Aspergillus fumigatus monoclonal antibody combination and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to an aspergillus fumigatus monoclonal antibody combination and application thereof. The monoclonal antibodies specifically comprise murine monoclonal antibodies 3D2 and 1F10 for recognizing galactomannan antigens. The monoclonal antibody 3D2 is used as a coating antibody, the monoclonal 1F10 is used as a detection antibody, a double-antibody sandwich ELISA system is formed, and the detection sensitivity is remarkably improved by combining a biotin-avidin signal amplification technology. The invention also provides amino acid sequences and coding nucleotide sequences of heavy chain and light chain variable regions of the antibody, which can be used for 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 technical field of biological detection, and particularly relates to a monoclonal antibody combination of Aspergillus fumigatus and its application. Background Art

[0002] Invasive Fungal Disease (IFD) is a serious type of fungal infection, usually referring to deep infections caused by fungi invading human tissues, blood or organs. The incidence of invasive fungal diseases shows an increasing trend worldwide. Currently, there are more than 300 million fungal infection patients globally every year, and more than 1.5 million patients die from IFD. Therefore, the WHO and the US CDC have successively called for increased attention to invasive fungal diseases to address the threat posed by IFD to global health. According to statistics, more than 5 million people in China are also threatened by IFD every year. Among them, molds are one of the important pathogenic bacteria of invasive fungal diseases. However, the clinical diagnosis of invasive aspergillus infection is relatively difficult, and invasive aspergillosis usually has a poor prognosis and a high mortality rate, posing a great threat to the lives of patients.

[0003] Aspergillus fumigatus is the main pathogen of invasive aspergillus, with strong pathogenicity. When the body's immunity is low or the immune function is damaged, such as in cases of neutropenia or organ transplantation, invasive infections often occur. Aspergillus fumigatus is extremely common in the environment, and almost everyone has antibodies against aspergillus in their serum. Therefore, serological detection of specific antibodies has little significance for the early diagnosis of invasive aspergillus infection.

[0004] Traditional detection methods such as culture and microscopy are time-consuming, and the test results lack specificity with large differences in sensitivity. In recent years, some new laboratory detection technologies have been gradually applied to clinical practice, including molecular biology methods such as mass spectrometry, fluorescence quantitative PCR, and next-generation sequencing, as well as antigen detection methods such as the G test (1,3-β-D glucan detection) and the GM test (galactomannan detection). Among them, the GM test, as an antigen detection method specifically for aspergillus, has shown high sensitivity and specificity in the early screening of invasive Aspergillus fumigatus infection and has become one of the important tools widely used in clinical practice. The G test is suitable for the preliminary screening of various deep fungal infections.

[0005] Galactomannan is a specific polysaccharide in the cell wall of Aspergillus. When the mold grows, it is released into the blood or body fluid from the tip of the hypha. GM antigen (galactomannan antigen) is recognized as an early marker for invasive aspergillosis. The content of GM in blood or body fluid is positively correlated with the content of Aspergillus in tissues, which can indirectly reflect the degree of Aspergillus infection in patients. Therefore, the detection of Aspergillus-specific GM antigen can not only help clinicians detect and intervene early, but also dynamically monitor and evaluate the treatment effect, and has a certain guiding role for the prognosis of patients. GM detection has also been recommended as a detection item in multiple domestic and foreign authoritative guidelines and expert consensuses, which is of great significance for the diagnosis of invasive aspergillosis.

[0006] However, China still faces many challenges in the laboratory diagnosis of fungal infections. The development of fungal detection laboratories is extremely uneven, and some primary medical institutions do not even have the ability to carry out basic fungal detections. There are not many industrial antibodies for the detection of Aspergillus galactomannan on the market, and the existing types of early diagnosis reagents for Aspergillus are few, which limits the accuracy and popularity of diagnosis. Summary of the Invention

[0007] The present invention screened and obtained a specific murine monoclonal antibody combination against Aspergillus fumigatus galactomannan through hybridoma technology, and established an ELISA method applicable to the detection of galactomannan antigen based on this. This method is simple and fast, has high detection sensitivity, and has important application prospects in the laboratory detection of invasive aspergillosis.

[0008] To achieve the above object, the main technical solutions adopted by the present invention include: The present invention provides a monoclonal antibody combination against Aspergillus fumigatus. The monoclonal antibody combination against Aspergillus fumigatus specifically recognizes galactomannan antigen. The monoclonal antibody combination against Aspergillus fumigatus includes monoclonal antibody 3D2 and monoclonal antibody 1F10. The heavy chain variable region of monoclonal antibody 3D2 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.1-SEQ ID NO.3; The light chain variable region of monoclonal antibody 3D2 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.4-SEQ ID NO.6; The heavy chain variable region of monoclonal antibody 1F10 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.7-SEQ ID NO.9; The light chain variable region of monoclonal antibody 1F10 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown in SEQ ID NO.10-SEQ ID NO.12.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3D2 is as shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 3D2 is as shown in SEQ ID NO.14.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is as shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is as shown in SEQ ID NO.16.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3D2 is as shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3D2 is as shown in SEQ ID NO.18.

[0012] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is as shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F10 is as shown in SEQ ID NO.20.

[0013] In a second aspect, the present invention provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting Aspergillus fumigatus.

[0014] In some embodiments, the tool is used to detect galactomannan antigen derived from Aspergillus fumigatus.

[0015] In some embodiments, the tool includes reagents, kits, test strips and antibody chips.

[0016] In some embodiments, the monoclonal antibody combination is used to construct an ELISA detection system for detecting galactomannan antigen in a sample, and the ELISA detection system is not used for the diagnosis and treatment of diseases. In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0017] Beneficial effects The monoclonal antibody combination provided by the present invention comprises monoclonal antibodies 3D2 and 1F10 that specifically recognize galactomannan antigen. The heavy and light chain variable regions of the antibodies both contain clearly defined complementarity determining region (CDR) sequences, specifically as shown in SEQ ID NO.1 to SEQ ID NO.6 (corresponding to monoclonal antibody 3D2) and SEQ ID NO.7 to SEQ ID NO.12 (corresponding to monoclonal antibody 1F10), respectively. This antibody combination can efficiently and specifically bind to galactomannan antigen derived from Aspergillus fumigatus, and exhibits good pairing performance in a double antibody sandwich ELISA system. This combination not only has a high specificity for antigen recognition, avoiding cross-reaction with common fungi such as Candida albicans and Cryptococcus neoformans, but can also be used to construct an ELISA detection system with high sensitivity and good repeatability, especially a biotin-avidin signal amplification ELISA system, which is suitable for the highly sensitive detection of galactomannan antigen in clinical samples and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 WB detection results of different monoclonal antibodies and Aspergillus fumigatus; Figure 2 Biotin-avidin amplified ELISA detection sensitivity results; Figure 3 Results of detecting clinical samples by biotin-avidin amplified ELISA method; Figure 4 Results of detecting clinical samples by Bio-Rad kit. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The following further describes in detail the embodiments of the present application in conjunction with the drawings and examples. The detailed description and drawings of the following examples are used to exemplarily illustrate the principle of the present application, but cannot be used to limit the scope of the present application. The present application can be implemented in many different forms, not limited to the specific embodiments disclosed in the text, but including all technical solutions falling within the scope of the claims.

[0021] These embodiments are provided in order to make this application thorough and complete, and to fully convey the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, the components of materials, numerical expressions and values set forth in these embodiments should be construed as merely exemplary and not as limitations.

[0022] Example 1 1. Preparation of Aspergillus fumigatus antigen Whole cell antigen: The natural antigen of Aspergillus fumigatus was purchased from ViroStat, Inc. (BA132F01), and it is the whole cell antigen purified after inactivating and lysing the culture of Aspergillus fumigatus strain Ag-507.

[0023] Galactomannan antigen: The purification of galactomannan (GM) was completed by a commissioned service company. Specifically, the culture supernatant of the whole cell antigen of Aspergillus fumigatus was lysed by autoclaving at 121 °C for 20 min. The supernatant after centrifugation at 12,000 rpm was thoroughly mixed with 3 volumes of absolute ethanol solution and left to stand at 4 °C for 48 h; centrifuged at 8,000 rpm for 10 min at 4 °C, the supernatant was discarded, and the precipitate was thoroughly dried at 40 °C, resuspended with ultrapure water, centrifuged at 8,000 rpm for 10 min at 4 °C, the supernatant was retained and then 3 volumes of absolute ethanol were added, and left to stand at 4 °C for 2 h; centrifuged at 8,000 rpm for 10 min at 4 °C to discard the supernatant, the precipitate was washed 3 times with absolute ethanol and then centrifuged again to discard the supernatant, ultrapure water and 1 / 10 volume of activated carbon powder were added to fully dissolve the precipitate, adsorbed and decolorized at room temperature for 2 h, filtered and centrifuged to obtain the purified galactomannan antigen. The absorption peak was detected at 220 nm - 320 nm with an ultraviolet-visible spectrophotometer to determine whether it contained impurities such as proteins and nucleic acids. Since the purified polysaccharide antigen has a small molecular weight, poor immunogenicity and cannot be directly coated, it was conjugated to KLH and BSA respectively to prepare complete antigens for mouse immunization and coating screening.

[0024] In this study, by preparing the whole cell antigen of Aspergillus fumigatus and galactomannan (GM) antigen, and conjugating GM to KLH and BSA carrier proteins, it provided key antigenic raw materials for the subsequent screening of monoclonal antibodies and the establishment of ELISA detection methods.

[0025] 2. Mouse immunization The whole cell antigen of Aspergillus fumigatus was mixed with an equal volume of Freund's complete adjuvant (200 μL) and subcutaneously injected into 6-week-old female BALB / c mice at multiple points with a dose of 30 μg / mouse. After a 2-week interval, the mice were subcutaneously immunized again at multiple points with GM antigen conjugated to KLH. Subsequently, by intramuscular injection, each of them was mixed with an equal volume of MF59 adjuvant and immunized 2 more times at week 4 and week 6 respectively. One week after the last immunization, the mouse sera were taken to detect the antibody titers. Mice with higher titers were selected and boosted by intraperitoneal injection with KLH-GM antigen. Three days later, the mouse spleens were taken for the preparation of hybridoma cells.

[0026] In this experiment, by using the whole cell antigen of Aspergillus fumigatus and GM antigen conjugated to KLH in stages and combining with different adjuvants for immunization, high-titer GM-specific antibodies were induced in mice, providing a high-quality source of spleen cells for the preparation of monoclonal antibodies.

[0027] 3. Screening of hybridoma cell lines All spleen cells from the immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then placed in HAT medium for screening culture. When the fused cells grew to 1 / 2 of the well bottom, positive clones of BSA-GM were screened by indirect ELISA. At the same time, the whole cell antigens of Candida albicans, Cryptococcus neoformans, Candida glabrata, etc. were used for coating and screening to screen out specific cell lines that did not react with Candida albicans, Cryptococcus neoformans, Candida glabrata but only with Aspergillus fumigatus. The positive cells were cloned to the monoclonal state by the limiting dilution method, and then the cell lines were expanded and cryopreserved.

[0028] In this experiment, monoclonal antibody cell lines that specifically recognize GM antigen and do not cross-react with other common fungi were obtained through cell fusion, HAT screening and ELISA detection, and were amplified and preserved.

[0029] 4. Screening of positive clones by indirect ELISA method: Coat the whole cell antigens of Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Candida glabrata, etc. and the conjugated BSA-GM antigen in a microplate (coating buffer: carbonate buffer, 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, made up to 1 L of pure water), with a coating concentration of 1-5 μg / mL, overnight at 4°C; block with 1% gelatin, 150 μL per well, block at 37°C for 2 hours, wash the plate once with the washing solution, and pat dry; add 50 μL of cell culture supernatant and react at 37°C for 30 min. Discard the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry, then add 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) per well, react at 37°C for 30 min, wash the plate 4 times again, pat dry, add 50 μL of TMB chromogenic solution per well and develop color at room temperature for 10 min, and finally add 50 μL of TMB stop solution (Beijing Meikewanda Biology, 1001SA) to terminate the reaction, and measure the OD 450 nm value.

[0030] The screening results are shown in Table 1 below.

[0031] Table 1: ELISA identification results of the screened monoclonal antibodies and the whole cell antigen of Aspergillus fumigatus.

[0032]

[0033] The monoclonal antibodies against Candida albicans were provided by Zhuhai Bomei Biotechnology Co., Ltd. Using the whole cell antigen of Aspergillus fumigatus for coating to detect the binding activity of the screened monoclonal antibodies, it was found that the titers of monoclonal antibody clones 2H8, 1F10, and 3D2 were relatively high. Since the whole cell antigen of Aspergillus fumigatus was collected and purified from the culture supernatant, the purity of GM might not be high enough, resulting in a low reading of the binding activity, but the screened monoclonal antibodies were specific to Aspergillus fumigatus. By the indirect ELISA method, coat the BSA-GM antigen and the whole cell antigens of various common fungi (such as Candida albicans, Cryptococcus neoformans, Candida glabrata) on the microplate respectively to screen for positive hybridoma cell lines that can recognize both GM or the antigen of Aspergillus fumigatus and do not cross-react with other non-target fungi.

[0034] 5. Preparation of monoclonal antibody ascites After expanding the culture of the screened monoclonal cell line, inject 0.2 mL (containing 2.5×10 6 cells) of female BALB / c mice pretreated with Freund's incomplete adjuvant into the abdominal cavity. About 10 days later, when the abdomen of the mice is significantly swollen, collect the ascites using a sterile syringe needle. Centrifuge the collected ascites at 3000 r / min for 10 minutes and collect the middle layer.

[0035] 6. Affinity chromatography purification of monoclonal antibodies Centrifuge the ascites at 12,000 r / min for 5 minutes. Take the supernatant and dilute it with 3 volumes of 0.01 M acetic acid - sodium acetate, pH 4.0 solution. Then add an equal volume of saturated ammonium sulfate solution, mix well, and let it stand overnight at 4°C. Centrifuge at 12,000 rpm for 20 min and discard the supernatant. Dissolve the precipitate thoroughly with 10 volumes of the binding buffer (20 mM PB, 150 mM NaCl, pH 7.4) based on the volume of the ascites. Filter the crude purified antibody solution through a 0.22 - μm filter, and pump it into a pre - packed Protein L affinity chromatography column equilibrated with the binding buffer through a peristaltic pump. Connect the protein purifier, wash with 5 - 10 column volumes of the binding buffer until the UV absorption peak is washed flat, and then elute with the elution buffer (0.1 M glycine, pH 2.7). Collect the elution peak. Adjust the collected sample to neutral with 1 M Tris - HCl, pH 9, load it into a dialysis bag (MW: 8000 - 14000), and dialyze it in 20 mM PBS, pH 7.4 solution at 2 - 8°C for 16 hours. Transfer the liquid in the dialysis bag to a centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and the supernatant is the purified monoclonal antibody. Aliquot the purified monoclonal antibody and store it at - 20°C.

[0036] 7. Identification of the reaction between the monoclonal antibody and the antigen by Western Blot Perform SDS - PAGE electrophoresis separation on the whole - cell antigen of Aspergillus fumigatus, and then transfer it to a 0.45 - μm nitrocellulose membrane. Block it overnight at 4°C with 5% skim milk powder (diluted with TBST). After washing 3 times with TBST, add the purified above - mentioned monoclonal antibody (1 μg / ml, diluted with the blocking solution), and at the same time add a monoclonal antibody against Candida albicans (Zhuhai Bomei) with the same concentration as a control. Incubate at room temperature for 1 h. After washing 3 times with TBST, add HRP - labeled goat anti - mouse IgG (diluted 1000 - fold with TBST), and react in the dark at room temperature for 1 h. After thorough washing, add DAB (Beijing Solarbio, DA1010) for color development.

[0037] The WB identification results are shown in Figure 1 , where M in the figure is the protein marker; lanes 1 - 6 are: 2B12, 2H8, 1F10, 2D12, 3D2, 5B10 with a concentration of 1 μg / ml each; lane 7 is the monoclonal antibody against Candida albicans with a concentration of 1 μg / ml.

[0038] GM is a polysaccharide polymer containing a mannose backbone and galactose side chains. During SDS-PAGE, abnormal migration may occur. The WB results showed that among the 6 anti-galactomannan (GM) monoclonal antibodies tested, 2H8, 1F10, and 3D2 specifically bound to the whole-cell antigen of Aspergillus fumigatus in the range of 43–95 kDa, presenting a diffuse band, indicating that they were able to recognize GM antigens in different polymerization forms, which was consistent with the literature reports. In contrast, 2B12, 2D12, and 5B10 did not show obvious binding signals. It may be due to the destruction of the antigen structure caused by heating and boiling, making 2B12, 2D12, and 5B10 unable to effectively recognize galactomannan, while 2H8, 1F10, and 3D2 had stronger antigen-binding ability or epitope recognition ability. Therefore, 2H8, 1F10, and 3D2 were selected for subsequent experiments.

[0039] 8. Double-antibody sandwich ELISA pairing HRP labeling of antibodies: Dilute the antibody to be labeled with carbonate coupling buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6) to a final concentration of 2 mg / mL. Take 0.5 mL of this dilution (containing a total of 1 mg of antibody), add it to a reaction tube containing 2 mg of HRP (dissolved in 0.5 mL of ultrapure water) and pre-mixed with 0.5 mL of 0.06 M sodium periodate solution, and gently pipette to mix well. Incubate at room temperature for 1 h, and mix regularly during incubation. Add 50 μL of 5 mg / ml sodium borohydride and mix for 15 min to terminate the labeling reaction. Finally, dialyze the labeled antibody overnight in a buffer of 0.01 M PBS, pH 7.4, add glycerol in a volume ratio of 1:1, and store in aliquots at -20 °C.

[0040] Screening of paired antibodies: Purified monoclonal antibodies were separately coated at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water, pH 9.6), 50 μL per well, and incubated overnight at 4°C. The next day, the coating solution was discarded, and blocking was performed with 1 - 2% BSA or gelatin, 150 μL per well, incubated at 37°C for 2 h. The blocking solution was then discarded. The galactomannan antigen to be tested and the negative control cryptococcal capsular polysaccharide antigen were diluted with PBS to 100 ng / mL and added to the enzyme - linked immunosorbent assay (ELISA) plate, 50 μL per well, and incubated at 37°C for 35 min. The plate was washed 4 times with PBST washing solution. Monoclonal antibodies labeled with HRP diluted 500 - fold, 1000 - fold, and 2000 - fold with PBS were added, 50 μL per well, and incubated at 37°C for 35 min. The plate was washed 4 times again. After blotting dry, 50 μL of TMB chromogenic solution was added per well and allowed to develop color at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meikewanda Biology, 1001SA) was added to terminate the reaction, and the OD 450 nm value was measured with an enzyme - linked immunosorbent assay reader. The antibody combination with a higher detection value for galactomannan antigen and no reaction with the control cryptococcal capsular polysaccharide antigen was selected as the optimal pair for the double - antibody sandwich.

[0041] For multiple monoclonal antibodies with high titers, coating and HRP labeling were performed separately to screen for paired combinations. Antibodies with high binding affinity have higher detection sensitivity when applied to detection. Purposefully selecting these high - titer antibodies can make the screening of paired monoclonal antibodies more efficient.

[0042] The screening process is shown in Table 2.

[0043] Table 2: Screening results of the reactivity of different paired combinations of monoclonal antibodies.

[0044]

[0045] 1000* indicates a 1000 - fold dilution.

[0046] The experimental results showed that the double - antibody sandwich ELISA system composed of coating with monoclonal antibody 3D2 and HRP labeling with monoclonal antibody 1F10 had a relatively high detection reading for galactomannan antigen and no cross - reaction with the non - target antigen of cryptococcal capsular polysaccharide. Therefore, it was determined as the best antibody combination for detecting galactomannan antigen in the present invention.

[0047] 9. Biotin conjugation with antibodies The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) conjugated with monoclonal antibody was 20:1. First, 2.0 mg of activated biotin was added to 360 μL of ultrapure water and dissolved to prepare a 10 mM biotin solution. 2 mg of antibody was reacted with 26.6 μL of 10 mM biotin by shaking at room temperature for 3 h (the reaction system was controlled at about 2 mL). Then, the biotin-antibody mixture was dialyzed with 0.01 M PBS to remove excess free biotin, and an equal volume of glycerol was added, and it was stored at -20 °C. The final concentration of the labeled antibody obtained was about 0.5 mg / ml.

[0048] In this experiment, the biotin-conjugated monoclonal antibody used was the best detection antibody screened in the previous stage - monoclonal antibody 1F10. After being labeled with biotin, this antibody was used to construct a biotin-avidin signal amplification system.

[0049] 10. Establishment of biotin-avidin amplification ELISA system The above-screened monoclonal antibody combinations were used for coating and biotin conjugation respectively, and then by exploring the concentrations of the coating antibody, biotin antibody, and the dilution factor of HRP-labeled avidin, the most suitable reaction conditions for the amplification system were determined.

[0050] Monoclonal antibody was coated in a microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, overnight at 4 °C; the plate was washed once with the washing solution (PBST, PBS containing 0.05% Tween-20, pH 7.4), patted dry, blocked with 3% sucrose and 1% BSA, 150 μL per well, at 37 °C for 2 hours, and stored dry after patting dry for later use; the galactomannan antigen was diluted to a concentration of 10 ng / mL with PBS, 50 μL was added to the microplate coated with monoclonal antibody, and at the same time, the cryptococcal 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 flicked out, and the plate was washed 4 times with PBST washing solution and patted dry; the biotin-conjugated monoclonal antibody was diluted to concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL with PBS, 50 μL / well was added to the microplate, and reacted at 37 °C for 30 min. The liquid in the wells was flicked out, the plate was washed 4 times with PBST washing solution and patted dry, then 50 μL / well of HRP-polymeric streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10000, 20000, 40000 times with PBS) was added, reacted at 37 °C for 30 min, washed 4 times again, patted dry, then 50 μL / well of TMB chromogenic solution was added and developed at room temperature for 10 min, and finally the reaction was terminated by adding the termination solution, and the OD was measured with an enzyme-labeled instrument. 450nm value. Select the coating concentration, biotin monoclonal antibody concentration, dilution of HRP-labeled avidin, etc. with the most obvious positive and negative differences as the optimal reaction conditions.

[0051] The experimental results showed the optimal combination: coating monoclonal antibody 3D2 at a concentration of 1 μg / ml; biotin-conjugated monoclonal antibody 1F10 at a concentration of 2 μg / ml; HRP-polymeric streptavidin diluted 20,000-fold.

[0052] 11. Sensitivity identification of biotin-avidin amplified ELISA Determine the optimal reaction conditions such as coating concentration, biotin monoclonal antibody concentration, HRP-avidin dilution, etc. Referring to the above detection steps, first gradient dilute the galactomannan antigen (GM) with PBS buffer solution, and the diluted concentrations are 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, 10 pg / mL respectively. At the same time, take the cryptococcal capsular polysaccharide as a control, add 50 μL per well at the same concentration for detection, and determine the detection sensitivity of this detection system to the galactomannan antigen.

[0053] See Figure 2 , indicating that the detection method of this application has high detection sensitivity, and the lowest detection limit is about 100 pg / ml.

[0054] 12. Clinical evaluation of biotin-avidin amplified ELISA Use the established ELISA detection method to detect normal human serum and serum samples of patients with invasive aspergillosis. At the same time, use a commercial kit Aspergillus antigen detection kit (Bio-Rad Platelia aspergillusAg) for parallel testing.

[0055] The sample collection was specifically implemented by the cooperating hospital. There were 30 normal human sera from healthy people in the physical examination center. There were 20 serum samples of patients with invasive aspergillosis, which were determined by clinicians according to the guidelines combined with clinical symptoms and clinical test results.

[0056] Pretreatment of serum samples There will be a certain level of aspergillus fumigatus antibodies in normal human serum, and the galactomannan antigen will form an antigen-antibody complex with it, which will cause false negatives when detecting low-concentration galactomannan. In addition, endogenous or exogenous interfering factors in the blood such as rheumatoid factor and complement will also have a greater impact on the detection. Therefore, it is necessary to pretreat the serum samples to be tested. The present invention adopts two treatment methods of EDTA + heating or glycine + neutralization, specifically: Method 1: Add 200 ul of serum sample to 0.1 M disodium EDTA (pH 4.5) solution, and the volume of the disodium EDTA solution is 1 / 2 of the volume of the serum sample. Then add 30 ul of 0.01 M PBS (pH 7.4), mix well, heat at 100 °C for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant for detection.

[0057] Method 2: Add 200 ul of serum sample to 0.1 M glycine (pH 2.7), and the volume of the glycine solution is 1 / 2 of the volume of the serum sample. Mix well and let stand at room temperature for 5 minutes, then add 30 ul of neutralization buffer 1 M Tris-HCl (pH 9.0), mix thoroughly for neutralization, centrifuge at 12,000 rpm for 10 minutes, and take the supernatant for detection.

[0058] By adding galactomannan to normal serum to simulate positive serum and evaluating the above two sample pretreatment methods, it was found that the detection value of the sample treated by Method 2 was consistent with the detection of galactomannan antigen without adding serum, that is, using 0.1 M glycine (pH 2.7) solution to dissociate the antigen-antibody complex in the serum sample, and then adding an alkaline buffer (such as Tris-HCl, pH 9.0) for neutralization to restore the pH value of the sample to the range suitable for subsequent detection. The antigen-antibody complex is a complex formed by galactomannan antigen and endogenous or exogenous antibodies.

[0059] 13. Detection of clinical samples by ELISA method First, detect 30 serum samples from healthy people undergoing physical examinations, pretreat the samples according to Method 2 above, and detect the above samples using the established biotin-avidin amplified ELISA system to determine the average value of the detection value of aspergillus antigen in normal people, and determine the Cut-off value for detection as twice the average value. Then, pretreat and detect 20 serum samples from patients with invasive aspergillosis infection in the same way. The specific ELISA method for detection is as follows: Coat the antibody at a concentration of 1 μg / mL, incubate overnight at 4 °C, wash the plate and then block; take 50 μL of the pretreated serum to be tested, add it to the enzyme-linked immunosorbent assay plate coated with monoclonal antibody, and at the same time add galactomannan diluted to 10 ng / mL as a positive control and 50 μL of PBS as a blank control respectively, and react at 37 °C for 30 min. After washing the plate, add biotin-conjugated monoclonal antibody at 2 μg / mL, 50 μL / well, and react at 37 °C for 30 min. After washing and patting dry, add 20,000-fold diluted HRP-polymer streptavidin, react at 37 °C for 30 min, then develop color and terminate the reaction, and measure the OD 450 nm value.

[0060] The test results are shown in Figure 3 and Figure 4 , Figure 3This is the detection result of the ELISA method of this application. The vertical axis is OD 450 nm reading value, and the dotted line is the Cut-off value. Figure 4 This is the detection result of the Bio-Rad kit. The vertical axis is the I index, and the dotted line is the Cut-off value (I = 0.50). The detection results of normal human sera are all negative by both methods, and the results of detecting positive sera are also exactly the same. The OD 450 values of all samples from normal healthy populations are lower than the cut-off value, indicating that no significant Aspergillus fumigatus galactomannan antigen was detected in the normal population. The OD 450 values of samples from patients with invasive aspergillosis are higher than the Cut-off value, indicating that the detection method of this application can effectively identify serum samples of patients with invasive mycosis. Thus, it shows that the detection method of this application has high sensitivity and consistency in the patient population.

[0061] In addition, different from the index determination method of sample OD value / critical value control OD value used by the Bio-Rad kit, the self-developed ELISA method established in this study uses a fixed threshold method based on the mean value of samples from negative populations. By detecting 30 serum samples from healthy people, calculating the average OD 450 nm value, and setting 2 times of this value as the Cut-off value for determining positive results. This method does not need to add a critical value control in each experiment, nor does it need to perform index conversion, and has the advantages of simple operation, reagent saving, and being suitable for rapid clinical screening. Generally speaking, the effects of the two methods in detecting real clinical samples are relatively consistent, and the detection method of this invention has good clinical detection performance.

[0062] 14. Gene sequence Cloning and sequencing of monoclonal antibody variable region genes Use the RNeasy Mini Kit kit (Cat. No. 74104) to extract total RNA from hybridoma cells, and reverse transcribe it into cDNA with RandomPrimers; design universal primers for mouse antibody variable regions, amplify VH and VL genes by 2 rounds of PCR, and introduce Age1 and Bsiw1 restriction enzyme sites into the primers of the 3rd round of PCR. After cutting and purifying the PCR products, ligate them to the pUC19 vector, transform the TOP10 strain, pick single colonies for sequencing after culturing at 37°C for 14 h, and obtain the gene sequences of the heavy and light chains of the monoclonal antibody.

[0063] The specific sequences are as follows: Monoclonal antibody 3D2: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.17: GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGAAGAAGCCCGGCGAGACCGTGAGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCGGCGGCTGGATCAAGTGGGTGAAGCAGACCCCCGGCAAGGGCCTGAGGTGGATGGGCTGGCTGTGGGAGAAGGAGGGCAGCAGGACCTACAGCCTGGTGGCCAGCGGCAGGTTCGACTTCAGCAGCGAGACCAGCGCCAGCAGCGTGTACCTGCAGATCAACAACCTGAAGAACGAGGACACCGCCACCTACTTCTGCGCCAGGTTCCCCAAGATCTACTACGAGGTGAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCGCC。

[0064] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.13: EVQLQQSGPELKKPGETVRISCKASGYTFTSGGWIKWVKQTPGKGLRWMGWLWEKEGSRTYSLVASGRFDFSSETSASSVYLQINNLKNEDTATYFCARFPKIYYEVSWGQGTTLTVSA。

[0065] CDR region annotation: The sequence of CDR-H1 of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.1: SGGWIK; The sequence of CDR-H2 of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.2: WLWEKEGSRTYSLVASG; The sequence of CDR-H3 of the heavy chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.3: FPKIYYEVS。

[0066] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.18: AACATCGTGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCCTGGGCGACAGGGTGACCATCAGCTGCAGCGCCCAGCAGGGCCTGAGCAACTACATGACCTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACTACACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCGCCGACTTCAGCCTGACCATCAGCAACCTGGAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGTACAGCAGCTTCCCCCACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0067] The amino acid sequence of the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.14: NIVLTQSPSSLSASLGDRVTISCSAQQGLSNYMTWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGADFSLTISNLEPEDIATYYCQQYSSFPHTFGGGTKLEIKRTV。

[0068] CDR region annotation: The sequence of CDR-L1 in the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.4: SAQQGLSNYMT; The sequence of CDR-L2 in the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.5: YTSSLHS; The sequence of CDR-L3 in the light chain variable region of monoclonal antibody 3D2 is shown in SEQ ID NO.6: QQYSSFPHT。

[0069] Sequence of monoclonal antibody 1F10: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.19: CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGGCCCGGCACCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACGCCTTCACCAACTACTTCATCGAGTGGATCAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCGTGATCAACCCCGGCAGCAGCCACACCCACTACAACGACAAGCAGAAGGACAAGGCCACCCTGACCGCCGACAGGAGCAGCAGCACCGCCTACATGCACCTGAGGAGCCTGACCAGCGACGACAGCGCCGTGTACTTCTGCGCCTTCAGCCCCTACGAGGACAGCAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC。

[0070] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.15: QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYFIEWIKQRPGQGLEWIGVINPGSSHTHYNDKQKDKATLTADRSSSTAYMHLRSLTSDDSAVYFCAFSPYEDSSWGQGTTLTVSS。

[0071] CDR region annotation: 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.

[0072] Light chain: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F10 is shown in SEQ ID NO.20: GAGATCGTGCTGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCCTGGGCGACAGGGTGACCATCAGCTGCAGCGCCCAGCAGGGCCTGAGCAACTACATGACCTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACTACAACAGCAGCCTGCACAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCGCCGACTTCAGCCTGACCATCAGCAACCTGGAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGGTGAGCAGCATCCCCCACACCTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0073] The amino acid sequence of the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.16 as follows: EIVLTQSPSSLSASLGDRVTISCSAQQGLSNYMTWYQQKPDGTVKLLIYYNSSLHSGVPSRFSGSGSGADFSLTISNLEPEDIATYYCQQVSSIPHTFGGGTKLELKRTV。

[0074] CDR region annotation: The sequence of CDR-L1 of monoclonal antibody 1F10 is shown in SEQ ID NO.10 as: SAQQGLSNYMT; The sequence of CDR-L2 of monoclonal antibody 1F10 is shown in SEQ ID NO.11 as: YNSSLHS; The sequence of CDR-L3 of monoclonal antibody 1F10 is shown in SEQ ID NO.12 as: QQVSSIPHT.

[0075] So far, the 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. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0076] Although some specific embodiments of the present application have been described in detail by way of examples, those skilled in the art should understand that the above examples are only for illustration purposes and not for limiting the scope of the present application. Those skilled in the art should understand that the above embodiments can be modified or partial technical features can be equivalently replaced without departing from the scope and spirit of the present application.

Claims

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

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

14.

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

16.

4. The monoclonal antibody combination of Aspergillus fumigatus according to claim 3, wherein 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.

5. The monoclonal antibody combination of Aspergillus fumigatus according to claim 4, wherein 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.

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

7. The application according to claim 6, wherein The tool is used for detecting galactomannan antigen derived from Aspergillus fumigatus.

8. The application according to claim 7, wherein The tool includes reagents, kits, test strips and antibody chips.

9. The application according to claim 8, wherein The monoclonal antibody combination is used to construct an ELISA detection system, which is used for the detection of galactomannan antigen in samples, and the ELISA detection system is not used for the diagnosis of diseases.

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

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