A monoclonal antibody combination for Aspergillus fumigatus and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody combination for Aspergillus fumigatus and its application. Specifically, it includes mouse monoclonal antibodies 3D2 and 1F10 that recognize galactomannan antigens. Monoclonal antibody 3D2 is used as a coating antibody, and monoclonal 1F10 is used as a detection antibody to form a double-antibody sandwich ELISA system. Combined with biotin-avidin signal amplification technology, the detection sensitivity is significantly improved. The present invention also provides the amino acid sequence and encoding nucleotide sequence of the heavy chain and light chain variable regions of the antibody, which can be used for the development of detection tools such as reagents, kits, test strips or antibody chips. Experimental verification shows that the detection method shows good specificity and repeatability in clinical samples, and has important application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to an Aspergillus fumigatus monoclonal antibody combination and application thereof. Background Art

[0002] Invasive fungal disease (IFD) is a serious fungal infection that typically occurs when fungi invade human tissues, blood, or organs, causing deep infections. The incidence of IFD is increasing worldwide. Currently, over 300 million people contract fungal infections annually, with over 1.5 million dying from IFD. Consequently, the World Health Organization and the U.S. Centers for Disease Control and Prevention (CDC) have called for increased attention to IFD to address the global health threat it poses. According to statistics, over 5 million people in my country are also threatened by IFD annually. Fungi are a key pathogen of IFD, but clinical diagnosis of invasive Aspergillus infection is difficult, and invasive aspergillosis typically has a poor prognosis and a high mortality rate, posing a significant threat to patients' lives.

[0003] Aspergillus fumigatus is the primary pathogen of invasive Aspergillus infections. Its high pathogenicity often leads to invasive infections in immunocompromised or impaired individuals, such as those with neutropenia or organ transplantation. Aspergillus fumigatus is extremely common in the environment, and almost everyone in the population possesses antibodies to Aspergillus. Therefore, serological testing for specific antibodies is of limited value for the early diagnosis of invasive Aspergillus infections.

[0004] Traditional detection methods, such as culture and microscopy, are time-consuming, lack specificity, and exhibit wide variations in sensitivity. In recent years, novel laboratory testing technologies have been gradually adopted in clinical practice. These include molecular biology methods such as mass spectrometry, quantitative PCR, and next-generation sequencing, as well as antigen detection methods such as the G test (1,3-β-D glucan assay) and the GM test (galactomannan assay). The GM test, a specific antigen detection method for Aspergillus, demonstrates high sensitivity and specificity in the early screening of invasive Aspergillus fumigatus infections and has become a widely used and important tool in clinical practice. The G test, on the other hand, is suitable for initial screening of a variety of deep-seated fungal infections.

[0005] Galactomannan is a specific polysaccharide in the cell wall of Aspergillus, released from the tips of the hyphae into the blood or body fluids as the fungus grows. GM antigen (galactomannan antigen) is recognized as an early marker for invasive Aspergillus infection. GM levels in blood or body fluids are positively correlated with the level of Aspergillus in tissues and can indirectly reflect the severity of the patient's Aspergillus infection. Therefore, testing for Aspergillus-specific GM antigen not only aids in early clinical detection and guides intervention, but also allows for dynamic monitoring and evaluation of treatment efficacy and provides a certain indicator of patient prognosis. GM testing has also been recommended in multiple authoritative guidelines and expert consensus reports, both domestically and internationally, and is of great significance for the diagnosis of invasive Aspergillus.

[0006] However, my country still faces numerous challenges in the laboratory diagnosis of fungal infections. The development of fungal testing laboratories is extremely uneven, with some primary healthcare institutions lacking the capacity to conduct even basic fungal testing. There are few commercially available antibodies for Aspergillus galactomannan detection, and the availability of early diagnostic reagents for Aspergillus is limited, limiting the accuracy and accessibility of diagnostics. Summary of the Invention

[0007] The present invention screened a combination of mouse monoclonal antibodies specific for Aspergillus fumigatus galactomannan using hybridoma technology, and established an ELISA method applicable to galactomannan antigen detection based on this method. This method is simple, rapid, and has high detection sensitivity, and has important application prospects in laboratory detection of invasive Aspergillus infections.

[0008] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] The present invention provides an Aspergillus fumigatus monoclonal antibody combination, which specifically recognizes galactomannan antigens. The Aspergillus fumigatus monoclonal antibody combination includes monoclonal antibody 3D2 and monoclonal antibody 1F10. The heavy chain variable region of monoclonal antibody 3D2 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively.

[0010] The light chain variable region of monoclonal antibody 3D2 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0011] The heavy chain variable region of monoclonal antibody 1F10 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively;

[0012] The light chain variable region of monoclonal antibody 1F10 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.14.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.16.

[0015] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3D2 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3D2 is shown as SEQ ID NO.18.

[0016] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F10 is shown as SEQ ID NO.20.

[0017] In a second aspect, the present invention provides use of the above-mentioned monoclonal antibody combination in preparing a tool for detecting Aspergillus fumigatus.

[0018] In some embodiments, the means are used to detect galactomannan antigens derived from Aspergillus fumigatus.

[0019] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0020] In some embodiments, the monoclonal antibody combination is used to construct an ELISA detection system, and the ELISA detection system is used to detect galactomannan antigens in a sample. The ELISA detection system is not used for diagnosis and treatment of diseases.

[0021] In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0022] Beneficial effects

[0023] The Aspergillus fumigatus monoclonal antibody combination provided by the present invention comprises monoclonal antibodies 3D2 and 1F10 that specifically recognize galactomannan antigens. The heavy and light chain variable regions of these antibodies contain well-defined complementarity-determining region (CDR) sequences, as shown in SEQ ID NOs. 1 to 6 (corresponding to monoclonal antibody 3D2) and SEQ ID NOs. 7 to 12 (corresponding to monoclonal antibody 1F10), respectively. This antibody combination efficiently and specifically binds to galactomannan antigens derived from Aspergillus fumigatus and exhibits excellent pairing performance in a double-antibody sandwich ELISA system. This combination not only exhibits high antigen recognition specificity, avoiding cross-reactivity with common fungi such as Candida albicans and Cryptococcus neoformans, but can also be used to construct highly sensitive and reproducible ELISA detection systems, particularly biotin-avidin signal amplification ELISA systems, suitable for the high-sensitivity detection of galactomannan antigens in clinical samples, and has promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 The results of WB detection of different monoclonal antibodies against Aspergillus fumigatus;

[0026] Figure 2 This is the sensitivity result of biotin-avidin amplified ELISA test;

[0027] Figure 3 The results are from the biotin-avidin amplified ELISA method for testing clinical samples;

[0028] Figure 4 Results of clinical samples tested using the Bio-Rad kit. DETAILED DESCRIPTION

[0029] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0030] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0031] Example 1

[0032] 1. Preparation of Aspergillus fumigatus Antigen

[0033] Whole-cell antigen: Aspergillus fumigatus natural antigen was purchased from Germany's Weirun Seron Company (BA132F01), which is the whole-cell antigen purified after inactivation and lysis of the Aspergillus fumigatus Ag-507 strain culture.

[0034] Galactomannan antigen: Galactomannan (GM) purification is completed by a commissioned service company. Specifically, the supernatant of the Aspergillus fumigatus whole-cell antigen culture is sterilized at 121°C for 20 minutes and then lysed.

[0035] After centrifugation at 12,000 rpm, the supernatant was thoroughly mixed with 3 volumes of anhydrous ethanol solution and allowed to stand at 4°C for 48 hours. The supernatant was discarded and the precipitate was fully dried at 40°C. The precipitate was thoroughly resuspended in ultrapure water and centrifuged at 8,000 rpm for 10 minutes at 4°C. The supernatant was then added with 3 volumes of anhydrous ethanol and allowed to stand at 4°C for 2 hours. The supernatant was discarded. The precipitate was washed with anhydrous ethanol three times and centrifuged again. The supernatant was discarded. The precipitate was fully dissolved in ultrapure water and 1 / 10 volume of activated carbon powder. The precipitate was adsorbed and decolorized at room temperature for 2 hours. The purified galactomannan antigen was obtained by filtration and centrifugation. The absorbance peak was detected at 220 nm to 320 nm using a UV-visible spectrophotometer to determine whether it contained impurities such as proteins and nucleic acids. The purified polysaccharide antigen had a small molecular weight, poor immunogenicity, and could not be directly coated. Therefore, it was coupled to KLH and BSA to prepare complete antigens for mouse immunization and coating screening.

[0036] In this study, whole-cell antigens and galactomannan (GM) antigens of Aspergillus fumigatus were prepared, and GM was coupled to KLH and BSA carrier proteins, providing key antigen materials for subsequent monoclonal antibody screening and the establishment of ELISA detection methods.

[0037] 2. Mouse Immunization

[0038] Six-week-old female BALB / c mice were immunized with a whole Aspergillus fumigatus antigen mixed with an equal volume of Freund's complete adjuvant (200 μL) by subcutaneous injection at multiple sites at a dose of 30 μg per mouse. Two weeks later, mice were immunized again subcutaneously with a KLH-conjugated GM antigen. Subsequently, both antigens were mixed with an equal volume of MF59 adjuvant and administered intramuscularly twice at weeks 4 and 6. One week after the final immunization, mouse sera were collected for antibody titer determination. Mice with higher titers were selected for intraperitoneal booster immunization with KLH-GM antigen. Three days later, spleens were harvested for hybridoma cell production.

[0039] This study used whole-cell antigens of Aspergillus fumigatus and KLH-coupled GM antigens in stages, combined with different adjuvants for immunization, to induce mice to produce high-titer GM-specific antibodies, providing a high-quality source of spleen cells for the preparation of monoclonal antibodies.

[0040] 3. Screening of hybridoma cell lines

[0041] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for screening. When the fused cells reached half the bottom of the well, BSA-GM-positive clones were screened by indirect ELISA. Simultaneously, whole-cell antigens from Candida albicans, Cryptococcus neoformans, and Candida glabrata were coated for screening to identify specific cell lines that reacted only with Aspergillus fumigatus and did not react with these three fungi. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were then expanded and cryopreserved.

[0042] In this study, a monoclonal antibody cell line that specifically recognizes GM antigen and does not cross-react with other common fungi was obtained through cell fusion, HAT screening and ELISA detection, and then amplified and preserved.

[0043] 4. Screening of positive clones by indirect ELISA:

[0044] Microplates were coated with whole-bacterial antigens of Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Candida glabrata, and conjugated BSA-GM antigens (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water) at a coating concentration of 1-5 μg / mL and incubated at 4°C overnight; blocked with 1% gelatin, 150 μL per well, blocked at 37°C for 2 hours, washed once with washing solution, and patted dry; 50 μL cell culture supernatant was added and reacted at 37°C for 30 minutes. The liquid in the wells was shaken out, and the plate was washed 4 times with PBST. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again, and after patting dry, 50 μL / well of TMB color development solution was added to develop at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande Biological, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value.

[0045] The screening results are shown in Table 1 below.

[0046] Table 1: ELISA results of screening monoclonal antibodies and whole-cell antigens of Aspergillus fumigatus.

[0047]

[0048] Monoclonal antibodies targeting Candida albicans were provided by Zhuhai Bomei Biotechnology Co., Ltd. The monoclonal antibodies were screened for binding activity using whole-cell Aspergillus fumigatus antigens. Monoclonal antibody clones 2H8, 1F10, and 3D2 showed high titers. Because whole-cell Aspergillus fumigatus antigens were collected and purified from culture supernatants, the purity of the GM may have been insufficient, resulting in low binding activity readings. However, the screened monoclonal antibodies were specific for Aspergillus fumigatus. Using an indirect ELISA method, microplates were coated with BSA-GM antigen and whole-cell antigens from various common fungi (such as Candida albicans, Cryptococcus neoformans, and Candida glabrata) to screen for positive hybridoma cell lines that recognized either GM or Aspergillus fumigatus antigens while exhibiting no cross-reactivity with other non-target fungi.

[0049] 5. Preparation of Monoclonal Antibody Ascites

[0050] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0051] 6. Affinity chromatography purification of monoclonal antibodies

[0052] The ascites was centrifuged at 12000 r / min for 5 minutes, the supernatant was diluted with 3 volumes of 0.01 M sodium acetate, pH 4.0 solution, and an equal volume of saturated ammonium sulfate solution was added, mixed thoroughly, and allowed to stand at 4°C overnight. Centrifuge at 12,000 rpm for 20 minutes, discard the supernatant, and thoroughly dissolve the precipitate in 10 volumes of binding buffer (20 mM PB, 150 mM NaCl, pH 7.4) with 10 times the volume of ascites fluid. Filter the crude antibody solution through a 0.22 μm filter and pump it through a peristaltic pump onto a Protein L affinity chromatography prepacked column equilibrated with binding buffer. Connect the column to a protein purifier and wash with binding buffer for 5-10 column volumes until the UV absorbance peak flattens. Then, elute with elution buffer (0.1 M glycine, pH 2.7) and collect the elution peak. Adjust the collected sample to neutrality with 1 M Tris-HCl, pH 9, place it in a dialysis bag (MW: 8,000-14,000), and dialyze it against 20 mM PBS, pH 7.4, at 2-8°C for 16 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12,000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody. Purified monoclonal antibodies were stored in aliquots at -20°C.

[0053] 7. Western Blot Identification of Monoclonal Antibody-Antigen Reaction

[0054] The whole-cell antigens of Aspergillus fumigatus were separated by SDS-PAGE electrophoresis and transferred to a 0.45 μm nitrocellulose membrane. The membrane was blocked with 5% skim milk powder (diluted with TBST) at 4°C overnight and washed three times with TBST. The purified monoclonal antibody (1 μg / ml, diluted in blocking buffer) was added. The same concentration of Candida albicans monoclonal antibody (Zhuhai Bomei) was added as a control. The membrane was incubated at room temperature for 1 hour. After washing three times with TBST, HRP-labeled goat anti-mouse IgG (1000-fold diluted with TBST) was added. The membrane was reacted at room temperature in the dark for 1 hour. After thorough washing, DAB (Beijing Solebao, DA1010) was added for color development.

[0055] WB identification results refer to Figure 1 In the figure, M is a protein marker; lanes 1-6 are: 2B12, 2H8, 1F10, 2D12, 3D2, 5B10, all with a concentration of 1ug / ml; lane 7 is a Candida albicans monoclonal antibody with a concentration of 1ug / ml.

[0056] GM is a polysaccharide composed of a mannose backbone and galactose side chains, which may exhibit abnormal migration during SDS-PAGE. Western blot results showed that among the six anti-galactomannan (GM) monoclonal antibodies tested, 2H8, 1F10, and 3D2 specifically bound to whole-cell Aspergillus fumigatus antigens within the 43–95 kDa range, generating diffuse bands. This indicates that they recognize various aggregated forms of GM antigen, consistent with literature reports. In contrast, 2B12, 2D12, and 5B10 showed no significant binding signal. This may be due to the destruction of the antigen structure during heating and boiling, which prevents 2B12, 2D12, and 5B10 from effectively recognizing GM. This, in turn, leaves 2H8, 1F10, and 3D2 with stronger antigen-binding or epitope recognition abilities. Therefore, 2H8, 1F10, and 3D2 were selected for subsequent experiments.

[0057] 8. Double Antibody Sandwich ELISA Pairing

[0058] Antibody HRP labeling: Dilute the antibody to be labeled in carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L pure water, pH 9.6) to a final concentration of 2 mg / mL. Add 0.5 mL of this dilution (containing 1 mg of antibody) to a reaction tube containing 2 mg of HRP (dissolved in 0.5 mL ultrapure water) pre-mixed with 0.5 mL of 0.06 M sodium periodate solution. Gently pipette to mix. Incubate at room temperature for 1 hour, mixing regularly during incubation. Terminate the labeling reaction by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 minutes. Finally, dialyze the labeled antibody overnight against 0.01 M PBS, pH 7.4. Add glycerol in a 1:1 ratio and store in aliquots at -20°C.

[0059] Screening of paired antibodies: The purified monoclonal antibody was coated with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6) at a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and coated at 4°C overnight. The coating solution was discarded the next day, and the plate was blocked with 1-2% BSA or gelatin at 150 μL / well and incubated at 37°C for 2 hours. The blocking solution was discarded, and the galactomannan antigen to be tested and the negative control Cryptococcus neoformans capsular polysaccharide antigen were diluted with PBS at 100 ng / mL and added to the ELISA plate at 50 μL / well, incubated at 37°C for 35 minutes, and the plate was washed 4 times with PBST solution. 500, 1000, and 2000-fold diluted HRP-labeled monoclonal antibodies were added at 50 μL / well and incubated at 37°C for 35 min. The plate was then washed four times and patted dry before adding 50 μL / well of TMB color development solution. The color was developed at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader. 450 The antibody combination with a high galactomannan antigen detection value and no reaction with the control Cryptococcus neoformans capsular polysaccharide antigen was selected as the optimal pairing for the double antibody sandwich.

[0060] Multiple monoclonal antibodies with high titers are individually coated and HRP-labeled to screen for paired combinations. High-binding antibodies have higher sensitivity when used in detection. Purposefully selecting these high-titer antibodies can make the screening of paired monoclonal antibodies more efficient.

[0061] The screening process is shown in Table 2.

[0062] Table 2: Reactivity screening results of different monoclonal antibody pair combinations.

[0063]

[0064] 1000* means 1000-fold dilution.

[0065] The experimental results showed that the double-antibody sandwich ELISA system composed of monoclonal antibody 3D2 for coating and monoclonal antibody 1F10 for HRP labeling had a high detection read value for galactomannan antigen and had no cross-reaction with non-target antigens of Cryptococcus neoformans capsular polysaccharide. Therefore, it was determined to be the optimal antibody combination for detecting galactomannan antigen in the present invention.

[0066] 9. Biotin-antibody conjugation

[0067] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) to monoclonal antibody conjugation was 20:1. First, 2.0 mg of activated biotin was dissolved in 360 μL of ultrapure water to prepare a 10 mM biotin solution. Then, 2 mg of antibody was reacted with 26.6 μL of 10 mM biotin at room temperature for 3 hours with shaking (the reaction volume was kept around 2 mL). The biotin-antibody mixture was then dialyzed against 0.01 M PBS to remove excess free biotin. An equal volume of glycerol was then added and stored at -20°C. The final labeled antibody concentration was approximately 0.5 mg / mL.

[0068] The biotin-conjugated monoclonal antibody used in this experiment was the best detection antibody screened in the early stage - monoclonal antibody 1F10. This antibody was labeled with biotin and used to construct a biotin-avidin signal amplification system.

[0069] 10. Establishment of Biotin-Avidin Amplified ELISA System

[0070] The above-screened monoclonal antibody combinations were used for coating and biotin coupling, and then the most suitable reaction conditions for the amplification system were determined by exploring the coating antibody, biotin antibody concentration and the dilution of HRP-labeled avidin.

[0071] Microplates were coated with monoclonal antibodies (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a coating concentration gradient of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and incubated at 4°C overnight. The plates were washed once with washing buffer (PBST, PBS containing 0.05% Tween-20, pH 7.4), patted dry, and blocked with 3% sucrose and 1% BSA, 150 μL per well, incubated at 37°C for 2 hours, patted dry, and stored dry. Galactomannan antigen was diluted to a concentration of 10 ng / mL in PBS and 50 μL was added to the monoclonal antibody-coated microplate. Cryptococcus neoformans capsular polysaccharide was diluted to 10 ng / mL as a negative control and reacted at 37°C for 30 min. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry. The biotin-conjugated monoclonal antibody was diluted with PBS to a concentration of 1μg / mL, 2μg / mL, and 4ug / mL, and 50μL / well was added to the microplate. The reaction was incubated at 37°C for 30 minutes. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry. HRP-polymer streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10,000, 20,000, and 40,000 times with PBS) was added at 50μL / well. The reaction was incubated at 37°C for 30 minutes. The plate was washed 4 times, patted dry, and TMB color development solution was added at 50μL / well. The color was developed at room temperature for 10 minutes. Finally, the stop solution was added to terminate the reaction. The OD was measured using a microplate reader. 450The coating concentration, biotin monoclonal antibody concentration, and HRP-labeled avidin dilution with the most obvious positive and negative differences were selected as the optimal reaction conditions.

[0072] The experimental results showed that the optimal combination was: coated monoclonal antibody 3D2, concentration of 1ug / ml; biotin-conjugated monoclonal antibody 1F10, concentration of 2ug / ml; HRP-polymer streptavidin 20,000 times diluted.

[0073] 11. Sensitivity Determination of Biotin-Avidin Amplified ELISA

[0074] Determine the optimal coating concentration, biotin monoclonal antibody concentration, HRP-avidin dilution and other reaction conditions. Referring to the above detection steps, first perform a gradient dilution of galactomannan antigen (GM) with PBS buffer solution. The concentrations after dilution are 1μg / mL, 100ng / mL, 10ng / mL, 1ng / mL, 100pg / mL, and 10pg / mL, respectively. At the same time, take Cryptococcus neoformans capsular polysaccharide as a control and add 50μL to each well at the same concentration for detection to determine the detection sensitivity of the detection system for galactomannan antigen.

[0075] See also Figure 2 , indicating that the detection method of the present application has a high detection sensitivity, and the minimum detection limit is around 100pg / ml.

[0076] 12. Clinical Evaluation of Biotin-Avidin Amplified ELISA

[0077] Normal human serum and serum samples from patients with invasive Aspergillus infection were tested using a well-established ELISA assay. A commercially available Aspergillus antigen detection kit (Bio-Rad Platelia aspergillus Ag) was also tested in parallel.

[0078] Sample collection was carried out by the cooperating hospital. A total of 30 samples of normal human serum were collected from healthy people at the physical examination center. A total of 20 samples of serum were collected from patients with invasive aspergillus infection. The samples were determined by clinical physicians based on the guidelines, clinical symptoms, and clinical test results.

[0079] Pretreatment of serum samples

[0080] Normal people have a certain level of Aspergillus fumigatus antibodies in their serum, and galactomannan antigens will form antigen-antibody complexes with them, which can cause false negatives when detecting low concentrations of galactomannan. In addition, endogenous or exogenous interfering factors in the blood, such as rheumatoid factor and complement, can also have a significant impact on the detection. Therefore, it is necessary to pre-treat the serum sample to be tested. The present invention adopts two treatment methods: EDTA + heating or glycine + neutralization, specifically:

[0081] Method 1. Take 200 μl of serum sample and add 0.1 M disodium EDTA (pH 4.5) solution (the volume of the disodium EDTA solution should be 1 / 2 of the serum sample volume). Then add 30 μl of 0.01 M PBS (pH 7.4), mix thoroughly, heat at 100°C for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and collect the supernatant for analysis.

[0082] Method 2: Add 0.1M glycine (pH 2.7) to 200µl of serum sample, with the volume of the glycine solution being half the volume of the serum sample. Mix thoroughly and incubate at room temperature for 5 minutes. Then, add 30µl of 1M Tris-HCl (pH 9.0) neutralization buffer, mix thoroughly, and centrifuge at 12,000 rpm for 10 minutes. Collect the supernatant for analysis.

[0083] The two sample pretreatment methods described above were evaluated by adding normal serum to simulate positive galactomannan serum. Method 2 demonstrated consistent detection values for samples treated with galactomannan antigen detection without serum. This method involves using a 0.1M glycine solution (pH 2.7) to dissociate the antigen-antibody complex in the serum sample, followed by neutralization with an alkaline buffer (such as Tris-HCl, pH 9.0) to restore the sample pH to a range suitable for subsequent testing. Antigen-antibody complexes are formed between galactomannan antigens and endogenous or exogenous antibodies.

[0084] 13. ELISA method for testing clinical samples

[0085] First, 30 serum samples from healthy individuals were pretreated according to Method 2 above. These samples were then tested using an established biotin-avidin amplified ELISA system. The average Aspergillus antigen detection value for the healthy population was determined, and the cut-off value was determined as twice the average. Twenty serum samples from patients with invasive Aspergillus infection were pretreated and tested using the same procedures. The ELISA protocol was as follows: 1 μg / mL antibody was coated overnight at 4°C, the plate was washed, and then blocked. 50 μL of the treated serum was added to the antibody-coated ELISA plate. A positive control of galactomannan diluted to 10 ng / mL and a blank control of 50 μL of PBS were added simultaneously. The plates were incubated at 37°C for 30 minutes. After washing, 50 μL / well of biotin-conjugated monoclonal antibody (2 μg / mL) was added and the plate was incubated at 37°C for 30 minutes. After washing, pat dry and add 20,000-fold diluted HRP-polymer streptavidin. After reacting at 37°C for 30 minutes, color development and termination of the reaction were performed. OD was measured using a microplate reader. 450 nm value.

[0086] The test results can be found in Figure 3 and Figure 4 , Figure 3 The detection results of the ELISA method of this application, the vertical axis is OD 450 nm reading, the dotted line is the Cut-off value, Figure 4 The results of the Bio-Rad kit test are shown in Figure 2. The vertical axis is the I index and the dotted line is the cut-off value (I = 0.50). Both methods tested normal human serum and the results of the positive serum were completely consistent. 450 The values were all lower than the cut-off value, indicating that no significant Aspergillus fumigatus galactomannan antigen was detected in the normal population. The OD values of samples from patients with invasive Aspergillus infection were 450 The value is higher than the cut-off value, indicating that the detection method of the present application can effectively identify serum samples of patients with invasive fungal diseases. This shows that the detection method of the present application has high sensitivity and consistency in the patient population.

[0087] In addition, unlike the index determination method of sample OD value / critical value control OD value used by Bio-Rad kit, the self-developed ELISA method established in this study uses a fixed threshold method based on the mean value of negative population samples. By testing 30 healthy human serum samples, the OD 450 The cut-off value for determining a positive result was set as 2 times the average value of nm. This method does not require the addition of a critical value control for each experiment, nor does it require index conversion. It has the advantages of being easy to operate, saving reagents, and being suitable for rapid clinical screening. Overall, the two methods are relatively consistent in detecting real clinical samples, and the detection method of the present invention has excellent clinical detection performance.

[0088] 14. Gene sequence

[0089] Monoclonal antibody variable region gene cloning and sequencing

[0090] Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0091] The specific sequence is as follows:

[0092] Monoclonal antibody 3D2:

[0093] Heavy chain:

[0094] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO. 17: GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGAAGAAGCCCGGCGAGACCGTGAGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCGGCGGCTGGATCAAGTGGGTGAAGCAGACCCCCGGCAAGGGCCTGAGGTGGATGGGCTGGCTGTGGGAGAAGGAGGGCAGCAGGACCTACAGCCTGGTGGCCAGCGGCAGGTTCGACTTCAGCAGCGAGACCAGCGCCAGCAGCGTGTACCTGCAGATCAACAACCTGAAGAACGAGGACACCGCCACCTACTTCTGCGCCAGGTTCCCCAAGATCTACTACGAGGTGAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC.

[0095] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.13:

[0096] EVQLQQSGPELKKPGETVRISCKASGYTFTSGGWIKWVKQTPGKGLRWMGWLWEKEGSRTYSLVASGRFDFSSETSASSVYLQINNLKNEDTATYFCARFPKIYYEVSWGQGTTLTVSA.

[0097] CDR region annotation:

[0098] The sequence of the heavy chain variable region CDR-H1 of monoclonal antibody 3D2 is shown in SEQ ID NO.1: SGGWIK; the sequence of the heavy chain variable region CDR-H2 of monoclonal antibody 3D2 is shown in SEQ ID NO.2: WLWEKEGSRTYSLVASG; the sequence of the heavy chain variable region CDR-H3 of monoclonal antibody 3D2 is shown in SEQ ID NO.3: FPKIYYEVS.

[0099] Light chain:

[0100] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.18:

[0101] AACATCGTGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCCTGGGCGACAGGGTGACCATCAGCTGCAGCGCCCAGCAGGGCCTGAGCAACTACATGACCTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACTACACCAGCAGCCTGCAC AGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCGCCGACTTCAGCCTGACCATCAGCAACCTGGAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCCACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0102] The amino acid sequence of the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO. 14: NIVLTQSPSSLSASLGDRVTISCSAQQGLSNYMTWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGADFSLTISNLEPEDIATYYCQQYSSFPHTFGGGTKLEIKRTV.

[0103] CDR region annotation:

[0104] The sequence of the light chain variable region CDR-L1 of monoclonal antibody 3D2 is shown in SEQ ID NO.4: SAQQGLSNYMT; the sequence of the light chain variable region CDR-L2 of monoclonal antibody 3D2 is shown in SEQ ID NO.5: YTSSLHS; the sequence of the light chain variable region CDR-L3 of monoclonal antibody 3D2 is shown in SEQ ID NO.6: QQYSSFPHT.

[0105] Sequence of monoclonal antibody 1F10:

[0106] Heavy chain:

[0107] The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.19:

[0108] CAGGTGCAGCTGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCACCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACGCCTCACCAACTACTTCATCGAGTGGATCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCAGCAGCCACACC CACTACAACGACAAGCAGAAGGACAAGGCCACCCTGACCGCCGACAGGAGCAGCAGCACCGCCTACATGCACCTGAGGAGCCTGACCAGCGACGACAGCGCCGTGTACTTCTGCGCCTTCAGCCCCTACGAGGACAGCAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC.

[0109] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.15:

[0110] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYFIEWIKQRPGQGLEWIGVINPGSSHTHYNDKQKDKATLTADRSSSTAYMHLRSLTSDDSAVYFCAFSPYEDSSWGQGTTLTVSS.

[0111] CDR region annotation:

[0112] The sequence of monoclonal antibody 1F10 CDR-H1 is shown in SEQ ID NO.7: NYFIE; the sequence of monoclonal antibody 1F10 CDR-H2 is shown in SEQ ID NO.8: VINPGSSHTHYNDKQKD; the sequence of monoclonal antibody 1F10 CDR-H3 is shown in SEQ ID NO.9: SPYEDSS.

[0113] Light chain:

[0114] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.20:

[0115] GAGATCGTGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCCTGGGCGACAGGGTGACCATCAGCTGCAGCGCCCAGCAGGGCCTGAGCAACTACATGACCTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACTACAACAGCAGCCTGCAC AGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCGCCGACTTCAGCCTGACCATCAGCAACCTGGAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGGTGAGCAGCATCCCCACACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0116] The amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.16:

[0117] EIVLTQSPSSLSASLGDRVTISCSAQQGLSNYMTWYQQKPDGTVKLLIYYNSSLHSGVPSRFSGSGSGADFSLTISNLEPEDIATYYCQQVSSIPHTFGGGTKLELKRTV.

[0118] CDR region annotation: the sequence of monoclonal antibody 1F10 CDR-L1 is shown in SEQ ID NO.10: SAQQGLSNYMT; the sequence of monoclonal antibody 1F10 CDR-L2 is shown in SEQ ID NO.11: YNSSLHS; the sequence of monoclonal antibody 1F10 CDR-L3 is shown in SEQ ID NO.12: QQVSSIPHT.

[0119] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0120] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody combination against Aspergillus fumigatus, characterized in that: The Aspergillus fumigatus monoclonal antibody combination specifically recognizes galactomannan antigens, and the Aspergillus fumigatus monoclonal antibody combination includes monoclonal antibody 3D2 and monoclonal antibody 1F10. The heavy chain variable region of the monoclonal antibody 3D2 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; The light chain variable region of the monoclonal antibody 3D2 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively; The heavy chain variable region of the monoclonal antibody 1F10 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively; The light chain variable region of the monoclonal antibody 1F10 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

2. The monoclonal antibody combination against Aspergillus fumigatus according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3D2 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 3D2 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination against Aspergillus fumigatus according to claim 2, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination against Aspergillus fumigatus according to claim 3, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3D2 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3D2 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination against Aspergillus fumigatus according to claim 4, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.

20.

6. Use of the monoclonal antibody combination according to claim 1 in preparing a tool for detecting Aspergillus fumigatus.

7. The use according to claim 6, characterized in that The tool is used to detect galactomannan antigens derived from Aspergillus fumigatus.

8. The use according to claim 7, characterized in that The tools include reagents, test kits, test strips and antibody chips.

9. The use according to claim 8, characterized in that The monoclonal antibody combination is used to construct an ELISA detection system, which is used to detect galactomannan antigens in a sample. The ELISA detection system is not used for diagnosing diseases.

10. The use according to claim 9, characterized in that The ELISA detection system is a biotin-avidin amplified ELISA system.

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