivity. The affinity between avidin and biotin is extremely strong; their binding affinity constant is at least 10,000 times higher than that of antigen and antibody. Therefore, they can bind rapidly, and the reaction is unaffected by external interference.

[0059] Furthermore, the marker is biotin, and the recognition signal includes a catalytic enzyme sequentially labeled with avidin and a substrate compatible with the catalytic enzyme.

[0060] Furthermore, the catalytic enzyme is selected from horseradish peroxidase or alkaline phosphatase;

[0061] Accordingly, the substrate is 3,3',5,5'-tetramethylbenzidine or o-phenylenediamine.

[0062] When the substrate is 3,3',5,5'-tetramethylbenzidine, the color development time is not fixed because the batches of the colorimetric solution are different. Generally, the OD value of the highest concentration standard is around 2.0.

[0063] Furthermore, the molar number of the first bound molecule is greater than the molar ratio of the second bound molecule.

[0064] Furthermore, the molar ratio of the first bound molecule to the second bound molecule is 5:1-4, preferably 5:2-3.

[0065] Furthermore, the kit includes a reaction plate coated with a first binding molecule, a biotin-labeled second binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

[0066] A preferred embodiment of the kit of the present invention is to label streptavidin with horseradish peroxidase, and use this label to recognize the biotinylated second binding molecule, that is, to achieve more sensitive detection by means of a biotin-avidin amplification system.

[0067] Furthermore, the kit also includes one or more of the following: mesothelin standard, sample diluent, washing solution, and stop solution.

[0068] Furthermore, the washing solution is PBST.

[0069] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0070] (1) The single-domain antibody used in this invention can significantly improve the detection sensitivity of soluble mesothelin in human plasma;

[0071] (2) The present invention uses binding molecules targeting mesothelin I region and binding molecules targeting mesothelin II region to effectively capture free mesothelin at different cleavage sites, thereby achieving effective detection of free mesothelin.

[0072] (3) The binding molecule of the present invention includes the rabbit IgG Fc fragment, which increases the stability of single-domain antibodies and facilitates secondary antibody design;

[0073] (4) The free mesothelin detection kit provided by the present invention has high detection specificity, high sensitivity and good stability. Attached Figure Description

[0074] Figure 1 The standard curve for detecting human soluble mesothelin using the 6-2 antibody is shown.

[0075] Figure 2 The standard curve for detecting human soluble mesothelin using antibody pairs 4-8 is shown.

[0076] Figure 3The standard curve for detecting human soluble mesothelin using the 6-3 antibody is shown.

[0077] Figure 4 The standard curve for detecting human soluble mesothelin using antibody pairs 1-8 is shown.

[0078] Figure 5 The intra-assay precision of four antibody pairs for detecting human soluble mesothelin;

[0079] Figure 6 To determine the inter-batch precision of the 6-2 antibody pair in detecting human soluble mesothelin;

[0080] Figure 7 The recovery rate of human soluble mesothelin was determined by four antibody pairs.

[0081] Figure 8 The linearity of human plasma matrix for detecting human soluble mesothelin was determined by four antibody pairs. Detailed Implementation

[0082] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0083] Example 1: Mesothelin-binding molecular pool

[0084] Four single-domain antibodies targeting the mesothelin I region and four single-domain antibodies targeting the mesothelin II region were selected, and the rabbit IgG Fc fragment was added to obtain mesothelin-binding molecules numbered 1-8. The amino acid sequences involved are shown in Table 1.

[0085]

[0086]

[0087] Example 2: Free mesothelin detection kit

[0088] The free mesothelin assay kit includes the following materials:

[0089] The reaction plate was coated with a first binding molecule, a biotin-labeled second binding molecule, streptavidin-horseradish peroxidase coupler (Streptavidin-HRP), TMB chromogenic solution, mesothelin standard, sample diluent, second binding molecule and streptavidin-HRP diluent, washing solution and stop solution.

[0090] Coating solution: Dilute 20×PBS to 1×PBS, purchased from Sangon Biotech, catalog number: B548117-0500;

[0091] Termination solution: purchased from Solarbio, product number: C1058;

[0092] PBST: 1×PBS + 0.05% Tween (500ml PBS + 250μl Tween);

[0093] Blocking fluid: Thermo Blocker TM Casein, purchased from Thermo, item number: 37528;

[0094] First diluent: one-third Thermo Blocker TM Casein + two-thirds of 1×PBS;

[0095] Second dilution: one-third Thermo Blocker TM Casein+2 / 3 PBST;

[0096] TMB (3,3',5,5'-tetramethylbenzidine): purchased from Abcam, catalog number: ab171524;

[0097] Streptavidin-HRP: Purchased from Biolegend, item number: 405210;

[0098] Standard product: Purchased from ACRO, item number: MSN-H522a;

[0099] The mesothelin standard is the full-length protein of the expressed meso extracellular segment (296-580).

[0100] The reaction plate coated with the first bound molecule was prepared using the following method:

[0101] 1) Dilute the antibody to 3 μg / ml with coating buffer, coat 100 μl / well of enzyme-labeled reaction plate, and incubate overnight at 4°C.

[0102] 2) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0103] 3) Add 200 μl of blocking solution to each well and incubate in a 37°C biochemical incubator for 2 hours.

[0104] 4) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0105] Example 3: Preparation of Standard Curve

[0106] The standard curve was prepared using the kit provided in Example 2, and the steps are as follows:

[0107] Standard dilution buffer 1) Dilute the standard with 4 ng / ml of the first solution, set down 4 gradients (2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml) and 0 ng / ml, add to the reaction plate coated with the first binding molecule, add 100 μl of standard to each well, seal the plate with sealing film, and incubate at room temperature for 2 hours with a shaker at 750±50 rpm.

[0108] 2) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0109] 3) Dilute the biotin-labeled second binding molecule to a final concentration of 0.375 ng / ml using the second dilution buffer. Add 100 μl of standard to each well, seal the plate with sealing film, and incubate at room temperature for 2 hours using a shaker at 750 ± 50 rpm.

[0110] 4) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0111] 5) Dilute Streptavidin (HRP) with the second diluent at a ratio of 1:4000 and incubate at room temperature for 45 minutes with a shaker at 750±50 rpm.

[0112] 6) Wash 4 times with PBST, 400 μl / well, 15 seconds each time, and pat dry with absorbent paper.

[0113] 7) Add 100 μl of TMB developer per well and incubate at room temperature in the dark for 15-20 minutes.

[0114] 8) Add 50 μl of stop solution per well to terminate the reaction.

[0115] The first and second binding molecules were selected to target different regions. 32 antibody pairs were initially screened (positive and negative coatings were counted as 2 pairs). An OD value higher than 1 for the first well at a concentration of 4 ng / ml was recorded as 1, otherwise as 0. The pairings and results are shown in the table below:

[0116]

[0117] After initial screening, the four single-domain antibodies targeting the mesothelin I region (numbered 1-4) and the four single-domain antibodies targeting the mesothelin II region (numbered 5-8) in the table can be used to detect human soluble mesothelin when coated on both sides. All single-domain antibodies numbered 1-8 can be used to detect human soluble mesothelin.

[0118] Further standard curve compliance screening was performed on the above single-domain antibodies to identify antibody pairs that met the standard curve. The detection results are as follows: Figure 1-4 As shown, where Figure 1 The standard curve for the 6-2 antibody pair is shown. Figure 2 The standard curve is for 4-8 antibody pairs. Figure 3 The standard curve for the 6-3 antibody pair is shown. Figure 4 The standard curves for antibody pairs 1-8 are shown above. The standard curves obtained for these four antibody pairs show good linearity, meeting the requirements for antigen detection. The content of human soluble mesothelin in the sample to be tested can be calculated based on the detection curves of the standards.

[0119] Example 4: Performance Testing of Antibody Pairs

[0120] (1) Intra-assay precision of 4 antibody pairs: Three samples with known concentrations (high, medium, and low concentrations of 3, 1.6, and 0.8 ng / ml, respectively) were tested 20 times on the same plate. The intra-assay precision results are as follows: Figure 5 As shown, at high and medium concentrations, the precision CV values ​​of all four antibody pairs were less than 15%; at low concentrations, 4-8 and 6-2 performed better, but their precision CV values ​​were still less than 15%.

[0121] (2) Inter-batch precision of the 6-2 antibody pair: Three known concentrations were tested 20 times each, over at least 3 days. The results of the inter-batch precision are as follows: Figure 6 As shown, at various concentrations, the precision CV values ​​of the 6-2 antibody pair were all below 15%.

[0122] (3) Recovery rate of incorporation of 4 antibody pairs: The recovery rate was calculated for samples incorporating known concentrations into 3 plasma matrices from different sources. The results are as follows: Figure 7 As shown, at undiluted and 2-fold dilution, the linear recoveries of all four antibody pairs were in the range of 80-120%. However, as the dilution factor increased, only the linear recovery of antibody pair 6-2 remained consistently in the range of 80-120%.

[0123] (4) Linearity of human plasma matrix for 4 antibody pairs: Linearity was calculated from 3 human plasma samples containing high concentrations of human soluble mesothelin after 2-fold serial dilution. The results are as follows: Figure 8 As shown, in undiluted form, the linear recoveries of all four antibody pairs were between 80% and 120%. However, as the dilution factor increased, only the linear recovery of antibody pair 6-2 remained consistently between 80% and 120%, indicating that interference factors in the complex matrix do not affect the binding between the antigen and the capture / detection antibody.

[0124] Example 5: Minimum detection dose for the 6-2 antibody pair

[0125] The standard curve was prepared according to the method in Example 3, except that the standard was diluted with a first solution of 4 ng / ml, and six gradients were set down (2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml, 0.125 ng / ml, 0.0625 ng / ml) and 0 ng / ml. The experiment was repeated three times, and the results are shown in the table below:

[0126]

[0127] The results showed that the 6-2 antibody could lower the detection limit, had high sensitivity, and stable detection performance. The detection range of the standard curve was 0.0625-4 ng / ml, and the standard curve plotting in three experiments all met the requirements.

[0128] Although the invention has been illustrated and described with reference to specific embodiments, it should be understood that many other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be included in the appended claims.

Claims

1. A kit for detecting free mesothelin, characterized in that, The kit contains a mesothelin-binding molecule, which comprises a mesothelin single-domain antibody. The complementarity-determining region (CDR) of the single-domain antibody includes CDR1, CDR2, and CDR3, wherein CDR1, CDR2, and CDR3 are selected from any combination of the following. The sequence of CDR1 is shown in SEQ ID NO:1, the sequence of CDR2 is shown in SEQ ID NO:7, and the sequence of CDR3 is shown in SEQ ID NO:14; or, The sequence of CDR1 is shown in SEQ ID NO:5, the sequence of CDR2 is shown in SEQ ID NO:11, and the sequence of CDR3 is shown in SEQ ID NO:

18.

2. The kit according to claim 1, characterized in that, The sequence of the single-domain antibody is selected from SEQ ID NO:47 or 51.

3. The kit according to claim 2, characterized in that, The mesothelin binding molecules are used in pairs, and are divided into binding molecules that target mesothelin I region and mesothelin II region. The binding molecule that targets mesothelin I region contains a mesothelin single-domain antibody as shown in SEQ ID NO: 47, and the binding molecule that targets mesothelin II region contains a mesothelin single-domain antibody as shown in SEQ ID NO:

51.

4. The kit according to any one of claims 1-3, characterized in that, The mesothelin-binding molecule also includes the rabbit IgG Fc fragment.

5. The kit according to claim 4, characterized in that, The amino acid sequence of the rabbit IgG Fc segment is shown in SEQ ID NO:

54.

6. The kit according to claim 4, characterized in that, The kit uses the sandwich ELISA method.

7. A free mesothelin detection kit, characterized in that, The kit includes a first binding molecule and a second binding molecule, and the kit performs detection in the form of "support-first binding molecule-analyte-second binding molecule-label". The first binding molecule and the second binding molecule respectively contain mesothelin single-domain antibodies targeting mesothelin I and II regions, or respectively contain mesothelin single-domain antibodies targeting mesothelin II and I regions. The amino acid sequence of the mesothelin single-domain antibody targeting mesothelin I region is shown in SEQ ID NO: 47; the amino acid sequence of the mesothelin single-domain antibody targeting mesothelin II region is shown in SEQ ID NO:

51.

8. The kit according to claim 7, characterized in that, The label is selected from any one of fluorescent substances, digoxigenin-labeled probes, biotin, radioactive isotopes, electron-dense substances, chemiluminescent labels, ultrasound contrast agents, photosensitizers, or enzymes.

9. The reagent kit as described in claim 8, characterized in that, The marker is biotin.

10. The kit according to claim 8, characterized in that, The fluorescent materials include quantum dots and paramagnetic ion fluorescent microspheres.

11. The kit according to claim 7, characterized in that, The molar number of the first bound molecule is greater than the molar ratio of the second bound molecule.

12. The kit according to claim 11, characterized in that, The molar ratio of the first bound molecule to the second bound molecule is 5:1-4.

13. The kit according to claim 12, characterized in that, The molar ratio of the first bound molecule to the second bound molecule is 5:2-3.

14. The kit according to claim 7, characterized in that, The kit includes a reaction plate coated with a first binding molecule, a biotin-labeled second binding molecule, an avidin-labeled catalytic enzyme, and a substrate.

15. The kit as described in claim 7, characterized in that, The kit also includes one or more of the following: mesothelin standard, sample diluent, washing solution, and stop solution.

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