Candida mannan antibody combination and use thereof

Abstract

The application provides an anti-candida mannan monoclonal antibody combination and application thereof. The anti-candida mannan monoclonal antibody combination comprises a first antibody and a second antibody, the first antibody comprises a first light chain variable region and a first heavy chain variable region, and the second antibody comprises a second light chain variable region and a second heavy chain variable region. The amino acid sequence of the first light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:1-3. The amino acid sequence of the first heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:4-6. The amino acid sequence of the second light chain variable region comprises VL-CDR1-3 shown in SEQ ID NO:7-9. The amino acid sequence of the second heavy chain variable region comprises VH-CDR1-3 shown in SEQ ID NO:10-12. The monoclonal antibody combination can improve the sensitivity and accuracy of in-vitro detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to an anti-candida mannan monoclonal antibody combination and application thereof. BACKGROUND

[0002] Candida is a kind of opportunistic pathogenic fungi, which can cause severe systemic infection in people with low immune function. In recent years, the incidence of candida infection has increased significantly, especially in intensive care unit (ICU) patients, broad-spectrum antimicrobial drug users, long-term hospitalized patients and immunosuppressed populations, which has become an important pathogenic bacterium of blood stream infection and catheter-related infection. According to statistics, candida ranks fourth in blood-borne infection and third in catheter-related infection. Even with antifungal drug treatment, the mortality rate is still as high as more than 40%.

[0003] Invasive candidiasis can involve multiple organ tissues, and its clinical manifestations are complex and diverse, and early diagnosis is difficult, which is easy to delay treatment. Therefore, the development of rapid, sensitive and specific in vitro diagnostic methods is of great significance for improving patient survival rate and guiding clinical drug use.

[0004] Mannan is the main component of candida cell wall, accounting for about 7% of the dry weight of the fungus, which will be released into the blood during the infection process, and thus can be used as a biomarker for invasive candida infection. The detection of mannan as an auxiliary diagnostic indicator has been recommended by the domestic and foreign expert consensus and clinical guidelines. However, the existing detection methods have certain limitations, such as unstable sensitivity and specificity, cross interference by other fungal components or polysaccharide structures, short detection window period and high risk of missed detection.

[0005] Immune detection technology has been widely used in pathogen detection due to its good specificity and convenient operation. In the prior art, methods such as immunochromatography, enzyme-linked immunosorbent assay (ELISA), immunofluorescence and chemiluminescence immunoassay can be used for antigen detection. However, the core of the above methods is to use high-quality antibodies as recognition elements.

[0006] Monoclonal antibodies have become the basis of high-performance immune detection due to their specific targeting of specific antigen epitopes, high uniformity and strong batch stability. Usually, specific antibody cell strains are obtained through hybridoma technology, and further sequence analysis, expression vector construction and recombinant expression are carried out to realize large-scale stable production. However, there is still a lack of a high-affinity and high-specificity anti-candida mannan monoclonal antibody, which limits the development of high-performance diagnostic products based on this target.

[0007] Therefore, it is urgent to provide an anti-candida mannan monoclonal antibody with high affinity and high specificity, in order to construct an in vitro immune diagnostic tool with excellent performance, so as to meet the needs of early detection and accurate diagnosis of invasive candidiasis. SUMMARY

[0008] The application provides an anti-candida mannan monoclonal antibody combination and application thereof, aiming to solve the technical problems of low detection sensitivity, poor specificity, easy cross-reaction and poor antibody batch stability in existing in vitro diagnosis of candida infection, and is suitable for various immunodetection methods, and is used for improving early detection rate and diagnostic accuracy of candida infection.

[0009] In a first aspect, the application provides an anti-candida mannan monoclonal antibody combination, comprising a first antibody and a second antibody, wherein the first antibody comprises a first light chain variable region and a first heavy chain variable region, and the second antibody comprises a second light chain variable region and a second heavy chain variable region; wherein,

[0010] The amino acid sequence of the first light chain variable region comprises VL-CDR1 shown in SEQ ID NO:1, VL-CDR2 shown in SEQ ID NO:2 and VL-CDR3 shown in SEQ ID NO:3.

[0011] The amino acid sequence of the first heavy chain variable region comprises VH-CDR1 shown in SEQ ID NO:4, VH-CDR2 shown in SEQ ID NO:5 and VH-CDR3 shown in SEQ ID NO:6.

[0012] The amino acid sequence of the second light chain variable region comprises VL-CDR1 shown in SEQ ID NO:7, VL-CDR2 shown in SEQ ID NO:8 and VL-CDR3 shown in SEQ ID NO:9.

[0013] The amino acid sequence of the second heavy chain variable region comprises VH-CDR1 shown in SEQ ID NO:10, VH-CDR2 shown in SEQ ID NO:11 and VH-CDR3 shown in SEQ ID NO:12.

[0014] According to the application, the monoclonal antibody combination simultaneously comprises two rabbit-derived antibodies, which can recognize two epitopes of candida mannan, so as to realize higher binding affinity and stronger specificity, effectively reduce the risk of false negative and cross-reaction, and improve the sensitivity and accuracy of in vitro detection.

[0015] Specifically, the first antibody and the second antibody have independent light chain and heavy chain variable regions, respectively, and the CDR1, CDR2 and CDR3 sequences of each are verified by animal immunization, screening and cloning, and have stability and specific recognition ability. By clearly defining the six complementarity determining regions (CDRs) of the two antibodies at the structural level, the precise positioning and repeatability of the antigen recognition site are ensured, which helps to maintain the consistency and stability of the antibody function in subsequent recombinant expression.

[0016] In addition, the first antibody and the second antibody can exert a synergistic effect when binding to the mannan antigen, so that the antibody combination provided by the present application is significantly enhanced in antigen capture, detection signal strength and anti-interference ability compared with a single antibody, thereby effectively improving the sensitivity and accuracy of in vitro detection of Candida.

[0017] In some embodiments, the amino acid sequence of the first light chain variable region is as shown in SEQ ID NO: 13; and the amino acid sequence of the first heavy chain variable region is as shown in SEQ ID NO: 14.

[0018] In some embodiments, the amino acid sequence of the second light chain variable region is as shown in SEQ ID NO: 15; and the amino acid sequence of the second heavy chain variable region is as shown in SEQ ID NO: 16.

[0019] In some of the above embodiments, by limiting the full-length variable region sequence, not just the complementarity determining region, the natural combination relationship of the framework region (FR) and the CDR can be completely retained. The framework region plays an important role in maintaining the spatial conformation of the antibody variable region, stabilizing the correct folding of the CDR, and supporting the formation of the antigen binding surface; therefore, further limiting the full-length variable region sequence based on the CDR sequence can ensure that the antibody maintains a stable tertiary structure and high-level specific binding ability in recombinant expression and actual application; compared with the scheme relying only on CDR sequence limitation, this embodiment can reduce the structural disturbance or affinity decline caused by framework region difference, ensure the consistency between batches and the reproducibility of antibody function, and thus exhibit higher stability and reliability in actual detection application.

[0020] In some embodiments, the nucleotide sequence of the first light chain variable region is as shown in SEQ ID NO: 17; and the nucleotide sequence of the first heavy chain variable region is as shown in SEQ ID NO: 18.

[0021] In some embodiments, the nucleotide sequence of the second light chain variable region is as shown in SEQ ID NO: 19; and the nucleotide sequence of the second heavy chain variable region is as shown in SEQ ID NO: 20.

[0022] In some of the above embodiments, by explicitly defining the nucleotide sequence of the antibody variable region, it can be ensured that the antibody can be stably expressed and maintain the correct amino acid sequence in different recombinant expression systems. The nucleotide level limitation not only covers the translation product of the amino acid sequence, but also ensures the consistency of transcription and translation, thereby avoiding amino acid drift caused by codon differences or potential mutations; compared with the limitation only from the amino acid level, the nucleotide sequence limitation can provide a more direct technical basis for subsequent plasmid construction, vector splicing and expression optimization, and improve the process controllability and production stability.

[0023] In a second aspect, the application provides a use of the anti-candida mannan monoclonal antibody according to any of the embodiments of the first aspect in the preparation of a tool for detecting candida.

[0024] According to the application, by applying the above antibody combination to an in vitro diagnostic tool, a high-sensitivity and high-specificity candida detection system can be constructed; the antibody combination can recognize different binding sites of the candida mannan antigen at the same time during the detection process, realize multiple capture and signal enhancement, and thus significantly improve the sensitivity and accuracy of the detection; compared with a single antibody, the combination has obvious advantages in reducing false negatives, reducing cross-reactions, and improving the reliability of the detection results.

[0025] In some embodiments, the tool for detecting candida is used for detecting candida in an in vitro sample, and the in vitro sample includes at least one of serum, plasma, whole blood, bronchoalveolar lavage fluid, cerebrospinal fluid, urine or sputum.

[0026] In some of the above embodiments, by explicitly defining the sample type, a variety of common clinical body fluid sources can be covered, which helps to realize rapid and accurate detection in different clinical scenarios; serum, plasma and whole blood samples are convenient for routine hematology detection, cerebrospinal fluid and bronchoalveolar lavage fluid are suitable for the diagnosis of central nervous system or respiratory system fungal infection, and urine and sputum samples further expand the application range. Compared with the detection tool limited to a single type of sample, the tool described in the present embodiment has more extensive applicability in clinical promotion.

[0027] In some embodiments, the anti-candida mannan monoclonal antibody is used in an immunodetection method for detecting candida in an in vitro sample.

[0028] In some of the above embodiments, by introducing the antibody combination provided by the application into the immunodetection system, the principle of specific binding of antigen and antibody can be used to directly detect whether the candida mannan antigen exists in the in vitro sample.

[0029] In some embodiments, the immune detection method comprises at least one of enzyme-linked immunoassay, immunofluorescence detection, chemiluminescence immunoassay, immune microsphere method and immune colloidal gold method.

[0030] In some embodiments described above, different immune detection methods can utilize the high specific recognition performance of the antibody combination of the present application; enzyme-linked immunoassay (ELISA) is suitable for high-throughput detection in the laboratory; immunofluorescence and chemiluminescence methods have higher sensitivity and quantitative detection capability; immune microsphere method is convenient for realizing multiplex detection; and immune colloidal gold method is suitable for rapid and simple bedside detection; therefore, with the antibody combination of the present application, different detection platforms can be flexibly matched to meet the diversified needs of laboratory research and instant detection.

[0031] In some embodiments, the tool for detecting Candida comprises at least one of a reagent, a kit, a test strip and an antibody chip.

[0032] In some embodiments described above, the antibody combination provided by the present application can be prepared into different forms of detection products, and the reagent or kit is suitable for standardized detection in the laboratory, the test strip is convenient for realizing rapid screening and bedside detection, and the antibody chip can realize high-throughput, automated parallel analysis; through the expansion of various tool forms, the present embodiment can cover various different application scenarios, greatly improving the flexibility and promotion value of detection.

[0033] Compared with the prior art, the present application has at least the following beneficial effects:

[0034] 1. The anti-Candida mannan monoclonal antibody combination provided by the present application comprises two antibodies obtained by animal immunization and screening, and by clearly limiting the CDR sequences of the light chains and heavy chains variable regions, high-efficiency recognition of Candida mannan is realized, and higher affinity and specificity can be obtained in in vitro detection.

[0035] 2. The two antibodies are used together in the same detection system, and can form a synergistic effect in the antigen capture process, compared with the single antibody detection method, the risk of false negative and cross reaction can be effectively reduced, thereby improving the sensitivity and accuracy of the detection result.

[0036] 3. By further limiting the full-length variable region amino acid sequence and nucleotide sequence, the structural consistency and functional stability of the antibody in recombinant expression and batch production can be ensured, and the repeatability and reliability of the detection tool in clinical and industrial application can be improved. BRIEF DESCRIPTION OF DRAWINGS

[0037] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the application.

[0038] Figure 1 This is an SDS-PAGE protein electrophoresis chromatogram of monoclonal antibodies Ab1# and Ab3# in one embodiment of this application. Detailed Implementation

[0039] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0040] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0041] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0042] In this specification, unless otherwise specified, "parts" refers to "parts by weight".

[0043] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0044] The main process route for preparing monoclonal antibodies in this application is as follows: first, antibodies against Candida mannan are obtained through animal immunization; then, monoclonal antibody cell lines are obtained through hybridoma technology; then, monoclonal antibody sequences are obtained through sequencing; and finally, monoclonal antibody plasmids are constructed through genetic engineering technology, and monoclonal antibodies are expressed using a eukaryotic mammalian expression system.

[0045] I. Experimental Materials:

[0046] Candida albicans: BNCC186382;

[0047] C. tropicalis: , BNCC335988;

[0048] C. parapsilosis: , BNCC263246;

[0049] C. krusei: Candida krusei , BNCC294714;

[0050] C. glabrata: , BNCC337348; all purchased from BNCC;

[0051] YM broth medium: purchased from Nantong Kaiheng Biological Technology Development Co., Ltd;

[0052] Freund's adjuvant: purchased from SIGMA;

[0053] New Zealand white rabbits: purchased from China Food and Drug Inspection Research Institute (Daxing);

[0054] Candida mannoprotein: Candida albicans strain was cultured in YM broth liquid medium, centrifuged to collect the bacterial cells after 48 h of culture at 37°C and 180 rpm, and washed with physiological saline for 6 times. Three volumes of methanol were added for precipitation, and the bacterial cells were washed with 75% ethanol solution for 3 times to remove impurities, and then re-dissolved with ultrapure water to obtain the Candida mannoprotein.

[0055] II. Test method:

[0056] (1) Preparation of immunogen:

[0057] A: Culture of strains: Candida albicans, C. tropicalis, C. parapsilosis, C. krusei, and C. glabrata were respectively placed in liquid YM broth medium, and cultured in a constant temperature incubator shaker. The culture conditions were 37°C, rotation speed 180 r / min, and shaking culture for 48 h.

[0058] B: Inactivation of strains: the cultured strains were placed in an 80°C oven for heat inactivation, and the inactivation time was 3 h.

[0059] C: Collection of bacterial cells: the bacterial cells were collected by centrifugation (8000 rpm / min, 20 min), and the supernatant was discarded after centrifugation. The bacterial cells were mixed with physiological saline again, and centrifuged again (8000 rpm / min, 20 min). The washing process was repeated once.

[0060] D: Resuspension of bacterial cells: after two times of washing with physiological saline, the bacterial slurry after centrifugation was resuspended with physiological saline, and the concentration of the bacterial suspension was adjusted by counting under a microscope. The concentration of Candida albicans was adjusted to 5×10 71 x 10 7 1 x 10 7 1 x 10 7 1 x 10 7

[0061] E: Preparation of immunogen: each bacteria solution was mixed in the following ratio:

[0062] C. albicans suspension: C. tropicalis suspension: C. parapsilosis suspension: C. krusei suspension: C. glabrata suspension = 5:1:1:1:1.

[0063] After mixing evenly, the mixed bacteria suspension was subjected to ultrasonic crushing with an ultrasonic crusher until the bacteria were completely crushed, which was the prepared immunogen.

[0064] (2) Animal immunization:

[0065] A: The self-prepared immunogen was mixed with Freund's adjuvant in equal volume to the appropriate volume, and was completely emulsified, and New Zealand white rabbits were immunized by subcutaneous multi-point injection, each New Zealand white rabbit was injected with 1000 μL of immunogen, and was injected once every two weeks, and a total of 10 New Zealand white rabbits were immunized;

[0066] B: After 10 times of immunization, the New Zealand white rabbits were subjected to ear vein blood collection one week after the last immunization, and the blood was collected, the serum was separated, and the antibody titer in the serum was tested by ELISA (enzyme-labeled plate coated with Candida mannan), and the OD values of the serum diluted by different folds of different New Zealand white rabbits were as shown in Table 1.

[0067] Table 1

[0068]

[0069] According to Table 1, 1#, 3# rabbits had low antibody titers and were eliminated, No. 9 rabbit died in the middle, and 2#, 6#, 7#, 8#, and 10# rabbits had the optimal titer, and No. 10 rabbit was selected for monoclonal cell preparation.

[0070] (3) Preparation of monoclonal antibody:

[0071] A: Screening of hybridoma cells

[0072] a) The spleen of the New Zealand white rabbit screened was treated, the isolated spleen cells were respectively fused with myeloma cells, and the fused cells were screened and plated by limited dilution method;

[0073] ​b), screening monoclonal cell holes and culture expansion, using ELISA method to detect the supernatant after culture, and the four monoclonal cell holes with the highest OD value in the cell culture plate are used as target hybridoma cells (1#, 2#, 3#, 4#), which are cultured and expanded for frozen cell seed and monoclonal antibody purification and verification;

[0074] B: Preparation of monoclonal antibody

[0075] a), using RT-PCR to isolate antibody variable region genes from hybridoma cells: after homogenization of the optimal hybridoma cells, cell lysis solution is added for RNA extraction, and isopropanol is used to precipitate RNA from the water phase layer. After centrifugation, the precipitated RNA is washed to remove impurities, resuspended and reverse transcribed to obtain cDNA;

[0076] b), using the specific primers of New Zealand white rabbits known in the prior art (synthesized by Jinsu Biotechnology Co., Ltd.) for PCR gene amplification, using hybridoma cell cDNA as a template to amplify the heavy and light chain variable region genes of the antibody, 50 μL system containing 5 μL cDNA, HotStar Taq Plus enzyme, dNTPs and 0.5 μM specific primers, PCR amplification is carried out according to the following conditions: pre-denaturation, 94°C, 5 min; amplification, 94°C, 30 s, 55°C, 30 s, 72°C, 50 s, 35 cycles; annealing 72°C, 7 min; the obtained PCR product is identified by 1% agarose gel electrophoresis, the target fragment is recovered and sent for sequencing, and the obtained antibody gene sequence can be obtained;

[0077] c), construction of monoclonal antibody expression vector:

[0078] According to the antibody gene sequence obtained by sequencing in step b, the expression vector of the monoclonal antibody is constructed. Homologous recombination primers are used to add homologous recombination arms at both ends of the antibody heavy chain variable region gene and at both ends of the light chain variable region gene, respectively. Double enzymes are used to linearize the expression plasmid containing the rabbit-derived antibody heavy and light chain IgG1 constant region to generate homologous recombination arms; the variable region gene fragment with the homologous recombination arms and the linearized plasmid are connected by homologous recombination to form a complete expression vector, and the expression vector is pCDNA3.4. The recombinant product is transformed into TOP10 E. coli competent cells, and the plasmid is amplified; through the above experimental operation, the paired pCDNA3.4-antibody heavy chain plasmid and pCDNA3.4-antibody light chain plasmid can be obtained.

[0079] d), expression and purification of monoclonal antibody:

[0080] The paired monoclonal antibody heavy and light chain expression plasmids obtained in step c are added to the culture medium in a 1:1 ratio, mixed thoroughly, and then 4 times the mass of DNA of transfection reagent PEI is added, mixed, and then placed in the dark at room temperature for 10 min. Then the plasmids are added to the 293T cells while shaking the cell flask, and the cells are cultured in a 5% carbon dioxide, 37°C carbon dioxide incubator, and shaken for 5 days. Then the cell culture supernatant is collected, and the expressed cell culture supernatant is purified using affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows:

[0081] (1) The expressed antibody supernatant is centrifuged at 10,000 x g and room temperature for 30 min to remove the precipitate;

[0082] (2) The affinity purification column containing Protein A is washed with 10 times the volume of binding buffer (Binding Buffer) to ensure complete flow;

[0083] (3) The expression supernatant is passed through the purification column at a flow rate of 5 mL / min;

[0084] (4) The purification column is washed with 20 times the volume of binding buffer (Binding Buffer) to ensure complete washing;

[0085] (5) The purification column is eluted with 0.1M citric acid buffer at pH 3.0-3.5 until the elution peak drops to equilibrium, and the pH is adjusted to 7.0 with 1M Tris-HCl buffer at pH 9.0;

[0086] (6) The purified monoclonal antibody is concentrated using a concentration centrifugal column, PBS is used as the antibody storage buffer, and finally the concentration of the concentrated antibody is determined using an ultramicro UV spectrophotometer.

[0087] Thus, monoclonal antibody Ab1#, monoclonal antibody Ab2#, monoclonal antibody Ab3#, and monoclonal antibody Ab4# are obtained, respectively.

[0088] III. Test Part

[0089] 1. Screening of monoclonal antibodies:

[0090] a) ELISA indirect method verification: Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata cell lysate was heated at 121°C for 30 min, centrifuged at 12000xg for 10 min, and the supernatant was collected (the supernatant mainly contains soluble polysaccharides, denatured soluble proteins and other small molecules); the supernatant was diluted 10 times with pure water, and the enzyme-labeled plate was coated. Four strains of antibodies (Ab1#, Ab2#, Ab3#, Ab4#) were diluted at concentrations of 100, 10, 1, and 0 ng / mL, respectively, and the diluted solution was added to the enzyme-labeled plate, incubated at 37°C for 1 h, washed, and then added with enzyme-labeled secondary antibody dilution solution (5000 times dilution) for incubation at 37°C for 0.5 h. After 15 min of color development with TMB, the reaction was stopped, and the absorbance value (OD) was measured at 450 / 620 nm wavelength. The results are shown in Table 2.

[0091] Table 2

[0092]

[0093] According to the analysis of the recognition of antibodies to different strains in Table 2, it can be seen that the monoclonal antibodies Ab1#, Ab3#, and Ab4# have better recognition effect on the five strains of bacteria.

[0094] b) Confirmation of antibody pairing combination

[0095] From the relevant hospitals, 30 positive samples and 30 negative samples were collected; the selected monoclonal antibodies were used for sandwich method combination (forming a coated antibody-antigen-detection antibody complex), and the 60 samples and critical samples (candida mannans were diluted with purified water to a concentration of 100 pg / mL) were detected by fluorescence immunochromatography (coating conditions: 0.02M PBS buffer, coating concentration 2.0 mg / mL, drying temperature 50°C, drying time 12h; labeling conditions: 0.02M PBS buffer, antibody and fluorescent microspheres (particle size about 300 nm) mass ratio 1:1, labeling 12h; sample loading amount is 100μL, read the results after 15 min); calculate the ratio (I) of the sample T / C value and the critical sample T / C value, and use this parameter as the critical value (I≥1 is positive, I<1 is negative); select the antibody combination with high positive and negative coincidence rate and large sample gradient, and the results are shown in Table 3.

[0096] Table 3

[0097]

[0098] According to Table 3, Ab1# antibody coating + Ab3# antibody detection and Ab1# antibody coating + Ab4# antibody detection have the best sample positive and negative coincidence rate.

[0099] c) Verification of monoclonal antibody cross-reaction:

[0100] The sandwich method was carried out with two pairs of alternative monoclonal antibodies respectively (forming a coating antibody-antigen-detection antibody complex), and the fluorescence immunochromatography method was used to detect different concentrations of mannans, galactomannans, capsular polysaccharides, lipopolysaccharides, (1-3)-β-D-glucan and BSA respectively, and the anti-interference ability of different antibodies to these several cross-reactive antigens was observed. The cross-reactions are shown in Table 4.

[0101] Table 4

[0102]

[0103] According to Table 4, the results show that the monoclonal antibody Ab1# and the monoclonal antibody Ab3# have the best effect of Ab1# antibody coating + Ab3# antibody detection, and no cross-reaction; thus, the monoclonal antibody Ab1# and the monoclonal antibody Ab3# are screened for Candida mannans.

[0104] 2. Characterization of monoclonal antibody Ab1# and monoclonal antibody Ab3#

[0105] a) Monoclonal antibody Ab1#:

[0106] Light chain:

[0107] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 17: GACGTGGTGATGACCCAGACCCCTTCCAGCAAATCCGCCGCCGTGGGCGACACCGTCACCATCAAGTGCCAGGCCAGCCAGTTCATCTCCAGATGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACACCGCTTCTACACTGGAGAGCGGCGTTCCCTCCCGGTTCAAGGGCAGCGGCAGCGGCACTCAGTTCACCCTGACCATCAATGGAGTGCAGTGTGACGACGCCGCTACATACTACTGCCAGCAGTACGAATCCTTCCCCTGGACCTTTGGCGGGGGCACAAAACTGGAAATCAAA.

[0108] The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 13: DVVMTQTPSSKSAAVGDTVTIKCQASQFISRWLAWYQQKPGKAPKLLIYTASTLESGVPSRFKGSGSGTQFTLTINGVQCDDAATYYCQQYESFPWTFGGGTKLEIK.

[0109] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:1: QASQFISRW; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:2: YQQKPGKAPKLLIY; and the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:3: QQYESFPWT.

[0110] Heavy chain:

[0111] The nucleotide sequence of the heavy chain variable region is as SEQ ID Shown in NO.18: CAGGTGCAGCTGGAGGAGAGCGGCGGCGGCCTGGTGAAGCCTGGCGGAACCCTGACCCTGACCTGCAAGGCCAGCGGCCCTAGCATCTTCGGAAGCGACTACTACATTTGCTGGGTGAGACAGGCCCTGGAAAGGGACTGGAGCTGATCGCCTGCATCCACTCTAGCGGCACTACGTGGTATGC TTCCTGGGTGAACGGCAGGTTCACCATCTCCCGTCCCACCTCTCTGAATACAGTGGACCTGAAAATGACCTCACTGACCGCCGCCGACACCGCCATCTACTACTGCGCCAGAGACCTGGATTTCGACATCGGCCACAACTACTATTACGGATTTGACGTCTGGGGACAGGGCACCCTGGTTACCGTGAGCAGC.

[0112] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.14: QVQLEESGGGLVKPGGTLTLTCKASGPSIFGSDYYICWVRQAPGKGLELIACIHSSGTTWYASWVNGRFTISRSTSLNTVDLKMTSLTAADTAIYYCARDLDFDIGHNYYYGFDVWGQGTLVTVSS.

[0113] The amino acid sequence of the complementarity-determining region VH-CDR1 of the heavy chain variable region is shown in SEQ ID NO:4: KASGPSIFGS; the amino acid sequence of the complementarity-determining region VH-CDR2 of the heavy chain variable region is shown in SEQ ID NO:5: CIHSSGTTWY; and the amino acid sequence of the complementarity-determining region VH-CDR3 of the heavy chain variable region is shown in SEQ ID NO:6: ARDLDFDIGHNYYYGFDVW.

[0114] b) Monoclonal antibody Ab3#:

[0115] Light chain:

[0116] The nucleotide sequence of the light chain variable region is as SEQ ID Shown in NO.19: GCCATCAAGATGACACAGACCCCCAGCTCCGTTGTCCGCCGCCGTCGGAGGCACCGTGACCATTAACTGCCAGGCATCCGTGGTGAACGACGTGAATCTGGCCTGGTACCAGCAGAAGCCCGGCCAGCCTCCCAAACTGCTGATCTACGATGCCTCCA ATTCTGGCCTCCGGAGTGCCCAGCCGCTTCAGTGGCTCCGGCAGCGGCACTGATTTCACCCTGACAATTAACGGGGTGCAGTGCGACGACGCTGCCACATACTATTGCCAGCAGAGGTCCAATTGGCTGTACACCTTCGGCCAGGGCACCAAGCTGGAAATTAAG.

[0117] The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.15: AIKMTQTPSSVSAAVGGTVTINCQASVVNDVNLAWYQQKPGQPPKLLIYDASNLASGVPSRFSGSGSGTDFTLTINGVQCDDAATYYCQQRSNWLYTFGQGTKLEIK.

[0118] The amino acid sequence of the complementarity-determining region VL-CDR1 of the light chain variable region is shown in SEQ ID NO:7: QASVVNDVNL; the amino acid sequence of the complementarity-determining region VL-CDR2 of the light chain variable region is shown in SEQ ID NO:8: YQQKPGQPPKLLIY; and the amino acid sequence of the complementarity-determining region VL-CDR3 of the light chain variable region is shown in SEQ ID NO:9: QQRSNWLYT.

[0119] Heavy chain:

[0120] The nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 20: CAGCAGCTGAAAGAGTCCGGCGGCGGCCTGGTGAAGCCCGGAGGCTCTCTGAAACTGTGCTGCAAGGCCAGCGGCTTTACCTTTGGCGACTTCGCCATGTGTTGGGTGCGGCAGGCTCCCGGCAAAGGGCTGGAATGGATCGGCTGCATCAGCTGGAACTCCGCCACCGTTCACTACGCCAGCTGGGTCAATGGACGCTTCACCCTGTCTAGAGACAATGCCCAATCTACTGTGTGCCTCCAGCTGAACAGCCTGACCGCCGCCGACACCGCCACGTACTTCTGCGCCAAGAGCAACGGCGAGTACGGCAGCGGCTCTGAAACAGATTACTGGGGACAGGGCACCCTCGTGACAGTGAGCAGC.

[0121] The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 16: QQLKESGGGLVKPGGSLKLCCKASGFTFGDFAMCWVRQAPGKGLEWIGCISWNSATVHYASWVNGRFTLSRDNAQSTVCLQLNSLTAADTATYFCAKSNGEYGSGSETDYWGQGTLVTVSS.

[0122] The amino acid sequence of the complementarity determining region VH-CDR1 of the heavy chain variable region is shown as SEQ ID NO: 10: KASGFTFGD; the amino acid sequence of the complementarity determining region VH-CDR2 of the heavy chain variable region is shown as SEQ ID NO: 11: CISWNSATVHY; and the amino acid sequence of the complementarity determining region VH-CDR3 of the heavy chain variable region is shown as SEQ ID NO: 12: AKSNGEYGSGSETDY.

[0123] c) Molecular weight test of monoclonal antibodies Ab1#, Ab3#

[0124] The SDS-PAGE protein electrophoresis chromatogram thereof is shown as Figure 1 According to Figure 1 wherein the M lane is Marker, the No. 1 lane is monoclonal antibody Ab1#, and the No. 2 lane is monoclonal antibody Ab3#, both of which are reduced into two parts, one part with a molecular weight of 50 kDa and the other part with a molecular weight of 25 kDa.

[0125] 3. Development and verification of a Candida mannan detection kit based on the obtained anti-Candida mannan monoclonal antibodies (monoclonal antibody Ab1#, monoclonal antibody Ab3#)

[0126] Brief description of the kit development process:

[0127] Ab1# antibody was used as a coating antibody to coat the NC membrane, and Ab3# antibody fluorescent microspheres were used as a detection antibody. A series of optimization studies were conducted on the coating conditions of Ab1# antibody (coating buffer, coating concentration, drying temperature and time, etc.), the labeling conditions of Ab3# antibody (antibody to fluorescent microsphere ratio, labeling buffer, labeling time, etc.), sample loading amount, and result reading time after loading, etc. After the kit was formed, the performance of the kit (sensitivity, repeatability, stability, etc.) was also studied.

[0128] The coating conditions were determined as follows: 0.02M PBS buffer, coating concentration 2.0 mg / mL, drying temperature 50°C, and drying time 12h;

[0129] The labeling conditions were as follows: 0.02M PBS buffer, antibody to fluorescent microsphere (particle size about 300 nm) mass ratio 1:1, and labeling time 12h; the sample loading amount was 100 μL, and the result was read after 15 min of loading.

[0130] I) Sensitivity:

[0131] Candida mannan was diluted into samples at 9 concentration levels of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125 and 0 pg / mL, and a Candida mannan detection kit (fluorescent immunochromatography method) was used to determine the analytical sensitivity. When the T / C value of the sample to be tested and the T / C value of 0 concentration were greater than 2.0, the corresponding concentration value was considered to be the analytical sensitivity, and the determination results are shown in Table 5.

[0132] Table 5

[0133]

[0134] According to Table 5, when the T / C value of the sample to be tested and the T / C value of 0 concentration were greater than 2.0, the minimum concentration corresponding to the T / C value was 3.125 pg / mL. Therefore, the present application provides a Candida mannan detection kit (fluorescent immunochromatography method) with a detection sensitivity of 3.125 pg / mL for Candida mannan.

[0135] II) Repeatability:

[0136] The Candida mannan concentration in sample 1 (Candida mannan concentration is 50 pg / mL) and sample 2 (Candida mannan concentration is 500 pg / mL) was determined by using the Candida mannan detection kit (fluorescent immunochromatography method). The test results are shown in Table 6.

[0137] Table 6

[0138]

[0139] As can be seen from Table 6, the coefficient of variation CV% of the Candida mannan detection kit (fluorescent immunochromatography method) is less than 15%, indicating that the repeatability of the kit is good.

[0140] III) Stability:

[0141] The Candida mannan detection kit (fluorescent immunochromatography method) was stored at 2-8℃, and the kit was taken out after 0, 3, 6, 9, and 12 months of production, respectively. The Candida mannan concentration in sample 1 (Candida mannan concentration is 50 pg / mL) and sample 2 (Candida mannan concentration is 500 pg / mL) was determined. The test results are shown in Table 7.

[0142] Table 7

[0143]

[0144] As can be seen from Table 7, the bias of the Candida mannan detection kit (fluorescent immunochromatography method) is less than 15%, indicating that the stability of the kit is good.

[0145] Finally, it should be noted that the above text has described various embodiments of the present application in detail. In order to avoid obscuring the concept of the present application, some details known in the art are not described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description. The above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them;

[0146] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A combination of anti-C. glabrata mannan monoclonal antibodies, characterized in that, comprises a first light chain variable region and a first heavy chain variable region, and a second antibody comprising a second light chain variable region and a second heavy chain variable region; wherein, the amino acid sequence of the first light chain variable region comprises a VL-CDR1 as set forth in SEQ ID NO: 1, a VL-CDR2 as set forth in SEQ ID NO: 2, and a VL-CDR3 as set forth in SEQ ID NO: 3; the amino acid sequence of the first heavy chain variable region comprises a VH-CDR1 as set forth in SEQ ID NO: 4, a VH-CDR2 as set forth in SEQ ID NO: 5, and a VH-CDR3 as set forth in SEQ ID NO: 6; the amino acid sequence of the second light chain variable region comprises a VL-CDR1 as set forth in SEQ ID NO: 7, a VL-CDR2 as set forth in SEQ ID NO: 8, and a VL-CDR3 as set forth in SEQ ID NO: 9; the amino acid sequence of the second heavy chain variable region comprises a VH-CDR1 as set forth in SEQ ID NO: 10, a VH-CDR2 as set forth in SEQ ID NO: 11, and a VH-CDR3 as set forth in SEQ ID NO:

12.

2. The anti-C. glabrata mannan monoclonal antibody combination of claim 1, wherein, the amino acid sequence of the first light chain variable region is as set forth in SEQ ID NO: 13; and the amino acid sequence of the first heavy chain variable region is as set forth in SEQ ID NO:

14.

3. The anti-C. glabrata mannan monoclonal antibody combination of claim 1, wherein, the amino acid sequence of the second light chain variable region is as set forth in SEQ ID NO: 15; and the amino acid sequence of the second heavy chain variable region is as set forth in SEQ ID NO:

16.

4. The anti-C. glabrata mannan monoclonal antibody combination of claim 1, wherein, the nucleotide sequence of the first light chain variable region is as set forth in SEQ ID NO: 17; and the nucleotide sequence of the first heavy chain variable region is as set forth in SEQ ID NO:

18.

5. The anti-C. glabrata mannan monoclonal antibody combination of claim 1, wherein, the nucleotide sequence of the second light chain variable region is as set forth in SEQ ID NO: 19; and the nucleotide sequence of the second heavy chain variable region is as set forth in SEQ ID NO:

20.

6. Use of the anti-C. albicans mannan monoclonal antibody combination according to any one of claims 1-5 in the manufacture of a tool for detecting Candida.

7. Use according to claim 6, characterized in that, The tool for detecting Candida is used for detecting Candida in an in vitro sample, which comprises at least one of serum, plasma, whole blood, bronchoalveolar lavage fluid, cerebrospinal fluid, urine or sputum.

8. Use according to claim 7, characterized in that, The anti-Candida mannan monoclonal antibody combination is used in an immunoassay method for detecting Candida in an in vitro sample.

9. Use according to claim 8, characterized in that, The immunoassay method comprises at least one of enzyme-linked immunoassay, immunofluorescence assay, chemiluminescence immunoassay, immunomicrosphere method and immunocolloidal gold method.

10. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting Candida comprises at least one of a reagent, a kit, a test strip.

11. The use according to any one of claims 6 to 9, characterized in that, The tool for detecting Candida comprises an antibody chip.

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