Lectin composition for detecting abnormal sugar chain glycoprotein

Abstract

The invention relates to the technical field of detection reagent preparation, and discloses a lectin composition for abnormal carbohydrate chain glycoprotein detection, which is prepared from the following raw materials in parts by mass: 50 to 60 parts of main lectin, 30 to 40 parts of auxiliary lectin, 50 to 60 parts of lectin diluent, 0.5 to 1.5 parts of modifier, 100 parts of cross-linking agent and 160 to 200 parts of biotin labeling liquid, wherein the main agglutinin is the agglutinin of the orange lachnum. According to the invention, the variety and proportion of lectin are optimized, methoxy polyethylene glycol active ester is adopted for modification and crosslinking, and then a biotin labeling solution prepared by a specific process is used for labeling, so that the composition is finally obtained. The method has the beneficial effects that through synergistic interaction of multiple lectins, the recognition range of tumor-related abnormal sugar chains is remarkably expanded, and the capture efficiency and the detection sensitivity are improved; meanwhile, the defects of narrow detection range, poor stability and poor repeatability in the prior art are overcome.

Description

Technical Field

[0001] This invention relates to the field of detection reagent preparation technology, and specifically to a lectin composition for the detection of abnormal glycan glycoproteins. Background Technology

[0002] Glycosylation is one of the most important post-translational modifications of proteins, playing a crucial role in vital processes such as cell recognition, signal transduction, and immune response. Numerous studies have shown that abnormal glycosylation is closely related to the occurrence, development, invasion, and metastasis of various major diseases, especially malignant tumors. Disruptions in the glycosylation pathway in tumor cells lead to abnormal glycan structures in glycoproteins, forming so-called "abnormal glycan glycoproteins," such as core fucosylation, sialylation, and O-glycan truncation. These specific abnormal glycan structures have become highly promising biomarkers for early tumor diagnosis, prognostic assessment, and monitoring of treatment efficacy.

[0003] Currently, the main techniques for detecting abnormal glycan glycoproteins include mass spectrometry, chromatography, and immunological methods. Among these, lectin-based detection methods have attracted widespread attention due to their ability to specifically recognize glycan structures rather than the protein backbone, as well as their relatively low cost and ease of operation. Lectins are a class of non-immunogenic glycan-binding proteins that can bind to specific monosaccharide or oligosaccharide structures with high specificity and reversibility.

[0004] However, existing lectin-based detection technologies, especially lectin compositions for capturing targets, still have significant shortcomings. First, the use of a single lectin is a major technical bottleneck. Because a single lectin is typically specific to only one or a few glycan structures, and tumor-associated abnormal glycan types are highly heterogeneous, using a single lectin cannot comprehensively cover a wide range of important abnormal glycan structures, resulting in low detection sensitivity and high false negative rates, making it difficult to meet the clinical need for broad-spectrum screening.

[0005] Secondly, to address the issue of narrow coverage of a single lectin, a strategy of simply mixing multiple lectins has emerged in the field. However, this simple physical mixing has inherent drawbacks: different lectins may exhibit competitive or inhibitory binding rather than synergistic effects; if the binding affinity between lectins differs too much, lectins with excessively strong affinity may occupy the main binding sites, while lectins with moderate affinity cannot function effectively, resulting in a decrease rather than an increase in the capture efficiency of certain key abnormal glycans, affecting the accuracy and stability of the detection.

[0006] In addition, lectins are proteins and have poor stability in complex detection environments. They are easily hydrolyzed by proteases or aggregated and inactivated, which directly affects the shelf life of the kit and the repeatability of the test results.

[0007] Based on this, this application provides a lectin composition for the detection of abnormal glycan glycoproteins. Summary of the Invention

[0008] Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a lectin composition for detecting abnormal glycan glycoproteins. By adjusting the proportion of lectins, specificity and broad-spectrum activity are balanced, avoiding the problems of insufficient binding sites or excessive non-specific binding of a single lectin.

[0009] Technical solution To achieve the above objectives, the present invention provides the following technical solution: A lectin composition for detecting abnormal glycan glycoproteins comprises the following raw materials in parts by weight: 50-60 parts of main lectin, 30-40 parts of auxiliary lectin, 50-60 parts of lectin dilution solution, 0.5-1.5 parts of modifier, 100 parts of cross-linking agent, and 160-200 parts of biotin labeling solution; The main lectin was *Dictyophora indica* lectin.

[0010] Furthermore, the binding dissociation constant of lectin to abnormal glycan proteins is 10. -6 -10 -7 Between mol / L.

[0011] Furthermore, the auxiliary lectin is at least one of peanut lectin, lentil lectin, wheat germ lectin, type E red kidney bean lectin, and datura lectin.

[0012] Furthermore, the modifier is a methoxy polyethylene glycol active ester with a relative molecular weight of 5000-8000 Da.

[0013] Furthermore, the crosslinking agent is carbodiimide.

[0014] Furthermore, the biotin-labeled solution is specifically prepared by the following steps: A1. Add the biotin derivative to the activation solvent dimethyl sulfoxide and vortex for 10-20 minutes. Then add it to the lectin dilution solution at a volume ratio of 1:999 and stir at a speed of 5-10 rpm until homogeneous. Adjust the pH of the lectin dilution solution to 7.2-7.4 using NaOH solution. A2. Filter the system obtained in step A1 through a 0.22 μm filter membrane to remove any possible particulate impurities and microorganisms, dispense it into sterile centrifuge tubes, and store at 4°C in the dark to obtain the biotin-labeled solution.

[0015] In the above technical solution, the biotin derivative is pre-dissolved in an activating solvent to solve the problem of its low water solubility, and then diluted with phosphate buffer to avoid lectin denaturation that may be caused by high concentration of activating solvent.

[0016] Further, in step A1, the biotin derivative is N-hydroxysuccinimide biotin and / or long-arm biotin-NHS.

[0017] Furthermore, in step A1, the mass-to-volume ratio of the biotin derivative to the activating solvent is (2-4) mg: 1 mL.

[0018] Further, the lectin diluent contains 0.5-1.0 g of NaH₂PO₄·2H₂O, 2-3 g of Na₂HPO₄·12H₂O, and provides Zn. 2+ 0.01-0.02g of soluble zinc salt, providing Mg 2+ 0.2-0.4 g of soluble magnesium salt in phosphate buffer.

[0019] Furthermore, the lectin composition for detecting abnormal glycan glycoproteins is specifically prepared by the following steps: S1. Weigh the main lectin and auxiliary lectin according to the formula, add the lectin diluent, and stir the system at 30-90 rpm at 0-4℃ to obtain a mixed lectin solution. S2. Add the modifier and crosslinking agent of the specified mass to the mixed lectin solution obtained in step S1. React the system at room temperature for 4-5 hours, maintaining a stirring speed of 30-90 rpm during the reaction. Then, perform column chromatography separation using Sephadex G-50 dextran gel, collect the first elution peak, and obtain the pretreated mixed lectin. S3. Mix the biotin-labeled solution of the specified proportions with the pretreated mixed lectin and react at room temperature in the dark for 2-3 hours, stirring every 10-15 minutes during the reaction. Remove unbound free biotin by dialysis. The dialysate is a diluted lectin solution. Dialyze at 0-4°C for 12-24 hours, changing the dialysate 3-4 times to obtain the lectin composition.

[0020] Beneficial technical effects The technical solution of this invention adopts a multi-lectin synergistic enhancement method. The orange-yellow reticulocyte lectin has a high specific binding ability to the Galβ-1,3GalNAc structure and serves as the master lectin to ensure detection targeting. When combined with auxiliary lectins such as peanut lectin and wheat germ lectin, it can better identify other characteristic structures of tumor-related abnormal glycans. It works synergistically with the master lectin to expand the detection coverage and improve the capture efficiency of abnormal glycan glycoproteins.

[0021] In this invention, methoxylated polyethylene glycol active ester binds to lectins via a cross-linking agent, forming a hydrophilic protective layer on the surface of the lectin molecules. This reduces the hydrolytic effect of proteases on the lectin, extending its half-life in the detection system. After treatment, the solubility of the lectin is improved, preventing aggregation during reconstitution after drying, ensuring the homogeneity of the reaction system, and improving detection repeatability. Detailed Implementation

[0022] 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 are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0023] The raw materials used in the embodiments of this invention are shown below, and all reagents used are analytical grade.

[0024] Datura lectin (DSA), peanut lectin (PNA), lentil lectin (LCA), wheat germ lectin (WGA), E-type red kidney bean lectin (E-PHA), and L-type red kidney bean lectin (L-PHA) were all purchased from Vector Laboratories. Orange-yellow reticulocyte lectin (AOL) was purchased from Tokyo Chemical Industry Co., Ltd., Japan.

[0025] Methoxylated polyethylene glycol active ester (mPEG-NHS): purchased from Beijing Kailai Tiancheng Biotechnology Co., Ltd.

[0026] Carbodiimide (EDC): Purchased from Sigma-Aldrich.

[0027] N-hydroxysuccinimide biotin (NHS-Biotin): purchased from Thermo Fisher Scientific.

[0028] Sephadex G-50 dextran gel: purchased from Cytiva.

[0029] Example 1 A method for preparing a lectin composition for detecting abnormal glycan glycoproteins specifically includes the following steps: S1. Weigh out 50 parts by weight of *Dictyophora indicum* lectin (main lectin) and 40 parts by weight of auxiliary lectin. The auxiliary lectin is composed of peanut lectin and E-type red kidney bean lectin mixed at a mass ratio of 3:1. Add the weighed lectin to 60 parts by weight of lectin dilution solution. The lectin dilution solution contains 0.8g of NaH2PO4·2H2O, 2.5g of Na2HPO4·12H2O, and 0.015g of ZnCl2 (providing Zn). 2+ ), MgCl2 0.3g (providing Mg 2+ Add water to a final volume of 1 L to prepare a phosphate buffer solution. Place the system in a 2°C ice-water bath and stir at 50 rpm for 40 minutes to obtain a mixed lectin solution.

[0030] S2. Add 1.0 part of a modifier and 100 parts of a crosslinking agent to the mixed lectin solution obtained in step S1. The modifier is a methoxylated polyethylene glycol active ester with a relative molecular weight of 6000 Da, and the crosslinking agent is carbodiimide. React the system at room temperature (25±2℃) with a stirring rate of 50 rpm for 4.5 hours. Subsequently, perform column chromatography using a Sephadex G-50 dextran gel, collect the first elution peak, and obtain the pretreated mixed lectin.

[0031] S3. Mix 180 portions of biotin-labeled solution with the pretreated mixed lectin obtained in step S2, and react at room temperature in the dark for 2.5 hours, stirring every 12 minutes. The biotin-labeled solution was prepared by adding 3.0 mg of N-hydroxysuccinimide biotin to 1.0 mL of the activation solvent dimethyl sulfoxide, vortexing for 15 minutes, and then slowly adding it to the lectin dilution at a volume ratio of 1:999, stirring at 8 rpm until homogeneous, and adjusting the pH of the system to 7.3 using 0.1 M NaOH solution; finally, filter the resulting solution through a 0.22 μm filter membrane, aliquot, and store at 4°C in the dark. After the reaction, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 10 kDa, use the lectin dilution as the dialysate, and dialyze at 4°C for 20 hours, changing the dialysate 3 times during the process to completely remove unbound free biotin, thus obtaining the final lectin composition.

[0032] Example 2 A method for preparing a lectin composition for detecting abnormal glycan glycoproteins specifically includes the following steps: S1. Weigh out 55 parts by weight of *Dictyophora indicum* lectin (main lectin) and 35 parts by weight of auxiliary lectin, wherein the auxiliary lectin is a mixture of lentil lectin and wheat germ lectin at a mass ratio of 2:1. Add the weighed lectin to 55 parts by weight of lectin dilution solution, wherein the lectin dilution solution is a phosphate buffer solution containing 0.7 g of NaH2PO4·2H2O, 2.8 g of Na2HPO4·12H2O, 0.018 g of ZnCl2, and 0.25 g of MgCl2, and dilute with water to a final volume of 1 L. Place the system at 4°C and stir at 80 rpm for 30 minutes to obtain a mixed lectin solution.

[0033] S2. Add 1.2 parts of a modifier and 100 parts of a crosslinking agent to the mixed lectin solution obtained in step S1. The modifier is a methoxylated polyethylene glycol active ester with a relative molecular weight of 7500 Da, and the crosslinking agent is carbodiimide. React the system at room temperature (25±2℃) with a stirring rate of 80 rpm for 4 hours. Subsequently, perform column chromatography using a Sephadex G-50 dextran gel, collect the first elution peak, and obtain the pretreated mixed lectin.

[0034] S3. Mix 190 portions of biotin-labeled solution with the pretreated mixed lectin obtained in step S2, and react at room temperature in the dark for 3 hours, stirring every 15 minutes. The biotin-labeled solution was prepared by adding 3.5 mg of long-arm biotin-NHS to 1.0 mL of dimethyl sulfoxide and vortexing for 12 minutes. Then, add the lectin diluent at a volume ratio of 1:999, stir at 6 rpm until homogeneous, and adjust the pH of the system to 7.4 using 0.1 M NaOH solution. Finally, filter the resulting solution through a 0.22 μm filter membrane, aliquot, and store at 4°C in the dark. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 10 kDa, use the lectin diluent as the dialysate, and dialyze at 2°C for 18 hours, changing the dialysate 4 times during the process to obtain the final lectin composition.

[0035] Example 3 A method for preparing a lectin composition for detecting abnormal glycan glycoproteins specifically includes the following steps: S1. Weigh out 60 parts by weight of *Dictyophora indicum* lectin (main lectin) and 30 parts by weight of auxiliary lectin, wherein the auxiliary lectin is datura lectin. Add the weighed lectin to 50 parts by weight of lectin dilution solution, wherein the lectin dilution solution contains 1.0 g of NaH2PO4·2H2O, 2.2 g of Na2HPO4·12H2O, 0.012 g of ZnCl2, and 0.35 g of MgCl2, and dilute with water to a final volume of 1 L to prepare a phosphate buffer solution. Place the system in an ice-water bath at 0°C and stir at 90 rpm for 35 minutes to obtain a mixed lectin solution.

[0036] S2. Add 0.8 parts of a modifier and 100 parts of a crosslinking agent to the mixed lectin solution obtained in step S1. The modifier is a methoxylated polyethylene glycol active ester with a relative molecular weight of 5000 Da, and the crosslinking agent is carbodiimide. React the system at room temperature (25±2℃) with a stirring rate of 90 rpm for 5 hours. Subsequently, perform column chromatography using a dextran gel Sephadex G-50, collect the first elution peak, and obtain the pretreated mixed lectin.

[0037] S3. Mix 200 portions of biotin-labeled solution with the pretreated mixed lectin obtained in step S2, and react at room temperature in the dark for 2 hours, stirring every 10 minutes. The biotin-labeled solution was prepared by the following method: 2.5 mg of N-hydroxysuccinimide biotin and 1.0 mg of long-arm biotin-NHS were mixed and added to 1.0 mL of dimethyl sulfoxide, vortexed for 18 minutes, and then added to the lectin dilution solution at a volume ratio of 1:999. The mixture was stirred at 10 rpm until homogeneous, and the pH of the system was adjusted to 7.2 using 0.1 M NaOH solution. Finally, the resulting solution was filtered through a 0.22 μm filter membrane, aliquoted, and stored at 4°C in the dark. After the reaction was completed, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa. Using the lectin dilution solution as the dialysate, dialyzing was performed at 0°C for 24 hours, changing the dialysate 3 times during the process, to obtain the final lectin composition.

[0038] Comparative Example 1 The difference between this comparative example and Example 2 is that the mass ratio of lectin to biotin is 1:1.

[0039] Comparative Example 2 The difference between this comparative example and Example 2 is that the mass ratio of lectin to biotin is 1:5.

[0040] Comparative Example 3 The difference between this comparative example and Example 2 is that the E-type red kidney bean lectin in the auxiliary lectin is replaced by an equal mass of L-type red kidney bean lectin (L-PHA), while the remaining components and preparation steps are the same as in Example 2.

[0041] Comparative Example 4 The difference between this comparative example and Example 2 is that the auxiliary lectin composed of lentil lectin and wheat germ lectin is not used. Instead, carnation lectin (GNA, which is specific to high mannose structures) is used as the sole substitute, and the amount is kept at 40 parts. The remaining components and preparation steps are the same as in Example 2.

[0042] The performance of the lectin compositions prepared in the examples and comparative examples is now being tested. The specific test methods are as follows: Initial binding activity assay: Standard aberrant glycan glycoproteins (Galβ-1,3GalNAc) were immobilized on the chip surface using a surface plasmon resonance (SPR) instrument. The lectin compositions prepared in each example and comparative example were diluted to the same concentration and flowed through the chip, and their binding response values ​​were measured. The initial relative binding activity (%) of each sample was calculated using the response value of a commercially available lectin composition (reference standard) with recognized superior performance as 100% as a baseline.

[0043] Stability testing: The lectin compositions obtained from different groups were stored at 37°C, and samples were taken at 14 and 21 days. The binding activity of the detector against standard abnormal glycan proteins was compared to assess the accelerated stability of the products. The lectin compositions from different groups were subjected to 5 and 10 freeze-thaw cycles at -20°C and room temperature, respectively. After each cycle, their binding activity was measured to compare the freeze-thaw stability of the products. The activity evaluation index was set at 100% binding activity on day 0. The relative activity after different time points or cycles was determined, and a relative activity ≥85% was considered stable and qualified.

[0044] The specific results of the stability test are shown in Table 1.

[0045] Table 1

[0046] As shown in Table 1, the initial binding activity of all embodiments exceeded that of the reference product, and the relative activity remained above 94% in both accelerated and freeze-thaw stability tests. This fully demonstrates that the lectin composition provided by this invention, employing a specific combination of a primary lectin and a preferred auxiliary lectin, and with optimized modification, cross-linking, and biotin labeling processes, exhibits excellent detection sensitivity and physicochemical stability.

[0047] The results from Comparative Examples 1 and 2 show that insufficient labeling in Comparative Example 1 led to inadequate signal amplification and a significant decrease in initial binding activity. In Comparative Example 2, steric hindrance severely interfered with the binding of lectin to the target glycan, resulting in unsatisfactory initial binding activity. Furthermore, excessive biotin introduced non-specific adsorption, further reducing stability. Therefore, the lectin and biotin used in this technical solution can achieve the desired effect of satisfying the subsequent streptavidin signal amplification requirements without causing excessive steric hindrance due to over-labeling.

[0048] As shown in Comparative Example 3, using L-type red kidney bean lectin instead of E-type red kidney bean lectin resulted in poor synergy between the sugar chain structure it recognizes and the core structure primarily recognized by *Reticulitermes auricula* lectin, leading to a mismatch in affinity levels and significantly lower initial binding activity and stability compared to Example 2.

[0049] As shown in Comparative Example 4, the use of carnation lectin completely replaces the auxiliary lectin of this invention. The high-mannose structure specifically bound by carnation lectin has a weak correlation with the core features of tumor-associated abnormal glycan chains, and cannot form an effective synergistic effect with *Dictyophora indica* lectin. Instead, it may generate ineffective competition, leading to a sharp decline in initial binding activity and stability, resulting in the worst performance.

[0050] The results of Comparative Examples 3 and 4 show that not all lectins can effectively synergize with the main lectin. Only the lectin selected in this invention can form the optimal pairing with the *Dictyophora indica* lectin, achieving the goal of broad-spectrum, stable, and efficient detection.

[0051] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0053] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.

Claims

1. A lectin composition for detecting abnormal glycan glycoproteins, characterized in that, Includes the following quantities of raw materials: 50-60 parts of main lectin, 30-40 parts of auxiliary lectin, 50-60 parts of lectin dilution solution, 0.5-1.5 parts of modifier, 100 parts of cross-linking agent, and 160-200 parts of biotin labeling solution; The main lectin was *Dictyophora indica* lectin.

2. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The binding dissociation constant of the auxiliary lectin to the abnormal glycan is 10. -6 -10 -7 Between mol / L.

3. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The auxiliary lectin is at least one of peanut lectin, lentil lectin, wheat germ lectin, type E red kidney bean lectin, and datura lectin.

4. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The modifier is a methoxy polyethylene glycol active ester with a relative molecular weight of 5000-8000 Da.

5. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The crosslinking agent is carbodiimide.

6. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The biotin-labeled solution is prepared by the following steps: A1. Add the biotin derivative to the activation solvent dimethyl sulfoxide and vortex for 10-20 minutes. Then add it to the lectin dilution solution and stir at 5-10 rpm until homogeneous. Adjust the pH of the lectin dilution solution to 7.2-7.4 using NaOH solution. A2. Filter the system obtained in step A1 through a filter membrane to remove any possible particulate impurities and microorganisms, dispense it into sterile centrifuge tubes, and store it at 0-4℃ in the dark to obtain the biotin-labeled solution.

7. The lectin composition for detecting abnormal glycan glycoproteins according to claim 6, characterized in that, In step A1, the biotin derivative is N-hydroxysuccinimide biotin and / or long-arm biotin-NHS.

8. The lectin composition for detecting abnormal glycan glycoproteins according to claim 6, characterized in that, In step A1, the mass-to-volume ratio of the biotin derivative to the activating solvent is (2-4) mg: 1 mL.

9. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The lectin diluent contains 0.5-1.0 g of NaH₂PO₄·2H₂O, 2-3 g of Na₂HPO₄·12H₂O, and provides Zn. 2+ 0.01-0.02g of soluble zinc salt, providing Mg 2+ 0.2-0.4 g of soluble magnesium salt in phosphate buffer.

10. The lectin composition for detecting abnormal glycan glycoproteins according to claim 1, characterized in that, The lectin composition for detecting abnormal glycan glycoproteins is prepared by the following steps: S1. Weigh the main lectin and auxiliary lectin according to the formula, add the lectin diluent, and stir the system at 30-90 rpm at 0-4℃ to obtain a mixed lectin solution. S2. Add the modifier and crosslinking agent in the formula mass fraction to the mixed lectin solution obtained in step S1, and react the system at room temperature for 4-5 hours, maintaining a stirring rate of 30-90 rpm during the reaction; then perform column chromatography separation, collect the first elution peak, and obtain the pretreated mixed lectin. S3. Mix the biotin-labeled solution of the specified proportions with the pretreated mixed lectin and react at room temperature in the dark for 2-3 hours, stirring every 10-15 minutes during the reaction. Remove unbound free biotin by dialysis. The dialysate is a diluted lectin solution. Dialyze at 0-4°C for 12-24 hours, changing the dialysate 3-4 times to obtain the lectin composition.

Images