A monoclonal antibody combination for detecting Candida albicans enolase protein and its application

Abstract

The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody combination and application for the detection of Candida albicans enolase protein. The monoclonal antibody combination comprises monoclonal antibodies 5G7 and 5B1. The amino acid sequences of the CDR regions of monoclonal antibodies 5G7 and 5B1 are shown in SEQ ID NO.1-12. The antibody combination has high specificity and binding activity and can be used for the efficient detection of Candida albicans enolase. The colloidal gold detection test strip constructed based on the combination has good sensitivity and specificity, with a minimum detection limit of up to 500pg / ml, and does not cross-react with other common fungi. The present invention provides a reliable tool for the early diagnosis and rapid detection of invasive Candida infections, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody combination for detecting Candida albicans enolase protein and its application. 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. Invasive candidiasis (IC) is a severe systemic infection caused by Candida species that invade the bloodstream or deep tissues. It carries a high mortality rate and is a common clinical invasive fungal disease. Its main manifestations include candidemia and candidiasis, in which Candida can be isolated and cultured from the blood or other sterile sites, such as the pleural and peritoneal cavities. It can also lead to septic shock with multiple organ failure. Candida species are opportunistic, yeast-like fungi that are widely found on the human skin, mucous membranes, and intestines. Under conditions of immunosuppression (such as anti-rejection drugs for organ transplantation) or dysbiosis (such as diabetes, HIV infection, chemotherapy, or long-term antibiotic / corticoid use), these infections can cause superficial infections (such as oral candidiasis) or invasive infections (such as candidemia and deep organ infections).

[0003] Enolase, present in the cytoplasm and cell wall of Candida albicans, is one of the most abundant proteins in the bacterial system. Composed of 440 amino acids and approximately 47-48 kDa in size, it is a key enzyme in the Candida glycolytic pathway. During invasive infection, it is expressed and secreted in high quantities and is considered an immunogen that plays a crucial role in invasive infection. Enolase is highly conserved among fungi and is widely expressed in various species, including Candida albicans, Candida glabrata, and Candida tropicalis. It can adhere to host cells or induce immune evasion, contributing to fungal pathogenicity. When Candida invades the bloodstream or deep tissues, enolase is continuously expressed at high levels in fungal cells and released into the bloodstream or body fluids early in infection, often before the onset of clinical symptoms or a positive traditional blood culture result. It is a relatively sensitive marker for infection detection.

[0004] Studies have shown that when Candida albicans is cultured, the level of enolase increases with hyphal growth, suggesting that enolase detection can reflect the invasive growth of C. albicans and help assess the occurrence of IC and monitor treatment efficacy. Enolase research typically focuses on antibody detection, and commercially available C. albicans enolase IgG antibody detection kits are used for clinical diagnosis. However, because invasive Candida infections often occur in immunocompromised individuals, who rarely produce antibodies, antibody testing lacks sensitivity for diagnosing IC, necessitating the use of antigen detection or other complementary methods to improve detection accuracy.

[0005] Because enolase is only released during Candida albicans infection and is a highly conserved protein, enolase antigen detection has important diagnostic value for IC. However, there are currently no commercially available kits or monoclonal antibodies specifically targeting Candida albicans enolase, limiting the application and development of this marker in clinical diagnosis. Summary of the Invention

[0006] Given the lack of specific detection methods for Candida albicans enolase protein in the existing technology, and the lack of monoclonal antibodies or supporting detection kits available on the market for the detection of this protein, it is difficult to achieve early, rapid and accurate diagnosis of invasive Candida infections. The present invention provides a monoclonal antibody combination and application for the detection of Candida albicans enolase protein, which solves the technical problem of the lack of high-affinity and high-specificity monoclonal antibodies and their application in antigen detection in the existing technology.

[0007] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] A monoclonal antibody combination for detecting Candida albicans enolase protein, comprising monoclonal antibody 5G7 and monoclonal antibody 5B1.

[0009] The heavy chain variable region of monoclonal antibody 5G7 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively;

[0010] The light chain variable region of monoclonal antibody 5G7 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 5B1 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 5B1 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 a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO.14.

[0014] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5B1 is shown as SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 5B1 is shown as SEQ ID NO.16.

[0015] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G7 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G7 is shown as SEQ ID NO.18.

[0016] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5B1 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5B1 is shown as SEQ ID NO.20.

[0017] The second aspect is based on the use of the above monoclonal antibody combination in the preparation of a tool for detecting Candida albicans enolase protein.

[0018] In further embodiments, the kit includes colloidal gold test strips, reagents, kits, and antibody chips.

[0019] In a further embodiment, the colloidal gold test strip uses monoclonal antibody 5G7 as the capture antibody and monoclonal antibody 5B1 as the labeling antibody.

[0020] In a further embodiment, the colloidal gold test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper sequentially connected to a back plate.

[0021] In a further embodiment, a detection line and a quality control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 5G7, the quality control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5B1.

[0022] Beneficial effects:

[0023] The present invention provides a monoclonal antibody combination for detecting Candida albicans enolase protein, comprising monoclonal antibodies 5G7 and 5B1. Both antibodies have well-defined complementarity determining region (CDR) sequences. The heavy chain CDRs of 5G7 are shown in SEQ ID NOs. 1-3, and the light chain CDRs are shown in SEQ ID NOs. 4-6; the heavy chain CDRs of 5B1 are shown in SEQ ID NOs. 7-9, and the light chain CDRs are shown in SEQ ID NOs. 10-12. This antibody combination exhibits excellent antigen specificity and binding capacity.

[0024] The monoclonal antibody combination of the present invention can efficiently and specifically identify the Candida albicans enolase protein, significantly improving detection sensitivity and accuracy. Experimental results show that this antibody combination can not only be used to detect purified recombinant enolase protein, but can also effectively identify Candida albicans culture supernatant samples, and has no cross-reaction with other common fungi, showing good clinical application prospects. In addition, this antibody combination has been successfully applied to the development of colloidal gold test strips, achieving rapid and convenient detection of the target protein, providing a powerful tool for early diagnosis and efficacy monitoring of invasive Candida albicans infection.

[0025] In summary, the monoclonal antibody combination provided by the present invention has the advantages of strong specificity, high sensitivity, and good stability. It can be widely used in the development of various diagnostic platforms such as immunoassay-related kits, test strips, and antibody chips, and has significant practical value and promotion prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 This is an SDS-PAGE analysis diagram of the recombinant Candida albicans enolase protein;

[0028] Figure 2 This is a comparison chart of the test results of colloidal gold test strips and commercial test strips;

[0029] Figure 3 This is a schematic diagram of the assembly of the colloidal gold test strip;

[0030] Figure 4 It is the specific test result of the colloidal gold test strip;

[0031] Figure 5This is a graph showing the sensitivity of the colloidal gold test strip to the enolase recombinant protein;

[0032] Figure 6 Figure 2 is a graph showing the binding activity of paired monoclonal antibodies. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Example 1

[0037] 1. Recombinant expression of Candida albicans enolase antigen

[0038] The Candida albicans enolase gene (Genbank#L10290.1) was synthesized by Qingke Biotechnology and cloned into the pET28a vector. The recombinant expression plasmid was confirmed by sequencing and transformed into Escherichia coli Rosetta (DE3) competent cells. Expression was induced with IPTG. Optimal expression conditions were selected. After large-scale culture, the harvested cells were fragmented and purified using various methods, including Ni affinity chromatography and molecular sieve separation, to obtain highly pure recombinant Candida albicans enolase protein.

[0039]

[0040] The recombinant protein of Candida albicans enolase was identified by induction and was efficiently expressed in a soluble form at 30°C and 200 rpm for 4 hours. Figure 1 The SDS-PAGE results showed that the purified Candida albicans enolase recombinant protein had a single band between 43-55 kDa, with a purity of more than 85%, and was close to the expected size of 51.8 kDa (including the His tag), indicating that the Candida albicans enolase recombinant protein was effectively expressed.

[0041] 2. Mouse Immunization

[0042] Six-week-old female BALB / c mice were immunized intraperitoneally with cultured Candida albicans supernatant. Two weeks later, mice were immunized subcutaneously at multiple sites with 20 μg of recombinant Candida albicans enolase protein emulsified in Freund's incomplete adjuvant. Subsequently, mice were immunized twice more intramuscularly, mixing 20 μg of the antigen with an equal volume of MF59 adjuvant. 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 recombinant Candida albicans enolase protein as the antigen. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0043] 3. Screening of hybridoma cell lines

[0044] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and cultured in HAT medium for selection. When the fused cells reached half the bottom of the well, clones positive for the recombinant Candida albicans enolase protein were screened by indirect ELISA. Simultaneously, cells were coated with recombinant protein expressed in the same pET28a vector (pET28a-HPV16 / E7, i.e., HPV16 / E7 recombinant protein) to identify cell lines that specifically reacted with the recombinant Candida albicans enolase protein. Positive cells were cloned to a single clone by limiting dilution, expanded, and cryopreserved.

[0045] 4. Screening of positive clones by indirect ELISA:

[0046] Microwell plates were coated with recombinant Candida albicans enolase protein and recombinant protein expressed by the same pET28a vector (pET28a-HPV16 / E7, i.e., HPV16 / E7 recombinant protein, synthesized by Qingke Biotechnology). The coating buffer (carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) was 1 μg / mL at a coating concentration of 1 μg / mL and incubated at 4°C overnight. The plates were then blocked with 1% gelatin, 150 μL per well, at 37°C for 2 hours, washed once with washing buffer, and patted dry. 50 μL of 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 The positive cell lines that reacted only with the recombinant protein of Candida albicans enolase were selected for monoclonal antibody preparation.

[0047] 5. Preparation of Monoclonal Antibody Ascites

[0048] 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 12,000 rpm for 10 minutes, and the mid-layer was collected.

[0049] 6. Affinity chromatography purification of monoclonal antibodies

[0050] The ascites was centrifuged at 12000 r / min for 5 min, the supernatant was diluted with 3 volumes of 0.01 M sodium acetate, pH 4.0 solution, and then 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 PBS, 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 absorption 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. The concentration of the purified monoclonal antibody was measured using an ultra-micro spectrophotometer and stored at -20°C.

[0051] Characterization of purified monoclonal antibodies.

[0052] After purification, the monoclonal antibodies screened above were used to coat a microplate with Candida albicans enolase recombinant protein and an HPV16 / E7 control recombinant protein at a concentration of 1 μg / ml using the indirect ELISA method. The monoclonal antibodies to be tested were serially diluted to 10 μg / ml, 1 μg / ml, and 100 ng / ml, and the binding activity of the antibodies to the antigen was determined. The data are shown in Table 1 below:

[0053] Table 1: Identification of the binding activity of the screened monoclonal antibodies to the recombinant protein of Candida albicans enolase.

[0054]

[0055] The table shows that all positive monoclonal antibodies screened specifically reacted with the recombinant Candida albicans enolase protein and did not cross-react with the HPV16 / E7 recombinant protein expressed in the pET28a vector. Although different monoclonal antibodies have different epitopes and binding activities, all screened antibodies are specific for Candida albicans enolase.

[0056] 7. Preparation of colloidal gold test strips:

[0057] The 16 anti-C. albicans enolase monoclonal antibodies screened above were streaked onto nitrocellulose membranes of varying sizes (20 mm x 300 mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL in PBS, pH 7.4) were sprayed horizontally in lines at a rate of 0.8 μL / cm to form the test line (T line). At 6 mm intervals, goat anti-mouse IgG antibody (diluted to a concentration of 1 mg / mL in 0.01 M PBS, pH 7.4) was sprayed horizontally in lines at a rate of 0.8 μL / cm to form the control line (C line).

[0058] 8. Preparation of antibody-colloidal gold labeled complex:

[0059] Antibody Labeling: Prepare colloidal gold solution using the sodium citrate reduction method, ultimately obtaining a 1.0% gold particle solution. For labeling 1 mL of colloidal gold solution, add 0.2 M potassium carbonate solution in a gradient of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to a centrifuge tube containing 1 mL of colloidal gold solution to achieve a pH that allows efficient coupling of the antibody and colloidal gold. Mix thoroughly, then add 5 μg of the monoclonal antibody to be labeled. Mix quickly and incubate at room temperature for 10 minutes. Then, add 10 μL of 10% (w / v) bovine serum albumin (BSA) to block nonspecific binding sites, and continue incubating at room temperature for 10 minutes. Add 10 μL of 10% (w / v) polyethylene glycol 20,000 (PEG20,000) to enhance labeling stability. Mix thoroughly, centrifuge at 12,000 rpm for 10 minutes, and discard the supernatant. Resuspend the precipitate with resuspending solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose) to obtain the antibody-colloidal gold labeled complex and store it in the dark at 4°C until use.

[0060] 9. Screening of paired antibodies:

[0061] Nitrocellulose membranes streaked with different monoclonal antibodies were paired with colloidal gold-labeled monoclonal antibodies for reaction. The membranes were then tested with recombinant Candida albicans enolase protein (diluted to 50 ng / mL) and a control HPV16 / E7 recombinant protein (diluted to 50 ng / mL). The combination that showed a strong color response to the recombinant Candida albicans enolase protein and did not react with the control protein was selected as the optimal pair for further testing of Candida albicans and other fungi. The screening process is shown in Table 2.

[0062] Table 2: Results of screening paired monoclonal antibodies using recombinant Candida albicans enolase protein.

[0063]

[0064] - indicates negative, i.e. no color development; + / ++ / +++ indicates positive, i.e. a color reaction. The more + signs there are, the darker the color development, i.e. the stronger the positive reaction.

[0065] Table 2 shows the screening results using recombinant Candida albicans enolase protein diluted to a concentration of 50 ng / ml as a positive antigen. The results for HPV16 / E7 recombinant protein and the blank dilution were all negative and are not shown. The results showed that the combination of monoclonal antibody 5G7 as the capture antibody and monoclonal antibody 5B1 as the labeling antibody produced the deepest color development for recombinant Candida albicans enolase protein, making this the optimal pairing. This means that the combination of monoclonal antibody anti-5G7 as the capture antibody and monoclonal antibody 5B1 as the labeling antibody specifically recognizes recombinant Candida albicans enolase protein.

[0066] To further verify the practicality of this pairing combination, the supernatant of Candida albicans culture (colony count of 4.5x10 6 CFU / mL), and tested with a 10-fold dilution, and compared with the Candida albicans test strips commercialized by Dawn Biotechnology. Figure 2 , the results show that, Figure 2 The leftmost side of the figure shows the test strip of the present invention. "Bai Nian 10 times" means the supernatant of Candida albicans culture diluted 10 times (the colony count is 4.5 x 10 6 CFU / mL), Figure 2 The middle test strip is a commercialized Candida albicans test strip from Liming Biotechnology, which is also used to test the supernatant of Candida albicans culture diluted 10 times (the colony count is 4.5x 10 6 CFU / mL), Figure 2 The test strip on the far right is a commercialized Candida albicans test strip produced by Dawn Biotechnology, which detects 1 ug / ml of ENO (recombinant Candida albicans enolase protein).

[0067] The test strip of the present invention not only detects Candida albicans culture supernatant but also directly identifies purified recombinant Candida albicans enolase protein, indicating that the test strip's detection target is the Candida albicans enolase protein. Commercial test strips, on the other hand, only detect Candida albicans culture supernatant and are unable to identify recombinant Candida albicans enolase protein, indicating that their target is not the Candida albicans enolase protein. Therefore, the present test strip has a more specific and accurate detection mechanism. With Candida albicans enolase protein as an infection marker, the present invention can be used for early diagnosis of Candida albicans infection and rapid detection in specific scenarios, demonstrating greater potential for application.

[0068] Sample dilution optimization:

[0069] Diluent 1: 0.01 mol / L PBS (pH 7.4) containing 0.2% Triton X-100 and 0.9% NaCl;

[0070] Dilution 2: 0.01 mol / L PBS (pH 7.4) containing 0.2% Triton X-100, 0.9% NaCl, and 0.1% SDS;

[0071] Dilution 3: 0.01 M Tris (pH 8.0) containing 0.2% Triton X-100 and 0.9% NaCl;

[0072] Dilution 4: 0.01 M Tris (pH 8.0) containing 0.2% Triton X-100, 0.9% NaCl, and 0.1% SDS.

[0073] During the testing process, the sample diluent was optimized and compared to determine the optimal diluent formula: 0.01 mol / L PBS (pH 7.4) containing 0.2% Triton X-100, 0.9% NaCl, and 0.1% SDS. This diluent effectively improved detection sensitivity and specificity while reducing nonspecific binding interference.

[0074] 10. Preparation and assembly of Candida albicans test strips.

[0075] Preparation of colloidal gold pad: A 6 mm × 300 mm glass fiber membrane was used as the colloidal gold conjugate pad. After treatment with PBS containing 1% BSA and 1% Tween-20, the prepared colloidal gold-labeled antibody was diluted with diluent and sprayed onto the conjugate pad at a rate of 1.2 ml per strip. The membrane was then dried at 40°C for 2 hours and then used.

[0076] Assembly of test strips: see Figure 3 A 60mm × 300mm PVC backboard was used as the supporting substrate, and nitrocellulose membrane, colloidal gold pad, sample pad and absorbent paper were pasted on it in sequence. The nitrocellulose membrane was coated with a test line (monoclonal antibody 5G7) and a quality control line (goat anti-mouse IgG), and the colloidal gold pad was coated with monoclonal antibody 5B1. After drying at 40°C for 2 hours, the large board was cut into test strips with a width of 4.05mm using a strip cutter and loaded into a plastic card shell, so that the sample pad was exposed to the sample loading hole position, and the test line and quality control line were exposed to the observation window position, completing the assembly of the Candida albicans detection test strip.

[0077] 11. Sensitivity and specificity test of Candida albicans test strips

[0078] Positive samples include: Candida albicans enolase recombinant protein.

[0079] Negative samples include: Candida glabrata culture supernatant, Candida tropicalis culture supernatant, Cryptococcus neoformans culture supernatant, Aspergillus fumigatus culture supernatant, and blank dilution.

[0080] All samples were serially diluted using an optimized sample diluent (0.01 mol / L PBS pH 7.4 + 0.2% TX100 + 0.9% NaCl + 0.1% SDS). Culture supernatants of Candida glabrata, Candida tropicalis, Cryptococcus neoformans, and Aspergillus fumigatus were used as negative samples and uniformly diluted to a colony count of 1.0 x 10 6 CFU / mL

[0081] Detection method: After serial dilution of positive and negative samples, take 80 μL of each sample and add it dropwise to the sample well of the test strip. Let it stand at room temperature for 20 minutes and observe the results.

[0082] Result judgment criteria: If both the T line and the C line show clear red stripes, it is judged as positive; if only the C line shows color, it is negative; if the C line does not show color, it is judged as invalid.

[0083] The culture supernatants of Candida glabrata, Candida tropicalis, Cryptococcus neoformans, and Aspergillus fumigatus were all commercially available and diluted 10-fold for testing. Figure 4 , Figure 4 The diluent used was 0.01 mol / L PBS (pH 7.4) containing 0.2% Triton X-100, 0.9% NaCl, and 0.1% SDS. Candida glabrata, Candida tropicalis, Cryptococcus neoformans, and Aspergillus fumigatus were diluted 10-fold from the culture supernatant of Candida glabrata, Candida tropicalis, Cryptococcus neoformans, and Aspergillus fumigatus, respectively. All negative samples tested negative.

[0084] Sensitivity test results show that, see Figure 5 The test strips of the present invention have a detection limit of 500 pg / ml for recombinant Candida albicans enolase protein, demonstrating high sensitivity. Furthermore, in specificity testing, only Candida albicans-related samples tested positive, while all other fungal and control samples tested negative, further validating the high specificity of this paired combination.

[0085] 12. Identification of paired monoclonal antibodies

[0086] The recombinant protein of Candida albicans enolase was coated on an ELISA plate at a concentration of 1ug / ml. The paired monoclonal antibody was serially diluted to 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml and 1ng / ml according to the indirect ELISA method to identify the titer of the antibody.

[0087] The binding activity of the paired monoclonal antibodies 5G7 and 5B1 was tested by indirect ELISA. Figure 6The results showed that both monoclonal antibodies could bind well to the recombinant protein of Candida albicans enolase, and were still positive at a monoclonal antibody dilution concentration of 1 ng / ml, indicating that the two monoclonal antibodies had a high titer when binding to the recombinant ENO antigen. Figure 6 “mAb” is the abbreviation of “monoclonal antibody”, which means monoclonal antibody, Ctrl is the Aspergillus fumigatus galactomannan monoclonal antibody, 5G7 and 5B1 represent monoclonal antibody 5G7 and monoclonal antibody 5B1, respectively.

[0088] The preparation process of Aspergillus fumigatus galactomannan monoclonal antibody is as follows:

[0089] Whole cell antigen: Aspergillus fumigatus natural antigen was purchased from Veron Seron GmbH, Germany (BA132F01). It is a whole cell antigen purified by inactivation and lysis of the culture of Aspergillus fumigatus Ag-507. The supernatant of the whole cell antigen culture of Aspergillus fumigatus was sterilized at 121°C for 20 minutes and then lysed.

[0090] 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. Because the purified polysaccharide antigen has a small molecular weight, poor immunogenicity, and cannot be directly coated, it was conjugated to KLH and BSA to prepare a complete antigen for mouse immunization and coating screening. Subsequent mouse immunization and coating screening follow conventional techniques. Six-week-old female BALB / c mice were immunized subcutaneously with whole Aspergillus fumigatus antigen mixed with an equal volume of Freund's complete adjuvant (200 μL) at multiple sites. A dose of 30 μg / mouse was administered. Two weeks later, KLH-conjugated GM antigen was administered subcutaneously at multiple sites. 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 high titers were selected for intraperitoneal boosting with KLH-GM antigen. Three days later, spleens were harvested for hybridoma cell production. All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then 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-bacterial antigens from Cryptococcus neoformans and Candida glabrata were used for screening to identify specific cell lines that reacted only with Aspergillus fumigatus and did not react with these bacteria. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were then expanded and cryopreserved to obtain monoclonal antibodies against Aspergillus fumigatus galactomannan.

[0091] 13. Sequence of paired monoclonal antibody

[0092] 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.

[0093] Monoclonal antibody 5G7:

[0094] Heavy chain:

[0095] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO. 17: CAGGTGCAGCTGAAGCAGAGCGGCCCCGAGCTGAAGAAGCCCGGCGAGACCGTGAGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCAGGCAGGGCATGAACTGGGTGAAGCAGACCCCCGGCAAGGGCCTGAGGTGGATGGGCTGGCTGAACACCCTGACCGGCCAGCAGACCTACGTGGACGACTTCAGCGGCAGGTTCGACTTCAGCAGCGAGACCAGCGCCAGCAGCGTGTACCTGCAGATCAACAACCTGAAGAACGAGGACACCGCCACCTACTTCTGCGCCAGGGCCCCCGGCACCAACGTGTTCGACAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC.

[0096] Amino acid sequence:

[0097] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO.13:

[0098] QVQLKQSGPELKKPGETVRISCKASGYTFTRQGMNWVKQTPGKGLRWMGWLNTLTGQQTYVDDFSGRFDFSSETSASSVYLQINNLKNEDTATYFCARAPGTNVFDSWGQGTTLTVSS.

[0099] CDR region annotation:

[0100] The amino acid sequences of the heavy chain variable region of monoclonal antibody 5G7, including the complementarity determining region CDR-H1, are shown in SEQ ID NO. 1: RQGMN;

[0101] The amino acid sequences of the heavy chain variable region of monoclonal antibody 5G7, including the complementarity determining region CDR-H2, are shown in SEQ ID NO. 2: WLNTLTGQQTYVDDFSG;

[0102] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G7, including the complementarity determining region CDR-H3, is shown in SEQ ID NO. 3: APGTNVFDS.

[0103] Light chain:

[0104] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO.18:

[0105] GACATCCTGATGACCCAGAGCCCCAGCTTCAGGAGCGTGAGCGTGGGCGAGAAGGTGATCATGAGCTGCAAGAGCAGCAGGAGCCTGCTGTACAGCGAGAACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCATCCTGCTGATCAGCTGGGCCAGC ACCAGCGAGAGCGGCGTGCCCGACAGGTTCACCGGCAGGGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGAAGGCCGAGGACCTGGCCCTGTACTACTGCCAGCAGTACTACATGTACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0106] The amino acid sequence of the light chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO.14: DILMTQSPSFRSVSVGEKVIMSCKSSRSLLYSENQKNYLAWYQQKPGQSPILLISWASTSESGVPDRFTGRGSGTDFTLTISSVKAEDLALYYCQQYYMYPLTFGAGTKLELKRTV.

[0107] CDR region annotation:

[0108] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 5G7 are shown in SEQ ID NO. 4: KSSRSLLYSENQKNYLA;

[0109] The amino acid sequences of the complementary determining region CDR-L2 of the light chain variable region of monoclonal antibody 5G7 are shown in SEQ ID NO. 5: WASTSES;

[0110] The amino acid sequence of the complementary determining region CDR-L3 of the light chain variable region of monoclonal antibody 5G7 is shown in SEQ ID NO. 6: QQYYMYPLT.

[0111] Monoclonal Antibody 5B1:

[0112] Heavy chain:

[0113] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5B1 is shown in SEQ ID NO. 19: GAGGTGCAGCTGAAGCAGAGCGGCCCCGAGCTGAAGAAGCCCGGCGAGACCGTGAGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCCAGGGCTGGAAGTGGGTGAAGCAGACCCCCGGCAAGGGCCTGAGGTGGATGGGCTGGCTGAACACCCTGGAGGGCCAGCAGACCTACGTGTTCTTCTTCAGCGGCAGGTTCGACTTCAGCAGCGAGACCAGCGCCAGCAGCGTGTACCTGCAGATCAACAACCTGAAGAACGAGGACACCGCCACCTACTTCTGCGCCAGGGCCCCCGGCATCAACGTGTTCGACAGCTGGGGCCAGGGCACCACCCTGACCGTGAGCAGC.

[0114] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5B1 is shown in SEQ ID NO.15:

[0115] EVQLKQSGPELKKPGETVRISCKASGYTFTSQGWKWVKQTPGKGLRWMGWLNTLEGQQTYVFFFSGRFDFSSETSASSVYLQINNLKNEDTATYFCARAPGINVFDSWGQGTTLTVSS.

[0116] CDR region annotation:

[0117] The amino acid sequences of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 5B1 are shown in SEQ ID NO. 7: SQGWK;

[0118] The amino acid sequences of the complementarity determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 5B1 are shown in SEQ ID NO. 8: WLNTLEGQQTYVFFFSG;

[0119] The amino acid sequences of the complementarity determining region CDR-H3 of the heavy chain variable region of the monoclonal antibody 5B1 are shown in SEQ ID NO. 9: APGINVFDS;

[0120] Light chain:

[0121] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5B1 is shown in SEQ ID NO. 20: GAGATCGTGCTGACCCAGAGCCCCAGCTTCAGGAGCGTGAGCGTGGGCGAGAAGGTGATCATGAGCTGCAAGAGCAGCAGGAGCTTCCTGTACAGCGAGAACCAGAAGAACGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCATCCTGCTGATCAGCATGGCCAGCGTGAGCGAGAGCGGCGTGCCCGACAGGTTCACCGGCAGGGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCGTGAAGGCCGAGGACCTGGCCCTGTACTACTGCCAGCAGTACTACATGTACCCCATCACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0122] The amino acid sequence of the light chain variable region of monoclonal antibody 5B1 is shown in SEQ ID NO.16:

[0123] EIVLTQSPSFRSVSVGEKVIMSCKSSRSFLYSENQKNAWYQQKPGQSPILLISMASVSESGVPDRFTGRGSGTDFTLTISSVKAEDLALYYCQQYYMYPITFGAGTKLELKRTV.

[0124] CDR region annotation:

[0125] The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of the monoclonal antibody 5B1 are shown in SEQ ID NO. 10: KSSRSFLYSENQKNA;

[0126] The amino acid sequences of the complementarity determining region CDR-L2 of the light chain variable region of monoclonal antibody 5B1 are shown in SEQ ID NO. 11: MASVSES;

[0127] The amino acid sequence of the complementarity determining region CDR-L3 of the light chain variable region of monoclonal antibody 5B1 is shown in SEQ ID NO. 12: QQYYMYPIT.

[0128] 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.

[0129] 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 for detecting Candida albicans enolase protein, characterized in that: The monoclonal antibody combination includes monoclonal antibody 5G7 and monoclonal antibody 5B1, The heavy chain variable region of the monoclonal antibody 5G7 includes three complementarity determining regions: CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 5G7 includes three complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3). The amino acid sequence of CDR-L1 is shown in SEQ ID NO. 4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO. 5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6. The heavy chain variable region of the monoclonal antibody 5B1 includes three complementarity determining regions: CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.8, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

9. The light chain variable region of the monoclonal antibody 5B1 includes three complementarity determining regions CDR-L1, CDR-L2 and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

12.

2. The monoclonal antibody combination for detecting Candida albicans enolase protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5G7 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5G7 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting Candida albicans enolase protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5B1 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 5B1 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting Candida albicans enolase protein according to claim 3, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5G7 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5G7 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting Candida albicans enolase protein according to claim 4, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5B1 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5B1 is shown in SEQ ID NO.

20.

6. Use of the monoclonal antibody combination according to claim 1 in preparing a tool for detecting Candida albicans enolase protein.

7. The use according to claim 6, characterized in that The tools include colloidal gold test strips, reagents, test kits and antibody chips.

8. The use according to claim 7, characterized in that The colloidal gold test strip uses monoclonal antibody 5G7 as a capture antibody and monoclonal antibody 5B1 as a labeling antibody.

9. The use according to claim 8, characterized in that The colloidal gold test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and absorbent paper which are sequentially connected to a back plate.

10. The use according to claim 9, characterized in that A detection line and a quality control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 5G7, the quality control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5B1.

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