Antibody combination for detecting mycobacterial lipoarabinomannan, its application and kit

Abstract

The present invention discloses an antibody combination for detecting mycobacterial lipoarabinomannan, its application, and a kit, relating to the field of biological detection. The inventors of this application screened and identified various sample types, identifying capture antibodies suitable for LAM antigen capture and detection antibodies suitable for LAM antigen detection. This resulted in a superior antibody combination for LAM antigen detection, which exhibits high sensitivity and specificity and can be used to detect LAM antigens in purified LAM, standard strains of Mycobacterium tuberculosis, clinical isolates, pleural effusions, plasma, and other bodily fluids. This provides a new antibody combination and method for LAM antigen detection, as well as a new method and kit for the auxiliary diagnosis of active tuberculosis.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to an antibody combination for detecting mycobacterium lipoarabinomannan, an application thereof, and a kit. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (Mtb). The diagnosis of TB, especially extrapulmonary TB, remains difficult. Tuberculous pleurisy (TP) is one of the most common extrapulmonary TB forms, and its diagnosis remains a major clinical challenge. Non-sputum biomarkers are crucial for the diagnosis of extrapulmonary TB and non-sputum TB. Lipoarabinomannan (LAM), a cell wall component of Mycobacterium tuberculosis, is a highly promising non-sputum biomarker. LAM is an important glycolipid component of the mycobacterial cell wall with a molecular weight of 17.5 kDa. During Mycobacterium tuberculosis (Mtb) infection, soluble lipoarabinomannan (LAM) can be released through bacterial metabolism or degradation and detected in body fluids (such as sputum, blood, and urine), but only in patients with active TB. Based on this characteristic, LAM has been used as a target for the auxiliary diagnosis of active TB.

[0003] Enzyme-linked immunosorbent assays and lateral flow urine LAM tests have been developed for the detection of LAM. A low-cost, rapid lateral flow urine LAM test kit (LF-LAM; Determine TB-LAM; Alere, USA) has been approved by the WHO for the diagnosis of tuberculosis in patients with CD4-positive T lymphocyte counts less than 200 cells / μl or severe AIDS. This kit uses a rabbit anti-LAM polyclonal antibody to detect this antigen. Although this method has good specificity for the detection of mycobacterial LAM antigens and is characterized by its low cost and rapidity, it also has limitations. Its sensitivity is limited to 1 ng / ml, and this method is not recommended for TB screening in non-AIDS patients, who account for the vast majority (85%) of the TB population, limiting its global application.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The object of the present invention is to provide an antibody combination for detecting mycobacterial lipoarabinomannan, its application and kit.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a use of a composition in preparing a product for detecting mycobacterial lipoarabinomannan or tuberculosis, wherein the composition comprises a capture antibody and a detection antibody;

[0008] Wherein, the capture antibody comprises a combination of any one or more antibodies of BJRbL01, BJRbL03, BJRbL20, BJRbL52 and BJRbL76;

[0009] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL01 are shown in SEQ ID NOs: 1-3, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively;

[0010] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL03 are shown in SEQ ID NOs: 7-8 and 3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 9, 5, and 6, respectively.

[0011] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL20 are shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:9 and SEQ ID NOs:11-12, respectively.

[0012] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL52 are shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:9, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0013] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL76 are shown in SEQ ID NOs: 13-15, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 16-18, respectively;

[0014] The detection antibodies include a combination of any one or more of biotin-labeled BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio and BJRbL76-Bio antibodies;

[0015] In a second aspect, an embodiment of the present invention provides the use of a capture antibody in preparing a product having the following uses, wherein the capture antibody is the capture antibody described in any of the preceding embodiments, and the uses include: detecting any one or more of mycobacterial lipoarabinomannan, diagnosing or assisting in the diagnosis of tuberculosis.

[0016] In a third aspect, an embodiment of the present invention provides the use of a detection antibody in preparing a product having the following uses, wherein the detection antibody is the detection antibody described in the preceding embodiment, and the uses include: detecting mycobacterial lipoarabinomannan, diagnosing or assisting in the diagnosis of tuberculosis, or any one or more thereof.

[0017] In a fourth aspect, an embodiment of the present invention provides a kit comprising: the composition described in the aforementioned embodiment.

[0018] In a fifth aspect, an embodiment of the present invention provides a double-antibody sandwich ELISA detection method for Mycobacterium lipoarabinomannan, wherein the capture antibody used in the detection method is as described in any of the foregoing embodiments, and the detection antibody used in the detection method is as described in any of the foregoing embodiments; the detection method is not directly intended for the diagnosis or treatment of a disease.

[0019] In a sixth aspect, an embodiment of the present invention provides the use of a target antibody in the preparation of a product for detecting Candida albicans or its related diseases, wherein the target antibody is a combination of any one or more of BJRbL20, BJRbL52, BJRbL20-Bio and BJRbL52-Bio described in the aforementioned embodiments.

[0020] In a seventh aspect, an embodiment of the present invention provides an application of a target antibody in detecting Candida albicans, wherein the target antibody is the target antibody described in the aforementioned embodiment, and the application is not directly intended for diagnosis or treatment of a disease.

[0021] The present invention has the following beneficial effects:

[0022] The inventors of the present application used various types of samples for screening and identification, determined capture antibodies suitable for LAM antigen capture and detection antibodies suitable for LAM antigen detection, and formed a superior antibody combination for LAM antigen detection, which has high detection sensitivity and specificity and can be used for the detection of LAM antigens in purified LAM, standard strains of Mycobacterium tuberculosis, clinical isolates, pleural effusions, plasma and other body fluids, providing a new antibody combination and new method for LAM antigen detection, and providing a new method and new kit for the auxiliary diagnosis of active tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Sandwich ELISA method for screening LAM antigen detection paired antibodies;

[0025] Figure 2 Sandwich ELISA to identify LAM antibody combinations and detect LAM antigen sensitivity;

[0026] Figure 3 Sandwich ELISA to identify BJRbL01 as the capture antibody for detection of Mycobacterium standard strains;

[0027] Figure 4 Sandwich ELISA to identify BJRbL03 as the capture antibody for detection of Mycobacterium standard strains;

[0028] Figure 5 Sandwich ELISA to identify BJRbL20 as the capture antibody for the detection of Mycobacterium standard strains;

[0029] Figure 6 Sandwich ELISA to identify BJRbL52 as the capture antibody for detection of Mycobacterium standard strains;

[0030] Figure 7 Sandwich ELISA to identify BJRbL76 as the capture antibody for detection of Mycobacterium standard strains;

[0031] Figure 8 Sandwich ELISA to identify BJRbL01 as the capture antibody for the detection of mycobacterial isolates;

[0032] Figure 9Sandwich ELISA to identify BJRbL03 as the capture antibody for the detection of mycobacterial isolates;

[0033] Figure 10 Sandwich ELISA to identify BJRbL20 as the capture antibody for the detection of mycobacterial isolates;

[0034] Figure 11 Sandwich ELISA to identify BJRbL52 as the capture antibody for the detection of mycobacterial isolates;

[0035] Figure 12 Sandwich ELISA to identify BJRbL76 as the capture antibody for the detection of mycobacterial isolates;

[0036] Figure 13 Indirect ELISA to identify LAM antibodies and detect common pneumonia pathogens;

[0037] Figure 14 Sandwich ELISA to detect the characteristics of LAM antibodies recognizing Candida albicans;

[0038] Figure 15 Indirect ELISA and sandwich ELISA were used to detect LAM antigen added to pleural effusion;

[0039] Figure 16 Sandwich ELISA for detection of LAM antigen in pleural effusion of tuberculosis patients;

[0040] Figure 17 .ROC curve of LAM antigen detection in pleural effusion;

[0041] Figure 18 .Sandwich ELISA detection of LAM antigen standard curve;

[0042] Figure 19 .Back-calculation results of LAM concentration in pleural effusion;

[0043] Figure 20 Sandwich ELISA to detect LAM antigen in the plasma of tuberculosis patients;

[0044] Figure 21 .ROC curve for detection of LAM antigen in plasma;

[0045] Figure 22 .Back-calculation results of LAM concentration in plasma;

[0046] Figure 23 .Correlation analysis of LAM antigen detection results in plasma and pleural effusion. DETAILED DESCRIPTION

[0047] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0048] With the continuous advancement of immunological technology, the use of LAM antigen testing in the auxiliary diagnosis of non-HIV-infected patients with active TB is also expanding, and the types of body fluids have also been expanded to include pleural effusion and plasma. In these tests, the quality and selection of antibodies are particularly critical.

[0049] Body fluids such as serum, plasma, pleural effusion, cerebrospinal fluid, and urine are important non-sputum biological samples from tuberculosis patients. They are composed of a mixture of multiple substances. The indirect ELISA method is not suitable for the detection of components in body fluids, especially those with low content in body fluids. During indirect ELISA testing, a large amount of mixed substances in the body fluids will be coated onto the enzyme-labeled plate, interfering with the coating of the target molecule. In this protocol, rabbit anti-LAM monoclonal antibodies were further screened and identified to screen out a group of antibody combinations suitable for the detection of LAM antigens in body fluids, including capture antibodies and detection antibodies. This antibody combination was used to detect LAM antigens in body fluids such as pleural effusion and plasma from patients with tuberculous pleurisy. It was found that this combination can be used to detect LAM antigens in patient body fluids, providing a new antibody combination and new method for LAM antigen detection, and a new method, application, and kit for the auxiliary diagnosis of active tuberculosis based on LAM antigen detection.

[0050] Technical Solution

[0051] In one aspect, an embodiment of the present invention provides a use of a composition in preparing a product for detecting mycobacterial lipoarabinomannan or diagnosing tuberculosis, wherein the composition comprises a capture antibody and a detection antibody;

[0052] Wherein, the capture antibody comprises a combination of any one or more antibodies of BJRbL01, BJRbL03, BJRbL20, BJRbL52 and BJRbL76;

[0053] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL01 are shown in SEQ ID NOs: 1-3, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively;

[0054] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL03 are shown in SEQ ID NOs: 7-8 and 3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 9, 5, and 6, respectively.

[0055] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL20 are shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:9 and SEQ ID NOs:11-12, respectively.

[0056] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL52 are shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:9, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0057] The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL76 are shown in SEQ ID NOs: 13-15, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 16-18, respectively;

[0058] The detection antibodies include biotin-labeled antibodies: a combination of any one or more antibodies of BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio and BJRbL76-Bio.

[0059] In some embodiments, the amino acid sequences of the heavy and light chain variable regions and the light chain variable region of BJRbL01 are shown in SEQ ID NO: 19 and SEQ ID NO: 20.

[0060] In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL03 are shown in SEQ ID NO:21 and SEQ ID NO:22.

[0061] In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL20 are shown in SEQ ID NO:23 and SEQ ID NO:24.

[0062] In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL52 are shown in SEQ ID NO:25 and SEQ ID NO:26.

[0063] In some embodiments, the amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL76 are shown in SEQ ID NO:27 and SEQ ID NO:28.

[0064] In some embodiments, the heavy chain constant region of the antibody or its functional fragment is selected from the heavy chain constant region of any one of IgD, IgE, IgM, IgA and IgG; the light chain constant region of the antibody or its functional fragment is a κ or λ type light chain constant region.

[0065] In some embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29.

[0066] In some embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0067] In some embodiments, the functional fragment is selected from Fab, Fab', F(ab')2, Fv or ScFv.

[0068] In some embodiments, the capture antibody comprises any one or more of BJRbL01, BJRbL03, BJRbL20, and BJRbL76, and the detection antibody comprises any one or more of biotin-labeled antibodies BJRbL01-Bio, BJRbL20-Bio, and BJRbL52-Bio.

[0069] In some embodiments, the capture antibody comprises BJRbL01, and the detection antibody comprises any one or both of BJRbL01-Bio and BJRbL20-Bio.

[0070] In some embodiments, the mycobacterium comprises a slow-growing mycobacterium.

[0071] In some embodiments, the slow-growing mycobacterium includes at least one of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium gordonii, Mycobacterium marinum, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium gastricis, Mycobacterium xenopus, and Mycobacterium malmoe.

[0072] In some embodiments, the diagnosing tuberculosis includes: assisting in the diagnosis of tuberculosis.

[0073] In some embodiments, the tuberculosis comprises active tuberculosis.

[0074] In some embodiments, applicable samples of the detection method include: any one or more of biological fluid samples, environmental samples, and artificially prepared standards.

[0075] In some embodiments, the body fluid comprises any one of sputum, blood, urine, tissue fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, semen and vaginal fluid.

[0076] In some embodiments, the product includes: reagents and / or kits for sandwich ELISA detection.

[0077] In some embodiments, in the reagent or kit for sandwich ELISA detection, the coating concentration of the capture antibody is 0.1 to 5 μg / ml. The coating concentration can specifically be any one of, or any two of, 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, and 5 μg / ml.

[0078] In some embodiments, the product can detect LAM antigens in samples (including biological fluid samples) with a sensitivity of up to 62.5 pg / mL.

[0079] On the other hand, an embodiment of the present invention provides the use of a capture antibody in preparing a product having the following uses, wherein the capture antibody is the capture antibody described in any of the preceding embodiments, and the uses include: detecting any one or more of mycobacterial lipoarabinomannan, diagnosing or assisting in the diagnosis of tuberculosis.

[0080] In some embodiments, the mycobacterium and the suitable sample of the product are as described in any of the above embodiments.

[0081] On the other hand, an embodiment of the present invention provides the use of a detection antibody in preparing a product having the following uses, wherein the detection antibody is the detection antibody described in any of the preceding embodiments, and the uses include: detecting mycobacterial lipoarabinomannan, diagnosing or assisting in the diagnosis of tuberculosis.

[0082] In some embodiments, the mycobacterium and the suitable sample of the product are as described in any of the above embodiments.

[0083] On the other hand, an embodiment of the present invention provides a kit comprising the composition described in any of the aforementioned embodiments.

[0084] In some embodiments, it further comprises: at least one of a coating solution, a washing solution, a color developing solution A, a color developing solution B, a stop solution, and a blocking solution.

[0085] In some embodiments, the kit is suitable for detecting mycobacterial lipoarabinomannan or tuberculosis.

[0086] In addition, an embodiment of the present invention further provides a double antibody sandwich ELISA detection method for mycobacterial lipoarabinomannan, wherein the capture antibody used in the detection method is the capture antibody described in any of the preceding embodiments, and the detection antibody used is the detection antibody described in any of the preceding embodiments; the detection method is not directly intended for the diagnosis or treatment of a disease;

[0087] In some embodiments, the steps of the detection method can refer to the existing sandwich ELISA method. Compared with the existing technology, the contribution of this application mainly lies in the screening and identification of a combination of capture antibodies and detection antibodies that can be effectively used in the sandwich ELISA method to detect LAM antigens.

[0088] In some embodiments, the detection method is applicable to samples including any one or more of biological fluid samples, environmental samples, and artificially prepared standards. Artificially prepared standards may include negative and positive samples. The organism may be selected from mammals, specifically humans, and further, tuberculosis patients.

[0089] In some embodiments, the body fluid comprises any one of sputum, blood, urine, tissue fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, semen and vaginal fluid.

[0090] In some embodiments, during detection, the working concentration of the detection antibody is 0.1-5 μg / ml. The working concentration can specifically be any one of, or any two of, 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 3 μg / ml, 3.5 μg / ml, 4 μg / ml, 4.5 μg / ml, and 5 μg / ml.

[0091] On the other hand, an embodiment of the present invention provides the use of a target antibody or a combination thereof in the preparation of a product for detecting Candida albicans or a disease related thereto, wherein the target antibody is selected from: any one of BJRbL20, BJRbL52, BJRbL20-Bio and BJRbL52-Bio as described in any of the foregoing embodiments.

[0092] In some embodiments, the combination of the target antibodies can be: any two of BJRbL20, BJRbL52, BJRbL20-Bio and BJRbL52-Bio, including but not limited to BJRbL20 and BJRbL52, BJRbL20 and BJRbL20-Bio, BJRbL52 and BJRbL52-Bio; it can also be any three or four, which will not be repeated here.

[0093] In some embodiments, the Candida albicans-related diseases include diseases or symptoms caused or triggered by infection with Candida albicans, and the related diseases may specifically include but are not limited to vaginitis, balanitis, etc.

[0094] In addition, an embodiment of the present invention further provides the use of a target antibody or a combination thereof in detecting Candida albicans, wherein the target antibody or the combination thereof is the target antibody or the combination thereof described in any of the foregoing embodiments, and the application is not directly intended for the diagnosis or treatment of a disease.

[0095] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0096] The amino acid sequences of the heavy chain constant regions of the antibodies used in the examples are all shown in SEQ ID NO: 29, and the amino acid sequences of the light chain constant regions are all shown in SEQ ID NO: 30.

[0097] Example 1 Sandwich ELISA method to identify LAM antigen detection antibody combination

[0098] LAM antibodies were biotin-labeled to obtain BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, and BJRbL76-Bio. Sandwich ELISA was used to screen and identify paired antibodies for LAM antigen detection.

[0099] ELISA assay procedure: Capture antibodies were immobilized with 1 μg / ml of LAM antibodies BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76, respectively. Diluted with coating buffer, 100 μl / well was added to a 96-well ELISA plate. Cover with a lid or protective film and incubate at 4°C overnight. Wash three times with 1× Wash Buffer, add 200 μl / well of 5% skim milk powder to block nonspecific binding sites, and incubate at 37°C for 2 hours. After washing three times, add 5% skim milk powder to dilute the LAM antigen to a concentration of 1 μg / ml, 100 μl / well, and incubate at 37°C for 2 hours. After washing five times, the detection antibody JRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, or BJRbL76-Bio was added at a concentration of 1 μg / ml, 100 μl / well, and incubated at 37°C for 2 hours. After washing, a 1:5000 dilution of horseradish peroxidase-labeled avidin (avidin-HRP) was added at 100 μl / well and incubated at 37°C for 1 hour. Wash the plate five times with a plate washer, add 100 μl / well of color development solution, and incubate at 37°C for 30 minutes. Add 50 μl / well of stop solution, and measure the OD using a microplate reader. 450nm The test results were analyzed using Graphpad software.

[0100] LAM antigen detection paired antibody preliminary screening results ( Figure 1 ) The results showed that all LAM antibodies could function as both capture and detection antibodies. A single antibody could function as both a capture and detection antibody. Given the existence of independent, identical epitopes on the LAM antigen, using the sandwich ELISA method, after the capture antibody captures LAM, independent, duplicated epitopes remain on the LAM antigen, allowing for further binding of the same antibody.

[0101] The main sources and preparations of the reagents used in the examples are:

[0102] Coating buffer (BD OptEIA Coating Buffer, 51-2713KC) 0.1 M sodium carbonate, pH 9.5;

[0103] Wash buffer (BD OptEIA Wash Buffer, 51-9003739) 20× concentrated wash buffer should be diluted with deionized or distilled water to a 1× working solution;

[0104] Chromogenic reagent A (BD OptEIA Substrate Reagent A, 51-2606KZ) contains a hydrogen peroxide buffer;

[0105] Chromogenic solution B (BD OptEIA Substrate Reagent B, 51-2607KZ) contains an organic solvent of 3,3',5,5'tetramethylbenzidine (TMB);

[0106] Stop solution (BD OptEIA Stop Solution, 51-2608KZ) 1. Sulfuric acid;

[0107] Blocking solution: 5% skim milk, add 5 g of milk to 100 ml of 1× PBS solution.

[0108] Example 2 LAM Antibody Combination Detection of LAM Antigen Sensitivity

[0109] Assay process: The capture antibodies were immobilized with 1 μg / ml of the LAM antibodies BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76, respectively; the detection antigen was a 10-fold serial dilution of the LAM antigen (1000-0.001 ng / ml); and the detection antibodies were 1 μg / ml of the JRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, or BJRbL76-Bio antibodies, respectively. The remaining steps were the same as those in the sandwich ELISA assay in Example 1.

[0110] Results of sensitivity test of paired antibodies recognizing LAM antigens ( Figure 2 (and Table 1): Comprehensive analysis showed that BJRbL01, BJRbL03, BJRbL20, and BJRbL76 had good sensitivity as capture antibodies for detecting purified LAM antigen, with most showing sensitivity above 0.1 ng / ml. When used as a capture antibody, BJRbL52 had slightly lower sensitivity (1 ng / ml) compared to other antibodies in various combinations. When used as detection antibodies, BJRbL01-Bio, BJRbL20-Bio, and BJRbL52-Bio had good sensitivity (0.1 ng / ml) in various combinations. When used as detection antibodies, BJRbL03-Bio and BJRbL76-Bio had slightly lower sensitivity (1 ng / ml) in various combinations. These results suggest that BJRbL01, BJRbL03, BJRbL20, and BJRbL76 are suitable as capture antibodies, and BJRbL01-Bio, BJRbL20-Bio, and BJRbL52-Bio are suitable as detection antibodies.

[0111] Table 1. Sensitivity of antibody combinations for detecting LAM antigens

[0112]

[0113] Example 3 Identification of inactivated supernatant of Mycobacterium standard strains by LAM antibody combination

[0114] Assay procedure: The capture antibodies were immobilized with 1 μg / ml of the LAM antibodies BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76. The detection antigen was the inactivated supernatant of standard mycobacterial strains (1:50 dilution), including 11 slow-growing and 12 fast-growing mycobacteria. The detection antibodies were 1 μg / ml of JRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, BJRbL52-Bio, or BJRbL76-Bio. The remaining steps were the same as those for the sandwich ELISA assay described above.

[0115] Among them, the 11 slow-growing mycobacteria include: Mycobacterium tuberculosis (M.tb.H37Rv), Mycobacterium bovis (M.bovis), Mycobacterium kansasii (M.kansasii), Mycobacterium intracellulare (M.intracellulare), Mycobacterium gordonae (M.gordonae), Mycobacterium marinum (M.marinum), Mycobacterium avium (M.avium), Mycobacterium scrofulaceum (M.scrofulaceum), Mycobacterium gastri (M.gastri), Mycobacterium xenopi (M.xenopi) and Mycobacterium malmoense (M.malmoense).

[0116] Among them, the 12 fast-growing mycobacteria include: Mycobacterium abscessus (M.abscessus), Mycobacterium fortuitum (M.fortuitum), Mycobacterium aurum (M.aurum), Mycobacterium neoaurum (M.neoaurum), Mycobacterium smegmatis (M.smegmatis), Mycobacterium parafortuitum (M.parafortuitum), Mycobacterium salmoniphilum (M.salmoniphilum), Mycobacterium nonchromogenicum (M.nonchromogenicum), Mycobacterium vaccae (M.vaccae), Mycobacterium phlei (M.phlei), Mycobacterium confluentis (M.confluentis) and Mycobacterium gilvum (M.gilvum).

[0117] Results of antibody combination detection of Mycobacterium standard strains ( Figure 3-7And Table 2): Comprehensive analysis shows that the overall detection effect of BJRbL76-Bio as a detection antibody is not good, which again suggests that BJRbL76-Bio is not suitable for use as a detection antibody. Any one of BJRbL01, BJRbL03, BJRbL20, BJRbL52 and BJRbL76 is used as a capture antibody, and any one of BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio and BJRbL52-Bio is used as a detection antibody, which can detect 100% of slow-growing mycobacteria. All combinations do not recognize fast-growing mycobacteria, and the detection specificity is 100%. The ELISA results are based on the OD of the fast-growing mycobacteria group. 450 The critical value was obtained by multiplying the mean by 2.1.

[0118] Table 2. Positive rate of LAM antigen in the detection of slow-growing mycobacterium standard strains by antibody combination

[0119]

[0120] Example 4 LAM Antibody Combination Identification of Inactivated Supernatants of Mycobacterium Clinical Isolates

[0121] Assay procedure: Capture antibodies were immobilized with 1 μg / ml of the LAM antibodies BJRbL01, BJRbL03, BJRbL20, BJRbL52, and BJRbL76. The detection antigen was the inactivated supernatant of clinical mycobacterial isolates (1:100 dilution), including 30 clinical isolates of Mtb (MTB) and 10 isolates of M. abscessus (MA). The detection antibodies were 1 μg / ml of JRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, and BJRbL52-Bio. The remaining steps were the same as those for the sandwich ELISA assay described above.

[0122] Results of antibody combination detection of mycobacterial clinical isolates ( Figure 8-12 ): BJRbL01, BJRbL03, BJRbL20, BJRbL52 and BJRbL76 were used as capture antibodies, and BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio and BJRbL52-Bio were used as detection antibodies. The sensitivity and specificity of any combination were 100%. 450 The values ​​± standard deviation (SD) are shown in Table 3. In general, BJRbL01 was used as the capture antibody and BJRbL01-Bio was used as the detection antibody, with BJRbL20-Bio having the second best detection effect.

[0123] Table 3. Average OD values ​​of mycobacterial clinical isolates detected by paired LAM antibody sandwich ELISA 450 value

[0124]

[0125] Example 5: Detection of common pneumonia pathogens using LAM antibodies

[0126] (1) Indirect ELISA to identify the characteristics of LAM antibodies in recognizing common pneumonia pathogens

[0127] Clinically, tuberculosis infection needs to be differentiated from pneumonia caused by other pathogens. When using LAM antibodies to identify clinical samples from tuberculosis patients, it is important to confirm that LAM antibodies specifically recognize mycobacteria and do not react with other pneumonia pathogens. This example uses indirect ELISA to identify LAM antibodies that react with Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Candida albicans, and Cryptococcus neoformans.

[0128] Detection process: Dilute the inactivated bacterial supernatant with coating solution 1:100 and incubate at 4°C overnight. After washing, blocking, primary antibody reaction (BJRbL01, BJRbL03, BJRbL20, BJRbL52 and BJRbL76, 1μg / ml), secondary antibody reaction (horseradish peroxidase-labeled goat anti-rabbit IgG antibody 1:5000), add substrate for color development, and measure OD with a microplate reader. 450 value.

[0129] Test results ( Figure 13 ) showed that BJRbL01, BJRbL03, and BJRbL76 did not bind to Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Haemophilus influenzae, Candida albicans, and Cryptococcus neoformans. However, BJRbL20 and BJRbL52 bound to Candida albicans. This result not only screened for antibodies BJRbL01, BJRbL03, and BJRbL76 suitable for capturing LAM antigens, but also confirmed that BJRbL20 and BJRbL52 bind to different epitopes of LAM than BJRbL01, BJRbL03, and BJRbL76.

[0130] (2) Sandwich ELISA method to identify the characteristics of LAM antibody recognition and binding to Candida albicans

[0131] The above identification shows that BJRbL20 and BJRbL52 can recognize and bind to Candida albicans. In this example, the sandwich ELISA method was used to identify the epitope characteristics of LAM antibodies that recognize and bind to Candida albicans.

[0132] Detection process: BJRbL20 and BJRbL52 antibodies were diluted in the coating solution to 1 μg / ml and added to the ELISA plate at 4°C overnight. After washing and blocking, 1:100 diluted bacterial supernatant (Mtb.H37Rv, Candida albicans, Staphylococcus epidermidis) was added at 37°C for 2h, 1 μg / ml detection antibodies BJRbL20-Bio and BJRbL52-Bio were added (37°C, 2h), horseradish peroxidase-labeled avidin was added (1:5000; 37°C, 1h), substrate was added for color development, and OD was measured by microplate reader. 450 value.

[0133] Test results ( Figure 14 ) showed that BJRbL20 and BJRbL52, along with BJRbL20-Bio or BJRbL52-Bio, only recognized the LAM antigen in the supernatant of the culture of Mycobacterium tuberculosis H37Rv strain in a sandwich ELISA assay, but not the related components in the supernatant of inactivated Candida albicans. This suggests that BJRbL20 and BJRbL52 may recognize the same LAM antigen epitope; and that the LAM antibody may only recognize a single Candida albicans-related epitope, thus different detection by the sandwich ELISA method.

[0134] Example 6 Detection of LAM Antigens in Pleural Effusion of Tuberculous Pleurisy Patients Using LAM Antibody Combination

[0135] (1) Indirect ELISA and sandwich ELISA for detection of LAM antigen added to pleural effusion

[0136] The purified LAM antigen was diluted to 1 μg / ml in pleural effusion with negative LAM antigen test, and the difference between indirect ELISA and sandwich ELISA was compared.

[0137] Indirect ELISA: Coat the plate with 100 μl / well of pleural effusion sample (LAM antigen concentration: 1 μg / ml) or 100 μl / well of pleural effusion sample in a 1:1 ratio (LAM antigen concentration: 1 μg / ml) overnight at 4°C. After washing and blocking, add the BJRbL01-Bio antibody (1 μg / ml). Avidin-HRP recognizes the biotinylated antibody. The remaining steps are the same as for the indirect ELISA described above.

[0138] Sandwich ELISA: The capture antibody is BJRbL01 (1 μg / ml). 100 μl / well of pleural effusion sample (LAM antigen concentration is 1 μg / ml) is tested, and the detection antibody is BJRbL01-Bio antibody (1 μg / ml). Avidin-HRP recognizes the biotinylated antibody. The remaining steps are the same as those for the sandwich ELISA described above.

[0139] Test results ( Figure 15The sandwich ELISA method can detect LAM antigen, but the indirect ELISA method cannot. Considering that a large number of mixed components in pleural effusion bind to the ELISA plate, LAM antigen cannot be detected, or only minimally binds to the plate. This indicates that the indirect ELISA method is not suitable for detecting relevant components in body fluid samples.

[0140] (2) Detection of LAM antigen in pleural effusion of patients with tuberculous pleurisy

[0141] Methods: 160 patients with tuberculous pleurisy (mean age, 36.4 years; 49 women, 111 men) and 50 patients with malignant pleural effusion (mean age, 59.8 years; 25 women, 25 men) were enrolled as controls. Pleural fluid was collected from these patients and the LAM antigen content in the fluid was measured by sandwich ELISA.

[0142] Detection process: Dilute the capture antibody BJRbL01 to 1 μg / ml in the coating solution, add it to the enzyme-labeled plate, and incubate at 4°C overnight. After washing and blocking, add 1001 μl / well of pleural effusion sample or a serially diluted LAM antigen standard (0.0625-4 ng / ml) (incubate at 4°C overnight), set up a blank control; after washing, add 1 μg / ml of detection antibody BJRbL01-Bio (37°C, 2h), and after washing, add horseradish peroxidase-labeled avidin (1:5000; 37°C, 1h), add substrate for color development, and measure OD with an enzyme-labeled instrument. 450 The test results were statistically analyzed, and a receiver operating characteristic (ROC) curve was drawn to evaluate the performance of LAM antigen detection in pleural effusion for the auxiliary diagnosis of active tuberculosis. A standard curve for LAM antigen detection was drawn, and the LAM antigen content in pleural effusion samples was back-calculated.

[0143] Analysis of LAM antigen detection results in pleural effusion of patients with tuberculous pleurisy Figures 16-19 ): The LAM antigen level in the pleural effusion of patients with tuberculous pleurisy was significantly higher than that in patients with malignant pleural effusion, and the difference was statistically significant ( Figure 16 ). The average OD of malignant pleural effusion group 450 When the value + 2*SD was used as the detection cutoff value, the detection sensitivity was 30.6% (49 / 160) and the specificity was 94% (47 / 50). The area under the ROC curve (AUC) was equal to 0.7701 ( Figure 17 ); When the Youden index (maximum) was used to determine the critical value, the sensitivity was 70% and the specificity was 72%. The standard curve was drawn based on the test results of the LAM antigen standard ( Figure 18), using 2.1 times the blank control as the cutoff value, the results showed that the detection sensitivity of BJRbL01-BJRbL01-Bio reached 62.5 pg / ml. Because the standard sample was diluted in series, the range was narrower than the previous 10-fold serial dilution, resulting in a more precise detection sensitivity.

[0144] According to the standard curve, the content of LAM antigen in the pleural effusion of tuberculosis patients was calculated. 450 The value was lower than the critical value (the average OD 450 The samples with the value of OD + 2*SD) were considered as LAM antigen negative samples, and the concentration was recorded as 0; 450 The concentration of samples with values ​​higher than the critical value is back-calculated ( Figure 19 Among the 49 positive pleural effusion samples, the LAM antigen detection range was 118.0-6530.9 pg / ml, with significant heterogeneity among the samples. In 4 samples, the LAM concentration was higher than 3000 pg / ml ( Figure 19 Left), the LAM antigen concentration of most samples was low; further analysis showed only these samples ( Figure 19 Right). There were 3 positive samples in the control group, with LAM antigen concentrations ranging from 120.7 to 126.0 pg / ml ( Figure 19 ).

[0145] Example 6 Identification of LAM Antigens in Plasma of Tuberculosis Patients by Combination of LAM Antibodies

[0146] Among the 160 patients with tuberculous pleurisy, plasma samples were collected from 45 patients. Plasma samples from these patients were tested for LAM antigen. Plasma samples from 30 healthy controls (HCs) were used as controls. Plasma LAM antigen levels were measured using a sandwich ELISA. Correlation between LAM antigen detection in plasma and pleural effusion was analyzed.

[0147] Detection process: Dilute the capture antibody BJRbL01 to 1 μg / ml in the coating solution and add it to the ELISA plate. Incubate at 4°C overnight. After washing and blocking, add 1001 μl / well of plasma sample (incubate at 4°C overnight). After washing, add 1 μg / ml of the detection antibody BJRbL01-Bio (37°C, 2 hours). After washing, add horseradish peroxidase-labeled avidin (1:5000; 37°C, 1 hour). Add substrate for color development, and measure the OD value using a microplate reader. 450 The test results were statistically analyzed, and a receiver operating characteristic (ROC) curve was drawn to evaluate the efficacy of plasma LAM antigen detection in assisting the diagnosis of active tuberculosis. The LAM antigen content in pleural effusion samples was back-calculated.

[0148] Results of LAM antigen detection in the plasma of patients with tuberculous pleurisy ( Figure 20-22): The plasma LAM antigen level in patients with tuberculous pleurisy was significantly higher than that in healthy people, and the difference was statistically significant ( Figure 20 ). The average OD of the healthy control group 450 When the value + 2*SD was used as the detection critical value, the detection sensitivity was 37.8% (17 / 45) and the specificity was 93.3% (28 / 30). The area under the ROC curve (AUC) was equal to 0.8430 ( Figure 21 When the Youden index (maximum) was used to determine the critical value, the sensitivity was 73.58% and the specificity was 75%. The LAM antigen content in the plasma of tuberculosis patients was calculated to be 115.6-803.8 pg / ml ( Figure 22 ).

[0149] Correlation between LAM antigen detection in plasma and pleural effusion ( Figure 23 ):OD was detected by sandwich ELISA of plasma and pleural effusion samples 450 The results of correlation analysis showed that the LAM antigen detection results in plasma and pleural effusion were positively correlated, and the difference was statistically significant, with a correlation coefficient of 0.3527.

[0150] The sequence information involved in this application is shown in the following table.

[0151]

[0152]

[0153] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Use of the composition in preparing a product for detecting mycobacterial lipoarabinomannan or diagnosing tuberculosis, characterized in that: The composition includes a capture antibody and a detection antibody; the capture antibody includes BJRbL01, and the detection antibody includes a combination of any one or more of biotin-labeled BJRbL01-Bio, BJRbL03-Bio, BJRbL20-Bio, and BJRbL52-Bio antibodies; the amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL01 are shown in SEQ ID NOs: 1-3, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively; The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL03 are shown in SEQ ID NOs: 7-8 and 3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 9, 5, and 6, respectively. The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2, and HCDR3 of BJRbL20 are shown in SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:3, respectively. The amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO:9 and SEQ ID NOs:11-12, respectively. The amino acid sequences of the heavy chain complementary determining regions HCDR1, HCDR2 and HCDR3 of BJRbL52 are shown in SEQ ID NO:7, SEQ ID NO:10 and SEQ ID NO:3, respectively, and the amino acid sequences of the light chain complementary determining regions LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:9, SEQ ID NO:5 and SEQ ID NO:6, respectively.

2. The use according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL01 are shown in SEQ ID NO: 19 and SEQ ID NO:

20.

3. The use according to claim 1, characterized in that The amino acid sequences of the light and heavy chain variable regions and the light chain variable region of BJRbL03 are shown in SEQ ID NO: 21 and SEQ ID NO:

22.

4. The use according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL20 are shown in SEQ ID NO: 23 and SEQ ID NO:

24.

5. The use according to claim 1, characterized in that The amino acid sequences of the heavy chain variable region and light chain variable region of BJRbL52 are shown in SEQ ID NO: 25 and SEQ ID NO:

26.

6. The use according to claim 1, characterized in that The heavy chain constant region of the antibody is selected from the heavy chain constant region of any one of IgD, IgE, IgM, IgA and IgG; the light chain constant region of the antibody is a κ-type or λ-type light chain constant region.

7. The use according to claim 6, characterized in that The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 29; the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30.

8. The use according to any one of claims 1 to 7, characterized in that The mycobacteria include slow-growing mycobacteria.

9. The use according to claim 8, characterized in that The slow-growing mycobacteria include at least one of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium gordonii, Mycobacterium marinum, Mycobacterium avium, Mycobacterium scrofulae, Mycobacterium gastricis, Mycobacterium toadense, and Mycobacterium malmoe.

10. The use according to any one of claims 1 to 7, characterized in that: The diagnosis of tuberculosis includes: auxiliary diagnosis of tuberculosis.

11. The use according to any one of claims 1 to 7, characterized in that: The tuberculosis includes active tuberculosis.

12. The use according to any one of claims 1 to 7, characterized in that: The product includes: reagents and / or kits for sandwich ELISA detection.

13. The use according to any one of claims 1 to 7, characterized in that: Applicable samples of the product include: any one or more of biological fluid samples, environmental samples and artificially prepared standards.

14. The use according to claim 12, characterized in that In the reagent or kit for sandwich ELISA detection, the coating concentration of the capture antibody is 0.1-5 μg / ml.

15. A kit, characterized in that It includes: The composition according to any one of claims 1 to 14.

16. The kit according to claim 15, characterized in that The kit further comprises: at least one of a coating solution, a cleaning solution, a color developing solution A, a color developing solution B, a stop solution and a blocking solution.

17. The kit according to claim 15 or 16, characterized in that The kit is suitable for detecting mycobacterial lipoarabinomannan or tuberculosis.

18. A double antibody sandwich ELISA method for detecting mycobacterial lipoarabinomannan, characterized in that: The capture antibody used in the detection method is the capture antibody described in any one of claims 1 to 14, and the detection antibody used is the detection antibody described in any one of claims 1 to 14; The detection method is not intended for the direct purpose of diagnosing or treating a disease.

19. The detection method according to claim 18, characterized in that Applicable samples of the detection method include: any one or more of biological fluid samples, environmental samples and artificially prepared standards.

20. The detection method according to claim 19, characterized in that The body fluid includes any one of sputum, blood, urine, tissue fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, semen and vaginal fluid.

21. The detection method according to claim 18, characterized in that During detection, the working concentration of the detection antibody is 0.1-5 μg / ml.

Images