A combined reagent for detecting aspergillus galactomannan, a kit and a detection method thereof

Abstract

The application discloses a combined reagent for detecting aspergillus galactomannan, a kit and a detection method thereof, and belongs to the technical field of biomedical detection. The combined reagent comprises a serum sample processing liquid and a protein denaturant termination liquid. The serum sample processing liquid comprises Bacillus subtilis protease with a concentration of 0.05-0.50 mg / mL, CTAC with a concentration of 0.005-0.05 mg / mL, a surfactant and weak alkaline buffer with a pH of 9.3-9.7. The protein denaturant termination liquid comprises a phosphate buffer, a protease inhibitor PMSF and sodium casein, etc. Through the synergistic effect of the protease and the denaturant under mild weak alkaline conditions, the application can efficiently dissociate the antigen-antibody complex in the serum and irreversibly inactivate the antibody, while keeping the activity of the antigen. The method is simple to operate, does not need heating, centrifugation or strong corrosive reagents, the processed sample does not precipitate or gel, and the accuracy, sensitivity and reliability of the detection are significantly improved, so the method is suitable for clinical promotion.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a combined reagent, kit, and detection method for the detection of Aspergillus galactomannan. Background Technology

[0002] Aspergillus galactomannan (GM) is a unique component of the cell wall of Aspergillus fungi, which is released into the serum of infected patients during infection. In invasive Aspergillus infections, the level of Aspergillus galactomannan in the patient's serum is significantly elevated, thus it is used as an important serological marker for diagnosing invasive aspergillosis (IA). Immunological methods such as enzyme-linked immunosorbent assay (ELISA) are commonly used techniques for detecting this marker.

[0003] The general reagents for GM detection are built around the "antibody-antigen" immune reaction, while the main differences and innovations among different technology platforms and manufacturers often lie in the "sample pretreatment" stage. How to release GM from complex serum samples more efficiently, gently, and easily, while removing various interferences, is the key to improving detection performance (such as sensitivity, specificity, and operability).

[0004] Aspergillus galactomannan in serum often binds to antibodies produced by the host in vivo, forming stable antigen-antibody complexes. These complexes mask antigenic epitopes, severely hindering antibody capture and recognition of free antigens, leading to decreased sensitivity in immunological tests and even false negative results. Therefore, effective pretreatment of serum samples before testing to dissociate the complexes and release the Aspergillus galactomannan antigen, while simultaneously ensuring antibody inactivation to avoid subsequent interference, is crucial for improving test accuracy.

[0005] The existing method for dissociating polysaccharide antigen-antibody complexes mainly involves heat treatment of serum samples with EDTANa2. The procedure involves mixing the serum sample with an EDTANa2 solution, heating at 100-120°C for 3-6 minutes, followed by centrifugation at 4°C for 10-15 minutes, and then collecting the supernatant for immunological analysis. The principle behind this method is that EDTANa2 chelates serum proteins (including polysaccharide-binding proteins), and the high temperature denatures the proteins, thus dissociating the antigen-antibody complex. Centrifugation separates the denatured proteins from the dissociated polysaccharide antigens; the dissociated polysaccharide antigens remain in the supernatant, while the denatured proteins are present at the bottom of the centrifuge tube. This method relies on intense denaturation at temperatures above 100°C, is cumbersome and time-consuming, requires centrifugation equipment, and the high temperature may cause irreversible damage to the polysaccharide antigen structure.

[0006] Other methods include the combined use of strong acidic releasing agents and alkaline neutralizing agents. The procedure involves first treating the serum with a strong acidic releasing agent, causing the antigen-antibody complex to dissociate under strong acidic conditions, and then denaturing and inactivating the antibody under strong alkaline conditions, thus releasing the antigen. Finally, an alkaline neutralizing agent is used to neutralize the acidic serum mixture to meet the requirements of immunological detection. However, this method involves many reagents, and improper operation can lead to residual strong acid or alkali, affecting the test results. The reagents are also highly acidic or alkaline, posing safety risks and prone to degradation (e.g., evaporation upon opening reduces the effective concentration, and absorption of carbon dioxide from the air reduces alkalinity). Furthermore, due to the variability of different samples, their reactions to strong acids vary, and some samples may exhibit visible protein precipitation or gelation, affecting the immunological test results. While the above methods avoid heating and centrifugation, they still rely on strong acid and alkali treatment, resulting in complex operation, highly corrosive reagents, and potential impact on antigen activity. Drastic pH fluctuations (from strong acid to strong alkali) can also damage the antigenic epitopes of Aspergillus galactomannan, affecting detection sensitivity.

[0007] In summary, there is an urgent need in the field for a serum sample pretreatment method that is simple to operate, has mild conditions, high safety, good stability, can dissociate antigen-antibody complexes in serum, protects the antigen, and does not affect subsequent detection. Summary of the Invention

[0008] This invention provides a simple, mild, and highly safe combination reagent, kit, and detection method for the detection of Aspergillus galactomannan. The core concept lies in the discovery and utilization of the significant difference in tolerance between Aspergillus galactomannan and its specific antibodies to protein denaturants and proteases under weakly alkaline conditions. This led to the design of a novel "mild biochemical synergistic inactivation" technical route. This route completely abandons traditional drastic denaturation methods, achieving efficient and irreversible clearance of interfering antibodies through the synergistic effect of three mechanisms (Bacillus subtilis protease, CTAC, and weakly alkaline environment), while ensuring the complete recovery of the target antigen.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a combined reagent for the detection of Aspergillus galactomannan, comprising a serum sample processing solution and a protein denaturant termination solution;

[0011] The serum sample processing solution consists of the following components:

[0012] Bacillus subtilis protease at a concentration of 0.05-0.50 mg / mL;

[0013] CTAC at a concentration of 0.005-0.05 mg / mL;

[0014] The pH of the serum sample processing solution is 9.3-9.7. In some embodiments, the pH can be 9.3, 9.4, 9.5, 9.6, 9.7, etc. The serum sample processing solution of this scheme is generally weakly alkaline. The antigen-antibody complex dissociates under weakly alkaline conditions. At the same time, the antibody loses its activity under the combined action of the weakly alkaline environment and the protein denaturing agent (CTAC). Aspergillus galactomannan is not easily inactivated in weakly alkaline conditions, thereby achieving the separation of Aspergillus galactomannan from the bound antibody.

[0015] This invention also discloses a protein denaturant termination solution, adapted to terminate the reaction of serum sample processing solution under weakly alkaline conditions in this invention. Specifically, the protein denaturant termination solution comprises the following components:

[0016] Phosphate buffer solution with a pH of 6.0-7.0;

[0017] PMSF, a protease inhibitor, at a concentration of 0.001-0.005 mg / mL;

[0018] Sodium caseinate at a concentration of 0.25-3.0% (w / v);

[0019] EDTANa2 at a concentration of 0.001-0.010% (w / v).

[0020] In some implementations, the pH of the serum sample is 9.5 to achieve a higher recovery rate.

[0021] In some implementations, the serum sample processing solution may also contain a weakly alkaline buffer, such as CHES buffer, to obtain a serum sample processing solution with a more stable pH.

[0022] In some embodiments, the serum sample processing solution and / or the protein denaturant termination solution further contain a surfactant at a concentration of 0.02-0.2% (v / v), said surfactant being Tween 20 or Tween 80.

[0023] This improves the efficiency and stability of dissociation.

[0024] In some embodiments, the serum sample processing solution and / or the protein denaturant termination solution also contain a preservative, ProClin 300, at a concentration of 0.05-0.2% (v / v). This inhibits the growth of bacteria, fungi, and molds in the reagent, thus extending the reagent's shelf life.

[0025] The present invention also provides an Aspergillus galactomannan detection kit, which comprises the above-described combined reagents. The kit preferably comprises a kit A and a kit B, wherein the serum sample processing solution is added to kit A, and the protein denaturing agent stop solution is added to kit B.

[0026] In some embodiments, the B box also contains an HRP-labeled Aspergillus galactomannan antibody, preferably at a concentration of 100 ng / mL.

[0027] This invention provides a method for detecting Aspergillus galactomannan using the above-mentioned combined reagents or kits, comprising the following steps:

[0028] S1 sample processing:

[0029] 1) Mix the serum sample to be tested with the serum sample processing solution, incubate, and prepare the test solution;

[0030] S2 Termination Steps:

[0031] The HRP-labeled Aspergillus galactomannan antibody was diluted with the protein denaturant stop solution to prepare an enzyme-labeled antibody solution, and the test solution was added to the enzyme-labeled antibody solution, mixed well, and incubated to prepare a stop serum solution.

[0032] S3 testing steps:

[0033] The stop serum solution was measured using an enzyme-linked immunosorbent assay (ELISA).

[0034] Preferably, in step S1 sample processing, the volume ratio of serum sample to serum sample processing solution is 1:0.5~1.5.

[0035] In some embodiments, the ratio of serum sample to serum sample processing solution is 1:0.5, 1:0.75, 1:1, 1:1.25, or 1:1.5. As a further preferred embodiment, the volume ratio of serum sample to serum sample processing solution is 1:1.

[0036] Preferably, in the S1 sample processing, the reaction temperature is room temperature. In this scheme, the room temperature range is defined as 20~30℃.

[0037] Compared with the prior art, the beneficial effects of the present invention are:

[0038] The technical effect of this invention stems from its unique serum sample processing solution of "weakly alkaline CHES buffer + Bacillus subtilis protease + CTAC" and the combined reagent system consisting of a matching protein denaturing agent termination solution. Its beneficial effects are specifically and directly reflected in the following aspects:

[0039] 1. Efficient and irreversible removal of antibody interference was achieved under mild conditions. As shown in Example 1 (Tables 1 and 2), CTAC and Bacillus subtilis protease exhibited a significant synergistic inhibitory effect on the activity of Aspergillus galactomannan antibody in a weakly alkaline environment of pH 9.3-9.7, while the Aspergillus galactomannan antigen itself remained stable under these conditions. This allows the treatment solution to efficiently dissociate the antigen-antibody complex without the use of strong acids, strong alkalis, or high temperatures, ensuring that the released antibody is irreversibly inactivated and preventing it from interfering again in subsequent immunoassay steps.

[0040] 2. This scheme achieves synergistic effects through the three components: a weakly alkaline environment weakens antibody stability, CTAC disrupts antibody structure, and Bacillus subtilis protease hydrolyzes the main peptide chain of the antibody. The three components launch an "attack" on different weak points of the antibody, resulting in a synergistic dissociation effect of "1+1+1>3". Aspergillus galactomannan is well preserved under these mild conditions due to its polysaccharide properties.

[0041] 3. Antigen recovery rates close to theoretical values ​​were obtained. According to Example 3 (Table 4), using the complete scheme (combination 6) of this invention to process serum samples of different concentrations, the detection recovery rate remained stable between 98% and 100%. This indicates that this processing scheme effectively removes interference while maximally protecting the integrity and immune activity of the target antigen, thus laying a solid foundation for high accuracy in detection results.

[0042] 4. The "one-step" operation simplifies the process and eliminates secondary interference. Compared to the two-step "acid treatment-base neutralization" method in the prior art, this invention only requires mixing and incubating the sample with a single treatment solution. This not only simplifies the operation but also fundamentally avoids pH shifts or localized concentration unevenness that may be caused by improper neutralization, thereby eliminating the risk of detection errors introduced by these factors. The accompanying stop solution further ensures the compatibility of the treated sample with the subsequent detection system.

[0043] 5. The processing is free from precipitation and gelation, ensuring the uniformity and reliability of the detection. By avoiding a strong acid environment, the processing of this invention will not cause uncontrolled acid denaturation and precipitation of impurities in serum samples. As noted in the background section of the specification, this effectively prevents protein precipitation or gel formation in the sample, thereby avoiding potential obstacles to automated sample loading, liquid circuit systems, or the uniformity of immune reactions caused by these physical phenomena, ensuring a smooth detection process and reliable results.

[0044] 6. The reagent system is stable, facilitating standardization and quality control. Both the treatment and termination solutions of this invention are aqueous solutions with well-defined compositions and stable pH. The weakly alkaline CHES buffer and near-neutral phosphate buffer system, compared to strong acid and strong base reagents, exhibits lower volatility, stronger resistance to interference from atmospheric carbon dioxide, and a longer shelf life. This inherent stability facilitates large-scale reagent production and quality control, ensuring consistency in performance across different batches.

[0045] 7. Optimized parameters are clearly defined, resulting in high reproducibility. As shown in Examples 4, 5, and 6, through systematic optimization of key parameters such as pH, CTAC concentration, protease concentration, incubation time, and temperature, this invention has determined its optimal operating window (e.g., pH 9.3-9.7, CTAC 0.005-0.05 mg / mL). Within this window, the coefficient of variation (CV) of the detection results is less than 10% (see Table 8), demonstrating the excellent reproducibility and robustness of the method.

[0046] In summary, the core effect of the technical solution of this invention lies in the fact that, through a synergistic reaction system with precisely optimized components and processes, it achieves efficient removal of detection interferences and high-fidelity recovery of target antigens without the need for external force (heating, centrifugation) or the intervention of violent chemicals (strong acids, strong alkalis). This simplifies the operation while significantly improving the accuracy, precision and reliability of the detection. Detailed Implementation

[0047] This invention provides a polysaccharide antigen-antibody immune complex dissociation agent. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention. The embodiments of this 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 this invention. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0048] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments. Unless otherwise specified, all percentage concentrations in the present invention documents are weight-volume ratios.

[0049] 1. Example 1: Verification of the difference in tolerance between Aspergillus galactomannan and antibodies to basic and protein denaturing agents

[0050] 1.1 Experimental Methods

[0051] Aspergillus galactomannan (from Tianjin Zhidin Biotechnology Co., Ltd.) was dissolved in pure water and diluted to 10 μg / mL with 0.01M PBS (pH 7.2) as the intermediate stock solution for polysaccharides.

[0052] The Aspergillus galactomannan antibody (from Beijing Bochengda Biotechnology Co., Ltd.) was dissolved in 0.01 M PBS (pH 7.2) and diluted to 10 μg / mL as the intermediate antibody stock solution.

[0053] Solutions with different pH values ​​(pH 7.0, pH 8.0, pH 9.0, pH 10.0, pH 11.0, pH 12.0, pH 13.0) and different concentrations of CTAC (protein denaturant, 0, 0.005, 0.020, 0.100, 0.200 mg / mL) were prepared.

[0054] The above-mentioned polysaccharide intermediate stock solution and antibody intermediate stock solution were diluted 5 times with the above-mentioned pH solution and CTAC solution, and treated at room temperature for 2 h; the treated polysaccharide and antibody solutions were then diluted to 10 ng / mL with 0.1 M PBS (pH 7.2).

[0055] The polysaccharide solution of 10 ng / mL was detected using the Aspergillus galactomannan assay kit (chemiluminescence method) (from DANA (Tianjin) Biotechnology Co., Ltd.), and the antibody solution of 10 ng / mL was detected using an ELISA plate pre-coated with Aspergillus galactomannan.

[0056] 1.2 Results Analysis

[0057] Table 1: Effects of different pH treatments on the activity of Aspergillus galactomannan and its antibodies

[0058]

[0059] Table 2: Effects of different CTAC concentrations on the activity of Aspergillus galactomannan and its antibodies

[0060]

[0061] This embodiment reveals for the first time, using quantitative data, the fundamental differences in the tolerance of Aspergillus galactomannan and its specific antibody to weak alkalinity and protein denaturants. The data (see Tables 1 and 2) show that within the pH range of 8.0–11.0, antibody activity decreases sharply with increasing pH, while GM antigen activity remains stable. Similarly, at CTAC concentrations as low as 0.005 mg / mL, antibody activity is significantly impaired, while GM antigen activity is almost unaffected. This provides a crucial theoretical basis and precise starting point for designing mild treatment schemes that "target antibodies and protect antigens," whereas existing technologies have never considered or utilized this difference in biochemical tolerance between GM antigen and antibody. It should be noted that since the protein denaturant stop solution is used to dilute the HRP-labeled Aspergillus galactomannan antibody, CTAC cannot be added to the protein denaturant stop solution (due to concerns about long-term storage stability). Considering that the pretreated sample and the HRP-labeled Aspergillus galactomannan antibody enzyme-labeled antibody diluted with the protein denaturant stop solution need to be mixed in subsequent operations, a protein protection component needs to be added to the protein denaturant stop solution to neutralize the influence of CTAC.

[0062] Example 2: Preparation of a combined reagent for the detection of Aspergillus galactomannan.

[0063] 2.1 Preparation of serum sample processing solution

[0064] Bacillus subtilis protease at a concentration of 0.05~0.50 mg / mL;

[0065] A protein denaturing agent with a concentration of 0.005~0.05 mg / mL, wherein the protein denaturing agent is CTAC;

[0066] A surfactant with a concentration of 0.02~0.2% (v / v), wherein the surfactant is either Tween20 or Tween80;

[0067] ProClin300 at concentrations of 0.05–0.2% (v / v);

[0068] CHES buffer solution, pH 9.3-9.7.

[0069] 2.2 Prepare the protein denaturant termination solution.

[0070] Phosphate buffer solution with a concentration of 0.02–0.10 M;

[0071] PMSF, a protease inhibitor, was used at concentrations of 0.001–0.005 mg / mL.

[0072] Sodium caseinate with a concentration of 0.25–3.0% (v / v);

[0073] Tween 80 or Tween 20 at a concentration of 0.02~0.2% (v / v);

[0074] ProClin300 at concentrations of 0.05–0.2% (v / v);

[0075] EDTANa2 at concentrations of 0.001~0.010% (v / v)

[0076] The pH value is 6.0~7.0.

[0077] In the serum sample processing solution of this embodiment:

[0078] 1. CTAC is a protein denaturant. The activity of an antibody depends on its specific three-dimensional conformation (secondary, tertiary, and quaternary structures), and conformational stability is maintained by various intramolecular forces: electrostatic interactions (such as attraction between positive and negative charges), hydrophobic interactions (aggregation of hydrophobic groups), hydrogen bonds, disulfide bonds, etc. Protein denaturants disrupt these forces, leading to changes in antibody conformation and ultimately affecting its activity.

[0079] 2. Antibodies are long chains formed by amino acids linked by peptide bonds (-CO-NH-). Their biological activity depends on their intact three-dimensional conformation. Bacillus subtilis protease hydrolyzes proteins by breaking peptide bonds, thereby destroying antibody activity.

[0080] 3. Antibodies are a class of immunoglobulins (Ig) with a highly specific ability to recognize and bind antigens. Their biological activities (such as antigen-binding capacity and effector function) depend entirely on their intact and stable three-dimensional conformation. Alkaline solutions (high pH environment) disrupt the key forces maintaining antibody structure, leading to conformational abnormalities or disintegration, ultimately affecting their activity.

[0081] The binding of antigens and antibodies is the result of the combined effects of hydrogen bonds, electrostatic attraction, hydrophobic interactions, and van der Waals forces, with hydrophobic interactions and hydrogen bonds being the main contributors. Precise complementarity of spatial structures is a prerequisite for the effective functioning of all these forces. The combined action of three reagents (CTAC, Bacillus subtilis protease, and weakly alkaline buffer) leads to changes in the antibody's spatial structure and a weakening or disappearance of its complementarity, thereby significantly reducing the antibody's affinity for the antigen and achieving the dissociation of the antigen-antibody complex.

[0082] Example 3: Effect of different components in serum treatment solution on recovery rate

[0083] Six different combinations of treatment solutions were designed (Table 3) to treat serum samples of known concentrations, and the recovery rates were detected (Table 4).

[0084] Table 3: Serum sample processing solutions with different combinations

[0085]

[0086] Table 4: Recovery results of each component corresponding to Table 3

[0087]

[0088] Data from Example 3 shows that using CTAC alone or simply changing the pH resulted in unsatisfactory recovery rates (combinations 1-5, all with recovery rates below 90%). Only when CTAC was combined with Bacillus subtilis protease under specific weakly alkaline conditions (pH 9.5) (combination 6) did the recovery rates for all tested serum concentrations jump to over 97%. This fully demonstrates the superior effectiveness of the ternary synergistic system employed in this invention.

[0089] It is not a simple superposition of the functions of each component, but rather produces an unexpected synergistic effect.

[0090] Example 4: Optimization of various values ​​in serum sample processing solution.

[0091] 4.1 Validation of pH optimization for serum sample processing solution.

[0092] This embodiment is used to verify the determination of the preferred pH range in the blood processing solution. The pH of the serum sample processing solution plays a crucial role in the dissociation effect of the serum sample. In some embodiments, the overall pH of the serum sample processing solution is adjusted by adjusting a weakly alkaline buffer. Specifically, weakly alkaline buffers with pH values ​​of 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, and 11.0 are prepared. Based on this, the other components of the serum sample processing solution are kept at (0.05 mg / mL CTAC + 0.05 mg / mL protease + 0.02% Tween20 + 0.1% ProClin300). The same positive serum sample of known concentration (reference concentration approximately 12.63 ng / mL) is treated with the above processing solution, and then detected using a self-developed kit. The results (see Table 5) show that the recovery rate of Aspergillus galactomannan was highest (close to 100%) when the pH of the treatment solution was in the range of 9.3 to 9.7, with good repeatability. When the pH was below 9.3 or above 9.7, the recovery rate showed a significant decreasing trend. These experimental data strongly support the preferred pH setting of 9.3–9.7, with pH 9.5 being the optimal value (see Table 5).

[0093] It should be noted that in some embodiments, the weakly alkaline buffer solution is preferably CHES buffer solution. It is important to know that the pH buffering range of CHES buffer solution is typically between 8.6 and 10.0, which highly overlaps with the optimal pH range certified in this method, providing the most stable and effective environment. It should also be noted that when the concentration of CHES buffer solution is below 0.05 M, its buffering effect is negligible. Therefore, while ensuring the pH is within the optimal range, the optimal concentration of CHES buffer solution in this method is between 0.05 and 0.2 M.

[0094] Table 5: Effect of different pH values ​​on sample recovery rate

[0095]

[0096] 4.2 Optimization and validation of CTAC concentration in serum sample processing solution.

[0097] This embodiment is used to verify the determination of the preferred range of CTAC concentration in the blood processing solution. In some embodiments, CTAC concentrations of 0.001, 0.005, 0.01, 0.05, and 0.1 mg / mL were selected, while keeping other components consistent (0.05 mg / mL protease + 0.02% Tween20 + 0.1% ProClin300, pH 9.5). Positive serum samples of known concentrations (reference concentration approximately 12.63 ng / mL) were treated with the above sample processing solution, and then detected using a self-developed Aspergillus galactomannan detection kit (enzyme-linked immunosorbent assay). The results are shown in Table 6: When the CTAC concentration is in the range of 0.005 mg / mL to 0.05 mg / mL, the antigen recovery rate is the highest and most stable (mean approximately 12.76 ng / mL). When the concentration decreased to 0.001 mg / mL, the recovery rate dropped sharply to approximately 6.17 ng / mL, indicating incomplete dissociation. When the concentration increased to 0.10 mg / mL, the recovery rate also decreased to approximately 7.98 ng / mL, indicating that excessively high concentrations may cause interference. These results clearly demonstrate that limiting the CTAC concentration is necessary and that the concentration range of 0.005–0.05 mg / mL is optimal.

[0098] Table 6: Recovery rates at different CTAC concentrations

[0099]

[0100] 4.3 Optimization and validation of protease concentration in sample processing solution

[0101] This embodiment was used to confirm the optimal concentration range (0.05–0.5 mg / mL) of Bacillus subtilis protease. In some embodiments, Bacillus subtilis protease concentrations of 0.025, 0.05, 0.1, 0.3, 0.5, and 1.0 mg / mL were used to prepare serum sample processing solutions under fixed conditions (0.05 mg / mL CTAC + 0.02% Tween 20 + 0.1% ProClin 300, pH 9.5). Testing showed that within the protease concentration range of 0.05 mg / mL to 0.5 mg / mL, the antigen recovery rate was as high as approximately 12.75 ng / mL, and the results were stable. At concentrations below 0.05 mg / mL, the recovery rate was insufficient (approximately 6.47 ng / mL), indicating inadequate enzymatic hydrolysis. At excessively high concentrations (1.0 mg / mL), the recovery rate also decreased significantly (approximately 5.35 ng / mL). This data (see Table 7) effectively demonstrates that limiting the concentration of Bacillus subtilis protease is necessary and that the range of 0.05 mg / mL to 0.5 mg / mL is optimal.

[0102] Table 7: Recovery rates at different protease concentrations

[0103]

[0104] 4.4 Optimization and Validation of Surfactant Concentration in Sample Processing Solution

[0105] It should be noted that in some embodiments, in addition to Tween20, Tween80 can also be used as the surfactant to achieve the same technical effect. Specific verification is as follows:

[0106] 4.4.1 This embodiment is used to verify the optimal concentration range of the surfactant (Tween20) from 0.02% to 0.2% (v / v). In some embodiments, surfactants of 0.00, 0.01, 0.02, 0.05, 0.10, and 0.20% are used to prepare serum sample processing solutions under fixed conditions (0.05 mg / mL CTAC + 0.05 mg / mL Bacillus subtilis protease + 0.1% ProClin300, pH 9.5). Each sample is tested 10 times, and the coefficient of variation (CV) of the 10 test results is calculated. Conditions with low CV (<10%) are selected, and considering cost factors, surfactant concentrations exceeding 0.2% are not used.

[0107] Testing showed that within the surfactant concentration range of 0.02–0.2% (v / v), the coefficient of variation (CV) was <10%, and the results were stable. At concentrations below 0.02 mg / mL, the CV was >10%; furthermore, considering cost factors, surfactant concentrations exceeding 0.2% were no longer tested. Specific data (see Table 8) effectively demonstrate that limiting the surfactant (Tween20) concentration is necessary, and that the 0.02–0.2% (v / v) range is optimal.

[0108] 4.4.2 This embodiment is used to verify the optimal concentration range of the surfactant (Tween80) from 0.02% to 0.2% (v / v). In some embodiments, surfactants of 0.00, 0.01, 0.02, 0.05, 0.10, and 0.20% are used to prepare serum sample processing solutions under fixed conditions (0.05 mg / mL CTAC + 0.05 mg / mL Bacillus subtilis protease + 0.1% ProClin300, pH 9.5). Each sample is tested 10 times, and the coefficient of variation (CV) of the 10 test results is calculated. Conditions with smaller CVs (<10%) are selected, and considering cost factors, surfactant tests exceeding 0.2% are not performed.

[0109] Testing showed that within the surfactant concentration range of 0.02–0.2% (v / v), the coefficient of variation (CV) was <10%, and the results were stable. At concentrations below 0.02 mg / mL, the CV was >10%; furthermore, considering cost factors, surfactant concentrations exceeding 0.2% were no longer tested. Specific data (see Table 8) effectively demonstrate that limiting the surfactant (Tween80) concentration is necessary, and the 0.02–0.2% (v / v) range is optimal.

[0110] Table 8: Effect of different surfactant concentrations on CV

[0111]

[0112] 4.5 Optimization and validation of preservative (ProClin300) concentration in sample processing solution

[0113] This embodiment was used to verify the optimal concentration range of the preservative (0.05~0.2% (v / v)). In some embodiments, ProClin300 was used at concentrations of 0.00, 0.01, 0.02, 0.05, 0.1, and 0.2%, and serum sample processing solutions were prepared under fixed conditions (0.05 mg / mL CTAC + 0.05 mg / mL Bacillus subtilis protease + 0.02% Tween 20, pH 9.5). Testing showed that within the preservative concentration range of 0.05~0.2% (v / v), the colony count was 0, and the results were stable. At concentrations below 0.05%, the colony count (CFU / mL) was greater than 1, indicating insufficient preservative effect. Considering cost factors, tests were not conducted with preservative concentrations exceeding 0.2%. The specific data (see Table 9) effectively demonstrate that limiting the concentration of the preservative (ProClin300) is necessary and that the range of 0.05 to 0.2% (v / v) is optimal.

[0114] Table 9: Validation of the effect of different concentrations of preservatives on sample processing and preservation

[0115]

[0116] Example 5: Optimization of various values ​​of the protein denaturant termination solution

[0117] 5.1 Optimization and validation of phosphate buffer concentration in protein denaturant termination solution

[0118] Protein denaturant stop solutions were prepared using 0.01, 0.02, 0.05, 0.10, and 0.20 M phosphate buffer (pH 6.0–7.0), respectively. The other components of the protein denaturant stop solutions were (0.001 mg / mL PMSF + 0.25% sodium caseinate + 0.02% Tween 20 + 0.05% ProClin 300 + 0.001% EDTANa2). The enzyme-labeled antibody in the ELISA kit was then diluted with the above protein denaturant stop solutions. A self-developed Aspergillus galactomannan assay kit (ELISA) was used to detect positive serum samples at known concentrations (reference concentration approximately 5.49 ng / mL). The results are shown in Table 10. The highest recovery rate (approximately 5.50 ng / mL) was observed at concentrations between 0.02 and 0.10 M, and the results were stable.

[0119] Table 10: Recovery rates at different phosphate concentrations

[0120]

[0121] 5.2 Optimization and Validation of Sodium Casein Concentration in Protein Denaturant Termination Solution

[0122] Protein denaturant stop solutions were prepared using 0.20%, 0.25%, 1.00%, 3.00%, and 5.00% sodium caseinate, respectively. The other components of the protein denaturant stop solutions were (0.001 mg / mL PMSF + 0.25% sodium caseinate + 0.02% Tween 20 + 0.05% ProClin 300 + 0.001% EDTANa2). The enzyme-labeled antibody in the ELISA kit was then diluted with the above protein denaturant stop solutions. A self-developed Aspergillus galactomannan assay kit (ELISA) was used to detect positive serum samples at known concentrations (reference concentration approximately 5.49 ng / mL). The results are shown in Table 11. The best recovery rate (approximately 5.47 pg / mL) was observed within the concentration range of 0.25% to 3.0%.

[0123] Table 11: Optimization of Sodium Casein Concentration

[0124]

[0125] 5.3 Optimization and Validation of Protease Inhibitor Concentration in Protein Denaturant Termination Solution

[0126] Protein denaturant stop solutions were prepared using protease inhibitors (PMSF) at concentrations of 0.0005, 0.001, 0.002, 0.005, and 0.010 mg / mL, respectively. The other components of the protein denaturant stop solutions were (0.02M phosphate + 0.25% sodium caseinate + 0.02% Tween 20 + 0.05% ProClin 300 + 0.001% EDTA Na2, pH 6.0). The enzyme-labeled antibody in the ELISA kit was then diluted with the above protein denaturant stop solutions. A self-developed Aspergillus galactomannan assay kit (ELISA) was used to detect positive serum samples at known concentrations (reference concentration approximately 5.49 ng / mL). As shown in Table 12, the best results were observed within the concentration range of 0.001–0.005 mg / mL (approximately 5.50 ng / mL).

[0127] Table 12: Recovery rates at different PMSF concentrations

[0128]

[0129] Example 6: Validation of optimization of serum sample processing conditions.

[0130] 6.1 To verify the effect of volume ratio on sample recovery, 100 μL of serum sample (reference concentration approximately 5.49 ng / mL) was added to a mixing plate, followed by 50, 75, 100, 125, and 150 μL of serum sample processing solution, respectively. The mixture was shaken for 2 minutes and incubated at room temperature for 30 minutes before detection. As shown in Table 13, the highest recovery rate (99%) was achieved when the serum-to-processing solution volume ratio was 1:1 (i.e., 100 μL each).

[0131] Table 13: Recovery rates at different volume ratios

[0132]

[0133] 6.2 Validation of the effect of temperature on sample recovery rate

[0134] First, add 100 µL of serum sample to the mixing plate, then add 100 µL of sample processing solution, shake for 2 minutes to mix thoroughly, and incubate at 2~8℃, room temperature (20~30℃), and 37℃ for 30 min respectively; take 50 μL of the processed sample and detect it with the self-built Aspergillus galactomannan detection kit (enzyme-linked immunosorbent assay), and calculate the recovery rate. The results are shown in Table 14. The recovery rate was the highest under room temperature conditions (20~30℃).

[0135] Table 14: Recovery rates at different processing temperatures

[0136]

[0137] 6.3 Verification of the effect of incubation time on recovery rate

[0138] First, add 100 µL of serum sample to the mixing plate, then add 100 µL of sample processing solution, and shake for 2 minutes to thoroughly mix. Incubate at room temperature for 5, 10, 15, 20, 30, 40, 50, and 60 min, respectively. Take 50 μL of the processed sample and detect it using a self-developed Aspergillus galactomannan detection kit (ELISA), and calculate the recovery rate. The results are shown in Table 15. The recovery rate is ≥98% when the sample processing time is within the range of 15–30 min.

[0139] Table 15: Recovery rates at different incubation times

[0140]

[0141] Example 7: Verifying the effect of different protein denaturant stop liquid volumes on detection results

[0142] First, add 100 µL of serum sample to the mixing plate, then add 100 µL of sample processing solution, shake for 2 minutes to thoroughly mix, and incubate at room temperature for 30 min. Take 50 μL of the processed sample and detect it using a self-built Aspergillus galactomannan detection kit (enzyme-linked immunosorbent assay). The enzyme-labeled antibody working solution was prepared with protein denaturing agent stop solution, and the sample loading volumes were 20, 35, 50, 65, 80, and 100 µL, respectively. The recovery rate was calculated, and the results are shown in Table 16. The highest recovery rate was observed when the protein denaturing agent stop solution was loaded at a volume of 50 μL.

[0143] Table 16: Recovery rates for different termination liquid volumes

[0144]

[0145] Example 8: Kit Composition

[0146] This protocol also discloses a kit for detecting Aspergillus galactomannan. See Table 17 for details.

[0147] Table 17: Composition and Components of Self-Built Detection Kit

[0148]

[0149] Example 9: Detection Method and Clinical Sample Validation

[0150] This invention also discloses a method for detecting Aspergillus galactomannan, comprising:

[0151] S1 sample processing:

[0152] 1) Add 100µL of serum sample to the mixing plate.

[0153] 2) Add another 100µL of serum sample processing solution to the mixing plate.

[0154] 3) Shake for 2 minutes to mix thoroughly, then incubate at room temperature for 30 minutes.

[0155] 4) Take 50 μL of the processed sample for detection.

[0156] S2 Termination Steps:

[0157] 1) Before the test, remove the kit and place it at room temperature (20-25°C) for at least 30 minutes.

[0158] 2) Open the sealed bag containing the ELISA plate, take out the required ELISA strips according to the sample quantity, seal the unused strips with sealing film and put them back into the sealed bag, store at 2-8℃, and place at room temperature for 20 minutes before the next test.

[0159] 3) Prepare the working washing solution: Dilute the concentrated aspirate 20 times according to the component ratio of 1 part concentrated washing solution to 19 parts sterile deionized water or purified water.

[0160] 4) Dilute the HRP-labeled Aspergillus galactomannan antibody to 250 ng / mL using a protein denaturing agent stop solution to prepare the enzyme-labeled antibody working solution, which will be used as the enzyme-labeled antibody for the kit.

[0161] S3 testing steps:

[0162] The test solution was detected by enzyme-linked immunosorbent assay (ELISA), including...

[0163] a. To prepare a calibration curve set, the specific steps include: first, adding the enzyme-labeled antibody working solution to the ELISA plate pre-coated with Aspergillus galactomannan antibody; then, adding 50 μL of each Aspergillus galactomannan calibrator to the corresponding ELISA plate containing the enzyme-labeled antibody in the order of the standard ae, thus completing the preparation of the calibration curve set.

[0164] b. Prepare the test sample set. The specific steps include: first, add 50 μL of the pretreated test solution to the corresponding enzyme-labeled plate containing the enzyme-labeled antibody, mix well, and then incubate at 37℃ for 60 min to complete the preparation of the test sample set.

[0165] c. Remove the sealing film and wash the microplate. Add at least 300 μL of washing buffer to each well each time, let stand for 40 seconds, then remove the liquid from the wells. Repeat the washing process on absorbent paper to remove any residual liquid, for a total of 5 washes.

[0166] d. After washing, add 200 μL of substrate solution to each well. Do not seal the plate with a sealing membrane, and incubate at 37°C for 15 minutes in the dark.

[0167] e. Add 50 μL of stop solution to each well, following the same order as the substrate solution. After mixing, read the absorbance value at 450 nm within 5 minutes (reference wavelength 620 / 630 nm).

[0168] It should be noted that, using this kit and the above method to detect a group of clinical serum samples with known concentrations, the results are shown in Table 18. The recovery rates of all samples were between 96.7% and 101.1%, demonstrating high precision and good repeatability. This fully demonstrates the excellent performance of the solution, kit, and method provided by this invention, and they can be used for accurate and reliable detection of clinical samples.

[0169] Table 18: Clinical Sample Test Results

[0170]

[0171] This invention provides a serum sample processing solution, a protein denaturation termination solution, and their application in the detection of Aspergillus galactomannan, which are simple to operate, have mild conditions, and high safety. By optimizing the concentration of each component and the processing conditions, efficient and stable dissociation of antigen-antibody complexes and antibody inactivation are achieved, significantly improving the accuracy and reliability of the detection, making it suitable for widespread clinical use.

[0172] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A combined reagent for the detection of Aspergillus galactomannan, characterized in that: Includes serum sample processing solution and protein denaturing agent stop solution; The serum sample processing solution consists of the following components: Bacillus subtilis protease at a concentration of 0.05-0.50 mg / mL; CTAC at a concentration of 0.005-0.05 mg / mL; The pH of the serum sample processing solution is 9.3~9.7; The serum sample processing solution also contains a weakly alkaline buffer solution; The weakly alkaline buffer solution is CHES buffer solution; The protein denaturant termination solution consists of the following components: Phosphate buffer solution with a pH of 6.0-7.0; PMSF, a protease inhibitor, at a concentration of 0.001-0.005 mg / mL; Sodium caseinate at a concentration of 0.25-3.0% v / v; EDTANa2 at a concentration of 0.001-0.010% v / v.

2. The combined reagent according to claim 1, characterized in that: The pH of the serum sample processing solution is 9.

5.

3. The combined reagent according to claim 1, characterized in that: The serum sample processing solution and / or protein denaturant termination solution further contain a surfactant at a concentration of 0.02-0.2% v / v, wherein the surfactant is Tween 20 or Tween 80.

4. The combined reagent according to claim 1, characterized in that: The serum sample processing solution and / or protein denaturant termination solution also contain the preservative ProClin 300 at a concentration of 0.05-0.2% v / v.

5. A kit for detecting Aspergillus galactomannan, characterized in that: Includes box A and box B. The serum sample processing solution according to any one of claims 1 to 4 is added to box A; The B box contains a protein denaturant termination solution according to any one of claims 1 to 4.

6. The Aspergillus galactomannan detection kit as described in claim 5, characterized in that: The B box also includes HRP-labeled Aspergillus galactomannan antibody, with the HRP-labeled Aspergillus galactomannan antibody at 100 ng / mL.

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