A monoclonal antibody for OXA-48 enzyme and its application

Abstract

The present invention relates to the field of antibody technology, and in particular to an OXA-48 enzyme monoclonal antibody and application thereof. The present invention provides an OXA-48 enzyme monoclonal antibody, which has high sensitivity when used for immunoassay, high affinity and good specificity for antigens, is suitable as an immunodiagnostic reagent for in vitro diagnosis of OXA-48 enzyme, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular to an OXA-48 enzyme monoclonal antibody and application thereof. Background Art

[0002] Carbapenem antibiotics Carbapenemase is an ancient biological enzyme. Gene evolution analysis shows that the enzyme may have appeared 1 to 2 billion years ago, and then evolved and integrated into environmental bacteria. Carbapenemase is divided into three categories according to Ambler molecular classification: Class A is a serine proteinase, which is divided into KPC, GES, and PER genotypes. The most common Class A enzyme is KPC, which is found in Klebsiella pneumoniae; Class B is a metalloenzyme, which is divided into IMP, VIM, GIM, SPM, SIM and NDM genotypes, and the common VIM and IMP types are mainly found in Pseudomonas aeruginosa; Category D is OXA enzyme, which is divided into OXA-23 and OXA-48 genotypes. OXA-23 enzyme is common in Acinetobacter baumannii, while OXA-48 enzyme is mainly found in Enterobacteriaceae. Carbapenemase is the main drug resistance mechanism of bacteria. Its encoding gene is located on chromosomes or plasmids. Through clonal transmission or horizontal gene transfer, bacteria can resist β-lactam antibiotics such as meropenem and imipenem.

[0003] OXA-48 enzymes have shown increasingly important carbapenemase activity in carbapenem resistance, and reports of OXA-48 enzymes in Enterobacteriaceae are increasing year by year around the world. The rapid spread of OXA-48 enzyme Enterobacteriaceae in different ecosystems has brought great challenges to clinical practice.

[0004] There are many methods for laboratory detection of carbapenemases. They mainly include modified Hodge test, CarbaNP test, modified carbapenem inactivation test, enzyme inhibitor enhancement test, immunogold label test and molecular biology methods. Among them, immunodiagnostic reagents are the fastest growing sub-field of in vitro diagnostic reagents. The qualitative or quantitative detection of carbapenemases can be achieved by utilizing the specific binding between antigen and antibody. However, the sensitivity of antibodies disclosed in the prior art when used for immunodetection still needs to be improved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an OXA-48 enzyme monoclonal antibody or an antigen-binding fragment thereof, comprising the following complementary determining regions:

[0006] CDR1-VH: amino acid sequence is GYWMN;

[0007] CDR2-VH: amino acid sequence is QIYPGDDDTNFNGNFKG;

[0008] CDR3-VH: amino acid sequence is GDYYGTFDV;

[0009] CDR1-VL: amino acid sequence is KSSQSLLNSRNQKNYLA;

[0010] CDR2-VL: amino acid sequence is WASTRES;

[0011] CDR3-VL: The amino acid sequence is QQYYTYPYT.

[0012] Preferably, the OXA-48 enzyme monoclonal antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are sequentially shown in SEQ ID NOs. 1-4, and / or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are sequentially shown in SEQ ID NOs. 5-8.

[0013] Preferably, the OXA-48 enzyme monoclonal antibody further comprises a constant region.

[0014] Preferably, the constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

[0015] Preferably, the species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

[0016] Preferably, the OXA-48 enzyme monoclonal antibody antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

[0017] Furthermore, the present invention provides a detection reagent or a kit, which contains the OXA-48 enzyme monoclonal antibody or an antigen-binding fragment thereof.

[0018] Preferably, the detection reagent or kit is used for detecting OXA-48 enzyme by double antibody sandwich indirect ELISA method or immunochromatography method.

[0019] Preferably, the kit comprises an immunochromatographic detection card, wherein the immunochromatographic detection card comprises the OXA-48 enzyme monoclonal antibody or an antigen-binding fragment thereof.

[0020] Preferably, the immunochromatographic test card further comprises a marker.

[0021] Preferably, the marker is selected from colloidal gold, colloidal silver, colloidal carbon, magnetic microspheres, fluorescent microspheres, colored microspheres or quantum dots.

[0022] Preferably, the present invention provides an OXA-48 enzyme chromatography test strip, comprising a sample pad, a colloidal gold binding pad, a reaction membrane, a water absorbent pad and a bottom plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the colloidal gold binding pad, the reaction membrane and the water absorbent pad are sequentially pasted on the bottom plate; the detection line is coated with the OXA-48 enzyme monoclonal antibody or its antigen binding fragment.

[0023] Furthermore, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof, the detection reagent or kit, the kit for detecting OXA-48 enzyme or the detection test strip in at least one of the following aspects:

[0024] (1) Preparation of products for detecting OXA-48 enzyme;

[0025] (2) In vitro detection of OXA-48 enzyme for non-diagnostic and non-therapeutic purposes;

[0026] (3) Quality control of OXA-48 enzyme detection products.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The invention provides an OXA-48 enzyme monoclonal antibody, which has high sensitivity when used for immune detection, high affinity and good specificity for antigens, is suitable for being used as an immunodiagnostic reagent for in vitro diagnosis of OXA-48 enzyme, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the sensitivity test result of the immunochromatographic test strip developed using the OXA-48 enzyme monoclonal antibody of the present invention.

[0030] Figure 2 It is the specific detection result of the immunochromatographic test strip developed using the OXA-48 enzyme monoclonal antibody of the present invention.

[0031] Figure 3 It is the stability test result of the immunochromatographic test strip developed using the OXA-48 enzyme monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0034] Example 1 Preparation of monoclonal antibodies against OXA-48 enzyme

[0035] 1. Antigen materials

[0036] The antigen material is OXA-48 enzyme antigen protein, which is used for animal immunization after being identified by SDS-PAGE method.

[0037] 2. Animal immunization

[0038] Five BALB / c mice were immunized with OXA-48 enzyme antigen protein. The immunization scheme is shown in Table 1.

[0039] Table 1 Animal immunization scheme

[0040]

[0041] Seven days after each immunization, the sera of the immunized animals were tested by indirect ELISA to determine the level of immune response.

[0042] 3. Cell fusion and screening

[0043] Cell fusion and plating: Two mice with higher immune response levels were selected for fusion, and one round of cell fusion was performed using the electrofusion method. All cells in each round of fusion will be plated into 96-well plates, with a maximum of 15 96-well plates.

[0044] Initial screening: Use the indirect ELISA method to screen the supernatant of the fused cells and select the supernatant that is positive for the target protein.

[0045] Confirmation screening: For the positive clones obtained in the initial screening stage, the indirect ELISA method is used to screen the positive mother clone cells that are positive for the antigen protein and negative for the His-tag / counter-screening protein counter-screening.

[0046] Clone expansion and freezing: Transfer positive mother clone cells to 24-well plates for expansion, up to 20 strains, and collect 2 mL of supernatant from each expanded clone for indirect ELISA detection. Cryopreserve specific positive clone cells to avoid clone loss.

[0047] 4. Subclone screening

[0048] Subcloning: The positive mother clones were subcloned by limiting dilution method (up to 5 strains were selected) to ensure that these positive mother clones were derived from a single mother clone cell. Subclone screening was performed by indirect ELISA. The coating antigens were: A: OXA-48 protein; B: OXA-23 protein; C: His tag negative protein; the coating concentration was 1 μg / mL, 100 μL / well; the coating buffer was pH 7.4 phosphate buffer; the screening results are shown in Table 2.

[0049] Table 2

[0050]

[0051] 5. Epitope classification and subtype identification

[0052] The above cell lines were subjected to epitope classification test by ELISA method, and the subtypes were identified using subtype kit. The results are shown in Table 3.

[0053] Table 3

[0054]

[0055] 6. Screening of cell lines

[0056] The relative affinity of different cell lines was tested by ELISA method, and the results are shown in Table 4.

[0057] Table 4

[0058]

[0059] 7. Antibody Preparation

[0060] The 8F6A9 cell line with the best affinity was selected for ascites preparation of antibodies. After the produced antibodies were purified, the titer of the purified antibodies was detected by indirect ELISA, where the coating antigens were: A: OXA-48 protein; B: OXA-23 protein; C: His tag negative protein; the coating concentration was 1 μg / mL, 100 μL / well; the coating buffer was a phosphate buffer at pH 7.4. The test results are shown in Table 5.

[0061] Table 5

[0062]

[0063] The titer is the highest dilution at which S / B (signal / blank) is >= 2.1. The OD in the blank 450 is the average of two technical replicates.

[0064] 8. Antibody Sequencing

[0065] Sequencing of the light chain variable region and heavy chain variable region of the OXA-48 enzyme monoclonal antibody revealed that the complementary determining regions of the antibody are as follows:

[0066] CDR1-VH: amino acid sequence is GYWMN;

[0067] CDR2-VH: amino acid sequence is QIYPGDDDTNFNGNFKG;

[0068] CDR3-VH: amino acid sequence is GDYYGTFDV;

[0069] CDR1-VL: amino acid sequence is KSSQSLLNSRNQKNYLA;

[0070] CDR2-VL: amino acid sequence is WASTRES;

[0071] CDR3-VL: The amino acid sequence is QQYYTYPYT.

[0072] The amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0073] Example 2

[0074] The OXA-48 enzyme monoclonal antibody of Example 1 was used to develop an immunochromatographic test strip, and the preparation steps were as follows:

[0075] 1. Preparation of colloidal gold

[0076] The gold was prepared by reduction method, and the gold preparation conditions were 5 mL of 2% chloroauric acid + 10 mL of 0.01% trisodium citrate.

[0077] 2. Colloidal gold labeled antibodies

[0078] Physical adsorption method, by adjusting the pH value to make colloidal gold and OXA-48 enzyme monoclonal antibody (Bomi-M9203-8H11) combined. The specific labeling conditions are: add 10µL of 0.1M potassium carbonate solution per milliliter of colloidal gold, and the labeling concentration of monoclonal antibody is 10μg / mL.

[0079] 3. Testing line and quality control line

[0080] Test line: Take appropriate concentration (1.0 mg / mL) of the OXA-48 enzyme monoclonal antibody of Example 1 and coat it on a nitrocellulose membrane to prepare a test line, dry it at 37°C, and spray the membrane at a volume of 1.0 µL / cm.

[0081] Quality control line: Take 1.0 mg / mL goat anti-mouse IgG polyclonal antibody to prepare the quality control line on the fiber membrane, dry at 37℃, and spray the spot at a volume of 1.0 µL / cm.

[0082] The cellulose sample pad, the glass fiber colloidal gold binding pad (containing the colloidal gold labeled antibody), the nitrocellulose membrane (containing the detection line and the quality control line) and the cellulose absorbent pad are sequentially pasted on the bottom plate to prepare a test paper strip.

[0083] 4. Inspection methods

[0084] Return the test strips, sample diluent and sample to room temperature. The test strips are tested as follows:

[0085] a. Take out the test strip from the aluminum foil bag, mark the sample, and place it flat on a horizontal work surface;

[0086] b. Take the treated sample solution (in this example, 3 μg / mL, 1.5 μg / mL, 30 ng / mL, 15 ng / mL, 300 pg / mL and 150 pg / mL of OXA-48 enzyme) or the quality control product and add it directly to the sample pad;

[0087] c. Add 100µL (2-3 drops) of sample diluent (preparation method: prepare 0.01M Tris-HCl buffer, pH 8.5; add 1% BSA and 10% sucrose);

[0088] d. Read the results within 15 to 20 minutes.

[0089] 5. Interpretation of test results

[0090] a. Positive: Both the test line and the quality control line are colored.

[0091] b. Negative: Only one red quality control line appears in the detection window.

[0092] c. Invalid: No red quality control line appears in the detection window.

[0093] The commercially available A and B test strips were used as controls. Figure 1 shown.

[0094] It can be seen that the sensitivity of the immunochromatographic test strip developed using the OXA-48 enzyme monoclonal antibody of the present invention can be as low as 150 pg / mL.

[0095] Example 3

[0096] The specificity of the immunochromatographic test strip prepared in Example 2 was tested, and the specific steps were as follows: 5 different types of carbapenemase enzyme types (1068#KPC positive, 1024#NDM positive, 1017#VIM positive, 4007#IMP positive, 1029#OXA23 positive) of marketed reagents were selected and confirmed, and the immunochromatographic test strip prepared in Example 2 was used to detect 5 different types of carbapenemase enzyme types, and 60 μL of each enzyme type was added for detection, and the results were interpreted for 15-20 minutes.

[0097] The test results are as follows Figure 2 As shown, it can be seen that the detection results of the antibody of the present invention (OXA-48 type) for samples positive for different types of enzymes KPC, NDM, VIM, IMP, and OXA-23 are all negative, indicating that different types of enzymes will not interfere with the antibody of the present invention (OXA-48 type), indicating that the antibody of the present invention has good specificity.

[0098] Example 4

[0099] The stability of the immunochromatographic test strip prepared in Example 2 was tested by accelerated aging experiment. The specific steps are as follows: the immunochromatographic test strip prepared in Example 2 was placed in a 37±2°C incubator for 3, 6, 9, 12, 15, 18, and 21 days, and a positive sample was used for testing once at each time point. 60 μL of sample was added for testing, and the results were read for 15-20 minutes.

[0100] The test results are as follows Figure 3 As shown, accelerated aging was performed at 37 degrees (3 days, 6 days, 9 days, 12 days, 15 days, 18 days, and 21 days) to detect positive samples. The results showed no difference from the results at room temperature, indicating that the antibody of the present invention has good stability.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-OXA-48 enzyme monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The complementarity determining regions include: CDR1-VH: amino acid sequence is GYWMN; CDR2-VH: amino acid sequence is QIYPGDDDTNFNGNFKG; CDR3-VH: amino acid sequence is GDYYGTFDV; CDR1-VL: amino acid sequence is KSSQSLLNSRNQKNYLA; CDR2-VL: amino acid sequence is WASTRES; CDR3-VL: The amino acid sequence is QQYYTYPYT.

2. The anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-OXA-48 enzyme monoclonal antibody comprises light chain framework regions FR1-L, FR2-L, FR3-L and FR4-L whose sequences are sequentially shown in SEQ ID NO.1-4, and / or heavy chain framework regions FR1-H, FR2-H, FR3-H and FR4-H whose sequences are sequentially shown in SEQ ID NO.5-8.

3. The anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The anti-OXA-48 enzyme monoclonal antibody also includes a constant region.

4. The anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.

5. The anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to claim 3, characterized in that: The species origin of the constant region is cow, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

6. The anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The anti-OXA-48 enzyme monoclonal antibody antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv.

7. A detection reagent or kit, characterized in that: It contains the anti-OXA-48 enzyme monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 6.

8. A kit for detecting OXA-48 enzyme, characterized in that The kit comprises an immunochromatographic detection card, and the immunochromatographic detection card comprises the anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

9. An OXA-48 enzyme chromatography test strip, characterized in that: The invention comprises a sample pad, a colloidal gold binding pad, a reaction membrane, a water absorbent pad and a bottom plate; the reaction membrane is provided with a detection line and a quality control line, and the sample pad, the colloidal gold binding pad, the reaction membrane and the water absorbent pad are sequentially pasted on the bottom plate; the detection line is coated with the anti-OXA-48 enzyme monoclonal antibody or the antigen binding fragment thereof according to any one of claims 1 to 6.

10. Use of the anti-OXA-48 enzyme monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the detection reagent or kit according to claim 7, the kit for detecting OXA-48 enzyme according to claim 8, or the detection test strip according to claim 9 in at least one of the following aspects: (1) Preparation of products for detecting OXA-48 enzyme; (2) In vitro detection of OXA-48 enzyme for non-diagnostic and non-therapeutic purposes; (3) Quality control of OXA-48 enzyme detection products.

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