Method for detecting specific immune globulin E of mosquito saliva protein in animal serum through capture method enzyme-linked immunosorbent assay

Abstract

The invention discloses a method for detecting mosquito salivary protein specific immune globulin E in animal serum through a capture method enzyme-linked immunosorbent assay, and belongs to the technical field of immunodetection. The monoclonal antibody specifically combined with the human or mouse IgE Fc fragment is used as the capture antibody, and the V5 tag is combined, so that the interference of IgG in a detection sample can be avoided, and the detection sensitivity breaks through the limit of an existing detection mode.

Description

Technical Field

[0001] The invention relates to a capture method enzyme-linked immunosorbent assay for detecting mosquito salivary protein-specific immunoglobulin E in animal serum, belonging to the technical field of immune detection. Background Art

[0002] Repeated mosquito bites can induce the host to produce mosquito salivary proteins (MSPs)-specific immunoglobulin E (IgE), sensitizing the host's skin mast cells. 1 When the mosquito bites the host again, the secreted MSP binds to the specific IgE on the surface of the host's sensitized skin mast cells, causing them to activate. The activated skin mast cells release a variety of pro-inflammatory mediators, causing immediate hypersensitivity reactions, manifested as erythema or wheal at the site of the mosquito bite, causing symptoms such as itching or pain. 2 Almost everyone will develop an allergic reaction to MSPs after repeated mosquito bites, but most people's allergic reactions are short-lived and disappear within minutes to hours after the bite. 3 Some people have more pronounced or prolonged allergic reactions to mosquito bites, a condition called mosquito allergy syndrome. 4 Mosquito allergy syndrome can seriously affect the patient's quality of life and even threaten his life. 4 It is now recognized that MSPs-specific IgE plays a key role in the pathogenesis of mosquito allergic reactions and mosquito allergy syndrome. Therefore, the detection of MSPs-specific IgE in host serum is of great significance for the diagnosis and prognosis of mosquito allergic reactions and mosquito allergy syndrome.

[0003] Although MSP-specific IgE plays a crucial role in the pathogenesis, diagnosis, and prognosis of mosquito allergy, efficient and specific methods for detecting MSP-specific IgE are currently lacking. This is primarily due to two main reasons: 1) It is difficult to collect sufficient MSPs, and preparing a single MSP is even more challenging. 2) The concentration and affinity of IgE in host serum are much lower than those of IgG, making it difficult to eliminate interference from MSP-specific IgG using conventional methods.

[0004] Several methods for detecting MSPs-specific IgE have been reported in the literature, including enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay. 5-8 Three limitations of the immunoblotting assay have limited its application: 1) interference from MSPs-specific IgG; 2) the requirement for large amounts of MSPs; and 3) the inability to accurately quantify MSPs-specific IgE titers.

[0005] ELISA is a powerful tool for detecting specific antigens or antibodies, with good detection sensitivity and specificity.9 At present, the ELISA method for detecting MSPs-specific IgE is mostly indirect ELISA. 10-12 . Although these methods are easy to operate, they cannot rule out the interference of IgG in serum. When MSPs-specific IgG and IgE coexist, specific IgG preferentially binds to the coated MSPs due to its absolute advantage in content and affinity. Therefore, the detection sensitivity of indirect ELISA is significantly lower than that of capture ELISA. Capture ELISA can specifically enrich IgE and remove IgG, thereby significantly improving the sensitivity and specificity of detection. However, capture ELISA still requires the use of a high amount of mosquito salivary protein 13 This greatly limits its promotion and application. The problem of time-consuming and labor-intensive collection of mosquito salivary proteins can be solved by in vitro expression systems, but the expression systems currently used in the literature are prokaryotic. The MSPs produced in prokaryotic expression systems may have significant differences in structure and function from the MSPs naturally synthesized by mosquitoes, thus affecting the detection of MSPs-specific IgE. 14 . Summary of the Invention

[0006] Problems to be solved by the invention

[0007] The existing technology cannot solve the problem of IgG interference when using indirect methods to detect MSPs-specific IgE, and cannot detect low concentrations of MSPs-specific IgE. At the same time, the current efficient MSPs expression method can quickly obtain a large amount of MSPs for detection. In the present invention, MSPs are expressed using a Drosophila S2 cell expression system and purified using a 6x His tag. The MSPs expressed in Drosophila S2 cells can ensure that they have the same structure and function as the salivary proteins naturally synthesized by mosquitoes. Furthermore, the present invention creatively utilizes the method of combining an antibody that specifically binds to the Fc fragment of IgE with a V5 tag to isolate IgG from contact with the detection system, and enables the detection sensitivity to break through the limits of existing detection methods.

[0008] Solutions for solving problems

[0009] [1] A method for detecting mosquito salivary protein-specific IgE, wherein the method comprises:

[0010] (a) a step of immobilizing a capture antibody;

[0011] (b) a step of binding the capture antibody to the target antibody in the sample to be tested;

[0012] (c) a step of binding the specific antigen to the target antibody in the sample to be tested;

[0013] (d) a step of binding of the specific antibody to the specific antigen;

[0014] (e) a step of detecting the captured antibody; and

[0015] (f) a color development step;

[0016] Optionally, the capture antibody is an antibody targeting the Fc fragment of the IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE;

[0017] The specific antigen is mosquito salivary protein (MSP); optionally, the MSP further comprises a tag sequence, and the tag is selected from the group consisting of a His tag, a Flag tag, an HA tag and a V5 tag, preferably a V5 tag.

[0018] [2] The method according to [1], wherein the specific antibody is an antibody against the tag, preferably an antibody against the V5 tag;

[0019] Optionally, the specific antibody is IgG, preferably mouse IgG.

[0020] [3] The method according to [1] or [2], wherein in step (e), a detection antibody is added to detect the captured antibody; the detection antibody is an antibody against the specific antibody, preferably an anti-mouse IgG antibody.

[0021] [4] The method according to [3], wherein the detection antibody further comprises a tracer marker, wherein the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

[0022] [5] The method according to [1], wherein the sample comprises serum, plasma and / or whole blood.

[0023] [6] The method according to [1], wherein the method further comprises a closing step after step (a) and a terminating step after step (f);

[0024] Preferably, a washing step is further included after at least one of steps (b) to (e).

[0025] [7] The method according to any one of [1] to [6], wherein the specific antigen is obtained using a Drosophila cell expression system.

[0026] [8] A kit for detecting mosquito salivary protein-specific IgE, wherein the kit comprises: a capture antibody, a specific antigen, a specific antibody and / or a detection antibody;

[0027] Optionally, the capture antibody is an antibody targeting the Fc fragment of the IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE;

[0028] Optionally, the specific antigen is mosquito salivary protein (MSP); optionally, the MSP further comprises a tag sequence, and the tag is selected from the group consisting of a His tag, a Flag tag, an HA tag and a V5 tag, preferably a V5 tag;

[0029] The specific antibody is an antibody against the tag, preferably an antibody against the V5 tag; optionally, the specific antibody is IgG, preferably mouse IgG;

[0030] The detection antibody is an antibody against a specific antibody, preferably an anti-mouse IgG antibody.

[0031] [9] The kit according to [8], wherein the capture antibody further comprises a tracer marker, wherein the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

[0032]

[10] The kit according to [8] or [9], wherein the kit further comprises one or more of an ELISA plate, a washing solution, a sample diluent, a negative control, a positive control, a substrate color development solution, and a stop solution.

[0033] Effects of the Invention

[0034] The method of producing mosquito salivary protein-specific IgE provided by the present invention has the following advantages:

[0035] 1) Using monoclonal antibodies against the Fc fragment of human or mouse IgE as capture antibodies not only improves the capture efficiency of IgE and the specificity of detection, but also maintains the binding ability of the IgE F(ab)2 fragment to MSPs;

[0036] 2) Using high-purity MSPs expressed in the Drosophila S2 system instead of MSPs obtained by collecting natural mosquito saliva eliminates the time-consuming and labor-intensive mosquito saliva collection process;

[0037] 3) A V5 tag was added to the carboxyl terminus of each recombinant MSP, which enables all MSPs to be detected using a single V5-tag monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 .Principles and operating steps of indirect ELISA and capture ELISA; (A) indirect ELISA, (B) capture ELISA.

[0039] Figure 2 Comparison of indirect ELISA and capture ELISA for the detection of mosquito salivary protein (MSP)-specific IgE in mouse serum. (A) Schematic diagram of the repeated mosquito bite model; (B)-(C) Detection of AAEL000749-specific IgE in mouse serum using indirect ELISA (B) and capture ELISA (C). In the figures, each C57BL / 6J mouse was bitten by one, five, or ten uninfected Aedes aegypti mosquitoes twice weekly for four weeks. Unbitten mice served as negative controls. Each group consisted of six mice, and serum was collected 7, 14, 21, and 28 days after the first mosquito bite.

[0040] Figure 3 Specificity of capture ELISA. (A) Purity and specificity of recombinant mosquito salivary proteins AAEL0006347 and AAEL000749 were verified by SDS-PAGE (i) and Western blotting targeting the V5 tag (ii), respectively; lane 1: AAEL006347, lane 2: AAEL000749. (B) Detection of AAAEL000749-specific IgE in mouse serum. C57BL / 6J mice were immunized twice with purified Aedes aegypti salivary proteins AAEL000749 or AAEL006347, two weeks apart. Simultaneously, BSA or adjuvant was injected as a control. Six mice per group. Serum was collected seven days after the second immunization, and the titer of AAEL000749-specific IgE was determined by capture ELISA.

[0041] Figure 4 Comparison of capture ELISA and indirect ELISA for the detection of MSP-specific IgE in human serum. (A) Schematic diagram of a human trial with repeated mosquito exposure. (B)-(C) Detection of AAEL000749-specific IgE in human serum using indirect ELISA (B) and capture ELISA (C). The figure shows four healthy adult volunteers recruited: Volunteers 1, 2, and 3, who had no history of Aedes aegypti bites, and Volunteer 4, who had a history of Aedes aegypti bites. Each volunteer was bitten by 10 uninfected Aedes aegypti mosquitoes twice weekly for four consecutive weeks. Serum was collected 7, 14, 21, and 28 days after the first mosquito bite.

[0042] Figure 5 Detection of AAEL000749-specific IgE in the serum of healthy individuals in areas with widespread Aedes aegypti distribution. (A) AAEL000749-specific IgE grading criteria. NC, negative control. (B) Percentage of individuals with different AAEL000749-specific IgE titers in the serum of healthy individuals in Xishuangbanna, according to different months. DETAILED DESCRIPTION

[0043] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0044] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0045] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0046] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0047] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0048] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0049] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0050] In this specification, when "normal temperature" or "room temperature" is used, the temperature may be 23±2°C.

[0051] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0052] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0053] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, Joseph Frank et al. "Molecular Cloning: A Laboratory Manual." (2001). (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0054] In the present invention, "MSPs", "MSP", and "mosquito salivary proteins" are used interchangeably to refer to a variety of biologically active proteins contained in mosquito saliva. These proteins play an important role in the blood-sucking process of mosquitoes, not only promoting blood-sucking but also regulating the host's immune and inflammatory responses, thereby affecting the transmission of mosquito-borne viruses and parasites.

[0055] In the present invention, "Fc" refers to the carboxyl terminal portion of an intact antibody obtained by papain hydrolysis, typically comprising the CH2, CH3, and CH4 domains of the antibody. In some specific embodiments, the antibody comprises IgE, particularly mosquito salivary protein-specific IgE.

[0056] The technical solution of the present invention is described in detail below:

[0057] In terms of concentration, the concentration of IgG in serum is tens of thousands times that of IgE (physiological basis), and the concentration of IgE in circulation remains at a low level, usually around 0.1 μg / mL, while that of IgG is 10 mg / mL. In terms of affinity, IgG obtains high affinity through affinity maturation, while the production of IgE lacks this optimization process (mechanistic basis). 18 Therefore, due to the advantages of IgG in serum in terms of concentration and affinity, it causes serious interference in the detection of IgE.

[0058] Currently, the existing technology mostly uses indirect methods to detect salivary protein-specific IgE. When traditional indirect ELISA detects allergen-specific IgE, high concentrations of IgG in the serum will competitively bind to antigens (such as MSP), resulting in false positives / false negatives. Although the existing capture method can separate IgE, it still cannot rule out the binding of free IgG to antigens in subsequent steps, and it cannot detect low concentrations of salivary protein-specific IgE. Based on these problems, the present invention creatively uses a method of combining an antibody that specifically binds to the Fc fragment of IgE with a V5 tag. After inserting the V5 tag into the allergen protein, it is combined with an anti-IgE Fc capture antibody to isolate the contact of IgG with the detection system, and the detection sensitivity breaks through the limit of existing detection methods.

[0059] <First Aspect>

[0060] The present invention provides a method for detecting mosquito salivary protein-specific IgE, which is a capture ELISA method, comprising:

[0061] (a) a step of immobilizing a capture antibody;

[0062] (b) a step of binding the capture antibody to the target antibody in the sample to be tested;

[0063] (c) a step of binding the specific antigen to the target antibody in the sample to be tested;

[0064] (d) a step of binding of the specific antibody to the specific antigen;

[0065] (e) a step of detecting the captured antibody; and

[0066] (f) Color development step.

[0067] In some optional embodiments, the specific capture antibody is an antibody targeting the Fc fragment of IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE. Exemplary examples include the monoclonal antibody Omalizumab, which can be purchased from MCE, Catalog No. HY-P9950, and the rabbit anti-mouse IgE antibody, which can be purchased from Nordic-MUbio, Catalog No. RAM / IgE(Fc) / 7S. The specific capture antibody can specifically bind only to IgE in the sample, and after washing, IgG and other proteins in the sample can be eliminated.

[0068] In some embodiments, the specific antigen is mosquito salivary protein (MSP). In some optional embodiments, the MSP further comprises a tag sequence, the tag being selected from the group consisting of a His tag, a Flag tag, an HA tag, and a V5 tag, preferably a V5 tag, the V5 tag comprising the sequence:

[0069] GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACG (SEQ ID NO: 1).

[0070] For example, the MSP sequence including the V5 tag is:

[0071] ATGATACTCCAATTCTGGGTCGTAACGTTTTCCGTGCTGTTCGCAGCTCGCGCCGATGAAAATCACTCAATATTGATCAAGCTGAACGACCTGGATCACCGCTTCACGCAAATGTTCAGCCAGCAATTTTACCGACACACCCAGCAAGTAACGGATCGAGTCTCGGCGCTGAAGATTTCCATTGACACCAATCTACTCGAACTGGACCAGCAGATTCAGCAAGCGCTGGATGGCATTCAATCCAACGAATCGTCATCGTCAGCTTCGGCAACGAAACCGCCCGGACTCACAACCATACCGATTGGATCCGAGCCTAGGGTTCCGGCTCTGTACGAACGGGAGCGCTACGGAGGAGATTGGTTGGTTGTGATGCATCGGTATGACGGCTCCGTAAAGTTCGATCGCACTTGGGCCGAGTACCGGGATGGGTTCGGAATGGTTGGGCAGGAGTTTTGGTACGGACTGGAGCGACTGCATCAGTTGACCAAGGAGAAATCGTACGAACTGATGGTTGAGATGGAGGACTTCAATGGGAGCTTGAAGTATGCTTGGTATGATAAATTTGTGGTGGGCCCCGAGGAACAACGTTACGCTTTGGTTGAGTTGGGGACGTTTAATGGAACGACAGATGGGGATTCCTTGAAGCCTCATAAAGGATCGGGATTCTCGACGTACGACAATGATGATTTCGGTTGCTCGAACAAGTACGCCAAAGGAGGATGGTGGTATTACAGTGGAAAGTGTTACGGATCGAGCCTTACTGGTATTTGGAAAAACGAACTTGCTTACTCATCGATAGTTTGGATGAAGTTCTCCGATGTTTCCAACACGCCGTTGAAGCTTGTAAGAATGATGATACGACCGAAAAATCTCGAGTCTAGAGGGCCCTTCGAAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACG(SEQ ID NO:2).

[0072] Amino acid sequence corresponding to MSP containing the V5 tag:

[0073] MILQFWVVTFSVLFAARADENHSILIKLNDLDHRFTQMFSQQFYRHTQQVTDRVSALKISIDTNLLELDQQIQQALDGIQSNESSSSASATKPPGLTTIPIGSEPRVPALYERERYGGDWLVVMHRYDGSVKFDRTWAEYRDGFGMVGQEFWYGLERL HQLTKEKSYELMVEMEDFNGSLKYAWYDKFVVGPEEQRYALVELGTFNGTTDGDSLKPHKGSGFSTYDNDDFGCSNKYAKGGWWYYSGKCYGSSLTGIWKNELAYSSIVWMKFSDVSNTPLKLVRMMIRPKNLESRGPFEGKPIPNPLLGLDST(SEQ ID NO:3).

[0074] In some exemplary embodiments, the MSP is obtained through a Drosophila cell expression system, more preferably through a Drosophila S2 expression system.

[0075] In some embodiments, the specific antibody is an antibody against the tag, preferably an antibody against the V5 tag. Exemplarily, the specific antibody is IgG, preferably mouse IgG. Exemplarily, the specific antibody can be purchased from MBL, catalog number: M167-3.

[0076] Furthermore, in step (e), a detection antibody is added to detect the captured antibody, wherein the detection antibody is an antibody against a specific antibody, preferably an anti-mouse IgG antibody, more preferably a goat anti-mouse IgG antibody.

[0077] In some exemplary embodiments, the detection antibody further comprises a tracer marker, and the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

[0078] In some embodiments, the detection antibody containing a tracer marker can be an HRP-labeled goat anti-mouse IgG antibody, which can be labeled with HRP using conventional technical means in the art, or can be obtained through commercial channels, for example, it can be purchased from MBL, product number: 330.

[0079] The sample to be tested in the present invention is serum, plasma and whole blood obtained directly, or can be a sample obtained by extracting human blood and separating the blood.

[0080] In some embodiments, a blocking step is further included after step (a) to prevent non-specific binding.

[0081] In some optional embodiments, a washing step is further included after at least one of steps (b) to (e), wherein the plate is washed 3 to 5 times, preferably 4 times, with PBST solution.

[0082] More specifically, the method for detecting mosquito salivary protein-specific IgE in the present invention comprises the following steps:

[0083] (1) In the capture antibody immobilization step, the monoclonal antibody Omalizumab or rabbit anti-mouse IgE antibody was diluted at a concentration of 4 μg / mL in 1× ELISA coating buffer, added to the high-binding microplate, and coated at 4°C overnight;

[0084] (2) Blocking: Block with 5% (w / v) skim milk powder in PBS at room temperature for 2 hours;

[0085] (3) Sample addition: Serum was diluted with PBS containing 0.5% (w / v) skim milk powder at an appropriate dilution (1:5 for human serum and 1:10 for mouse serum). 50 μL of diluted serum was added to each well. For the blank control, 50 μL of 0.5% skim milk was added. The cells were incubated at room temperature for 2 h.

[0086] (4) Washing: Wash the plate 3 to 5 times with PBST;

[0087] (5) Adding specific antigen: Add 0.1 μg / mL purified V5-tagged mosquito salivary protein AAEL000749 to 0.5% skim milk and incubate at room temperature for 1 h;

[0088] (6) Washing: Wash the plate 3-5 times with PBST;

[0089] (7) Adding specific antibodies: Add diluted anti-V5 tag antibodies to each well and incubate at room temperature for 1 hour; the diluted anti-V5 tag antibodies are diluted with 0.5% skim milk at a dilution ratio of 1:1500 to 1:2500;

[0090] (8) Washing: Wash the plate 3-5 times with PBST;

[0091] (9) Adding detection antibody: Add HRP-labeled goat anti-mouse IgG antibody (dissolved in 0.5% skim milk) to each well and incubate at room temperature for 40 minutes; the HRP-labeled goat anti-mouse IgG antibody is diluted with 0.5% skim milk at a dilution ratio of 1:4500 to 1:5500;

[0092] (10) Washing: Wash the plate 3-5 times with PBST;

[0093] (11) Color development: Add color developing solution and incubate at room temperature for 10 min; the color developing solution is ultra-sensitive TMB color developing solution;

[0094] (12) Termination: Add stop solution;

[0095] (13) Determination: Detect OD450nm using an enzyme-labeled instrument.

[0096] In the present invention, the "0.5% skim milk" is "PBS solution containing 0.5% (w / v) skim milk powder".

[0097] <Second Aspect>

[0098] A kit for detecting mosquito salivary protein-specific immunoglobulin E, comprising: a capture antibody, a specific antigen, a specific antibody and / or a detection antibody.

[0099] In some optional embodiments, the specific capture antibody is an antibody targeting the Fc fragment of IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE. The specific capture antibody includes the monoclonal antibody Omalizumab and a rabbit anti-mouse IgE antibody. The monoclonal antibody Omalizumab can be purchased from MCE, catalog number: HY-P9950, and the rabbit anti-mouse IgE antibody can be purchased from Nordic-MUbio, catalog number: RAM / IgE(Fc) / 7S.

[0100] In some optional embodiments, the specific antigen is mosquito salivary protein (MSP). Preferably, the MSP further comprises a tag sequence, and the tag is selected from a His tag, a Flag tag, an HA tag and a V5 tag, and the tag is preferably a V5 tag.

[0101] In some optional embodiments, the specific antibody is an antibody against the tag, preferably an antibody against the V5 tag. Exemplarily, the specific antibody is IgG, preferably mouse IgG. Exemplarily, the specific antibody can be purchased from MBL, catalog number: M167-3.

[0102] The detection antibody is an antibody against a specific antibody, preferably an anti-mouse IgG antibody.

[0103] The detection antibody further comprises a tracer marker, and the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

[0104] The kit further comprises one or more of an ELISA plate, a washing solution, a sample diluent, a negative quality control product, a positive quality control product, a substrate color development solution, and a stop solution.

[0105] In some embodiments, the sample diluent comprises a PBS solution containing skim milk or skim milk powder.

[0106] In some embodiments, the washing solution includes PBS, PBST, etc.

[0107] Example

[0108] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0109] Materials and Methods

[0110] 1. Ethics Statement

[0111] Blood samples for the detection of mosquito saliva protein (mosquito saliva protein)-specific IgE in serum were donated by healthy volunteers with informed consent and collected at Tsinghua University Hospital and Xishuangbanna Dai Autonomous Prefecture People's Hospital. The Medical Ethics Committees of Tsinghua University and Xishuangbanna Dai Autonomous Prefecture People's Hospital approved the collection and use of human blood samples.

[0112] 2. Experimental Animals

[0113] C57BL / 6J mice (Cat#Jax 000664, The Jackson Laboratory) were purchased from The Jackson Laboratory and bred in the SPF (specific pathogen-free) animal facility at Tsinghua University. Four-week-old female C57BL / 6J mice were used for animal experiments. All experiments were approved by and adhered to the guidelines of the Laboratory Animal Welfare and Ethics Committee of Tsinghua University. Aedes aegypti (Rockefeller strain) mosquitoes were maintained at 28°C and 80% humidity in a dedicated incubator (Cat#Model 818, Thermo Fisher) according to standard husbandry procedures.

[0114] 3. Mosquitoes bite mice repeatedly

[0115] To induce antibodies specific for mosquito salivary proteins, C57BL / 6J mice were bitten twice weekly by uninfected Aedes aegypti mosquitoes for four consecutive weeks. Before each bite, mice were anesthetized with an intraperitoneal injection of approximately 250 mg / kg of tribromoethanol. The anesthetized mice were placed in a mosquito cup (size: upper diameter 11.5 cm x lower diameter 9.3 cm x height 10.8 cm) and bitten by one, five, or ten mosquitoes. Unbitten mice served as negative controls. Successful blood collection was confirmed by observing the mosquito's abdomen for congestion. Serum was collected from the mice on days 7, 14, 21, and 28 after exposure.

[0116] 4. Immunization of mice with mosquito saliva protein

[0117] Four-week-old female C57BL / 6J mice were randomly divided into four groups of eight: immunized with AAEL000749, immunized with AAEL006347, immunized with bovine serum albumin (BSA, Cat# V900933, Sigma), and an adjuvant-only control group. Five micrograms of mosquito salivary protein or BSA mixed with an equal volume of aluminum adjuvant (Cat# vac-alu-250, InvivoGen) were subcutaneously injected into each group. A booster immunization was administered two weeks later. Serum samples were collected seven days after the second immunization.

[0118] 5. Mosquito bites on healthy volunteers

[0119] Four healthy adult volunteers were recruited, including one with a history of Aedes aegypti bites and three without a history of bites. Each volunteer was subjected to controlled bites by uninfected Aedes aegypti mosquitoes twice a week for 4 consecutive weeks. During each bite, 10 uninfected Aedes aegypti mosquitoes that had been starved for 24 hours were confined to a mesh blood feeding device (20×20×20 cm). 3 ) , volunteers exposed their forearms to mosquitoes for 5 minutes to allow them to feed. Successful feeding was confirmed by observing whether the mosquito's abdomen became engorged with blood. Adverse reactions to bites were monitored throughout the study, and volunteers were free to withdraw from the study at any time.

[0120] 6. Purification of recombinant mosquito salivary protein

[0121] The gene encoding the Aedes aegypti salivary protein was amplified using cDNA derived from mosquito salivary glands and inserted into the pMT / Bip / V5-His A vector (Cat# V4130-20, Invitrogen) to construct a salivary protein plasmid. The salivary protein expression plasmid was co-transfected with a hygromycin-resistant plasmid into Drosophila S2 cells to establish a cell line stably expressing the recombinant mosquito salivary protein. The recombinant protein, which carries a V5 tag and a 6x His tag at the C-terminus, was purified using a cobalt column (Cat# 635515, Clontech). The purified recombinant salivary protein was verified by V5 tag immunoblotting and its purity was assessed by SDS-PAGE.

[0122] 7. SDS-PAGE and Western Blot

[0123] Purified mosquito salivary protein samples were added to SDS PAGE loading buffer (Cat#P0015F, Beyotime) and denatured by heating at 100°C for 5 minutes. The samples were then separated by electrophoresis on a 12% SDS-PAGE (Cat#P01212, LABLEAD). The gel was stained with Coomassie Brilliant Blue (Cat#P0017F, Beyotime) for 1 hour and then destained until the protein bands were clearly visible. The samples were then transferred to a polyvinylidene fluoride (PVDF) membrane (18V for 55 minutes). The PVDF membrane was blocked with 5% skim milk and incubated with an HRP-conjugated V5 tag antibody (Cat#R961-25, Invitrogen) overnight at 4°C. The membrane was washed four times with PBST and developed using a chemiluminescent reagent (WBKLS0100, Millipore).

[0124] 8. Statistical analysis

[0125] Quantitative data with normal distribution and homogeneity of variance are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 10.0.0 software. Data from three or more groups were compared using one-way analysis of variance (ANOVA), two-way analysis of variance (ANOVA), and post-ANOVA multiple t-test. Statistical significance was set at p < 0.05. ns indicates no significance, * indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001.

[0126] Example 1: Principles and operating steps of capture ELISA and indirect ELISA

[0127] In this embodiment, "0.5% skim milk" refers to "PBS solution containing 0.5% (w / v) skim milk powder."

[0128] 1. Figure 1 A shows the principle and operation steps of indirect ELISA.

[0129] (1) Coating: Purified single mosquito salivary proteins were diluted in 1× ELISA coating buffer (Cat#C1050, Solarbio) at a concentration of 8 μg / mL (50 μL / well), added to high-binding microplates (Cat#3690, Corning), and coated overnight at 4°C;

[0130] (2) Blocking: Block with PBS solution (skim milk) containing 5% (w / v) skim milk powder (Cat#232100, BD) at 25°C for 2 h;

[0131] (3) Sample addition: Serum was diluted with 0.5% skim milk at an appropriate dilution (human serum was diluted 1:5, mouse serum was diluted 1:10), and 50 μL of diluted serum was added to each well. For the blank control, 50 μL of 0.5% skim milk was added and incubated at 25°C for 2 h.

[0132] (4) Washing: Wash the plate four times with PBS containing 0.02% (v / v) Tween 20 (Cat#P1379, Sigma);

[0133] (5) Add detection antibody: add 50 μL of HRP-labeled goat anti-mouse IgE antibody (Cat#SA5-10263, Invitrogen) diluted 1 / 1,000 (dissolved in 0.5% skim milk) to each well and incubate at 25°C for 1 h;

[0134] (6) Wash the plate 4 times with PBST;

[0135] (7) Color development: Add ultrasensitive TMB colorimetric solution (Cat#P0206, Beyotime) to detect bound antibodies at a volume of 50 μL / well and incubate at 25°C for 10 min;

[0136] (8) Termination: Add 50 μL / well of ELISA stop solution (Cat#C1058, Solarbio);

[0137] (9) Determination: Detect OD450nm using an enzyme-labeled instrument.

[0138] It is worth noting that the concentration and affinity of MSP-specific IgG in serum are much higher than those of MSP-specific IgE, which makes MSP-specific IgG preferentially bind to coated MSP, thereby interfering with the binding of MSP-specific IgE to coated MSP and significantly reducing the sensitivity of indirect ELISA.

[0139] 2. Figure 1B shows the principle and operation steps of the capture ELISA of the present invention.

[0140] (1) Coating: IgE-capturing monoclonal antibody Omalizumab (Cat#HY-P9950, MCE) (for human serum) or rabbit anti-mouse IgE antibody (Cat#RAM / IgE(Fc) / 7S, Nordic-MUbio) (for mouse serum) was diluted to 4 μg / mL (50 μL / well) in 1× ELISA coating buffer, added to a high-binding microplate, and coated overnight at 4°C.

[0141] (2) Blocking: Block with 5% (w / v) skim milk powder (Cat#232100, BD) in PBS at 25°C for 2 h;

[0142] (3) Sample addition: Serum was diluted with 0.5% skim milk at an appropriate dilution (human serum 1:5, mouse serum 1:10), and 50 μL of diluted serum was added to each well. For the blank control, 50 μL of 0.5% skim milk was added, and the cells were incubated at 25°C for 2 h.

[0143] (4) Washing: Wash the plate 4 times with PBST;

[0144] (5) Adding specific antigen: add 50 μL of 0.5% skim milk containing 0.1 μg / mL purified V5-tagged mosquito salivary protein AAEL000749 to each well and incubate at 25°C for 1 h;

[0145] (6) Washing: Wash the plate 4 times with PBST;

[0146] (7) Add specific antibody: add 50 μL of anti-V5 tag antibody (Cat#M167-3, MBL) diluted 1 / 2,000 (dissolved in 0.5% skim milk) to each well and incubate at 25°C for 1 h;

[0147] (8) Washing: Wash the plate 4 times with PBST;

[0148] (9) Add detection antibody: add 50 μL of HRP-labeled goat anti-mouse IgG antibody (Cat#330, MBL) diluted 1 / 5,000 (dissolved in 0.5% skim milk) to each well and incubate at 25°C for 40 min;

[0149] (10) Washing: Wash the plate 4 times with PBST;

[0150] (11) Color development: Add ultrasensitive TMB color development solution at 50 μL / well and incubate at 25°C for 10 min;

[0151] (12) Stop: add 50 μL / well of stop solution;

[0152] (13) Determination: Detect OD450nm using an enzyme-labeled instrument.

[0153] Example 2: Comparison of the effectiveness of capture ELISA and indirect ELISA in detecting MSP-specific IgE in mouse serum

[0154] (1) Sensitivity of different ELISAs

[0155] First, this example established a model of repeated mosquito bites in mice. C57BL / 6J mice were selected and grouped. Different numbers (1, 5, or 10) of uninfected Aedes aegypti mosquitoes were used to bite the exposed skin of the mice for 5 minutes twice a week for 4 weeks; the unbitten mice served as negative controls. Serum was collected in batches on the 7th, 14th, 21st, and 28th days after the first bite to detect the dynamic changes of mosquito salivary protein-specific IgE. This model can induce mice to produce specific IgG and IgE antibodies against MSP. The model achieves repeated MSP sensitization by controlling the number of mosquitoes and fixing the bite frequency and cycle, providing standardized samples for the optimization of subsequent antibody detection methods. ( Figure 2 A).

[0156] The protein encoded by the AAEL000749 gene (named AAEL000749) (NCBI protein sequence: MILQFWVVTFSVLFAARADENHSILIKLNDLDHRFTQMFSQQFYRHTQQVTDRVSALKISIDTNLLELDQQIQQALDGIQSNESSSSASATKPPGLTTIPIGSEPRVPALYERERYGGDWLVVMHRYDGSVKFDRTWAEYRDGFGMVGQEFWYGLERLHQLTKEKSYELMVEMEDFNGSLKYAWYDKFVVGPEEQRYALVELGTFNGTTDGDSLKPHKGSGFSTYDNDDFGCSNKYAKGGWWYYSGKCYGSSLTGIWKNELAYSSIVWMKFSDVSNTPLKLVRMMIRPKN, SEQ ID NO: 4) is abundant in mosquito saliva. 15,16 Therefore, AAEL000749 was selected as a representative MSP to verify the effectiveness of the two ELISA methods. The titer of AAEL000749-specific IgE in mouse serum was detected 1-4 weeks after repeated mosquito bites. The mouse serum samples were diluted 1:10 and tested. The indirect ELISA test in Example 1 showed that AAEL000749-specific IgE could not be detected in all mouse sera ( Figure 2B), while the capture ELISA was able to detect AAEL000749-specific IgE in all mouse sera collected 2-4 weeks after mosquito bites, and showed that the titer of AAEL000749-specific IgE in mouse sera increased significantly with the increase in the number and duration of mosquito exposure ( Figure 2 This indicates that the detection sensitivity of capture ELISA is significantly higher than that of indirect ELISA.

[0157] (2) Specificity of capture ELISA

[0158] Next, the inventors studied the specificity of capture ELISA.

[0159] Capture ELISA was used to detect the titer of AAEL000749-specific IgE in the sera of mice immunized with AAEL000749 protein (positive control, encoding gene Gene ID: 110675548), AAEL006347 protein (irrelevant salivary protein control, encoding gene Gene ID: 5567877), or bovine serum albumin (non-salivary protein control) and adjuvant-immunized mice. The purity and specificity of the purified AAEL000749 and AAEL006347 proteins were identified by SDS-PAGE and Western Blot. Figure 3 A). The results of capture ELISA showed that AAEL000749-specific IgE was detected in the sera of mice immunized with AAEL000749 protein, but not in the sera of mice immunized with other proteins or adjuvants. Figure 3 B), which shows that the capture ELISA has good detection specificity.

[0160] Example 3: Comparison of the effectiveness of capture ELISA and indirect ELISA in detecting MSP-specific IgE in human serum

[0161] The inventors recruited four healthy volunteers: Volunteers 1, 2, and 3 had no history of Aedes aegypti bites, while Volunteer 4 had a history of repeated exposure to Aedes aegypti mosquitoes and therefore served as a positive control. Each volunteer was bitten by mosquitoes twice a week for 4 weeks, with 10 uninfected Aedes aegypti mosquitoes biting the volunteer's forearm for 5 minutes each time ( Figure 4 A). Indirect ELISA and capture ELISA were used to detect AAEL000749-specific IgE in serum before and 1-4 weeks after mosquito bites. Indirect ELISA detected AAEL000749-specific IgE in serum samples of volunteers 1 and 4 2 and 4 weeks after mosquito bites. The titer at week 4 was unexpectedly lower than that at week 2 ( Figure 4B), which indicates that the indirect ELISA method lacks sensitivity and accuracy, and the results are unstable. In contrast, the capture ELISA method detected AAEL000749-specific IgE in the serum of all volunteers 2 weeks after mosquito exposure, and its titer increased with the passage of time and the number of exposures ( Figure 4 C). As expected, volunteer 4, who had a history of exposure to Aedes aegypti, showed significantly higher AAEL000749-specific IgE titers throughout the trial compared to the other volunteers. These results further demonstrate that capture ELISA has better detection sensitivity and accuracy than indirect ELISA.

[0162] Example 4: Using capture ELISA to monitor AAEL000749-specific IgE in the serum of healthy people in areas where Aedes aegypti is widely distributed

[0163] In this example, the capture ELISA method was used to dynamically monitor the titer of AAEL000749-specific IgE in the serum of healthy people in Xishuangbanna, where Aedes aegypti is widely distributed. The titer of AAEL000749-specific IgE in the serum was divided into four categories: high, medium, low and negative according to the corresponding OD450 value ( Figure 5 As expected, the proportion of people with high, medium, and low titers of AAEL000749-specific IgE in August, September, and October was significantly higher than that in January, February, and March ( Figure 5 B), which is consistent with the literature report that the mosquito population density in Xishuangbanna in August, September and October is much higher than that in January, February and March. 17 The monitoring results once again indicate that the capture ELISA of the present invention can be used as a powerful tool for monitoring MSP-specific IgE in human serum.

[0164] References

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[0180] 18. Gould HJ, Sutton BJ. IgE in allergy and asthma today. Nat Rev Immunol. 2008 Mar; 8(3): 205-17. doi: 10.1038 / nri2273. PMID: 18301424. It should be noted that although the technical solutions of the present invention are described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto. The above descriptions of various embodiments of the present invention are exemplary, not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles, practical applications, or technical improvements of the embodiments in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for detecting mosquito salivary protein-specific IgE, characterized in that: The method comprises: (a) a step of immobilizing a capture antibody; (b) a step of binding the capture antibody to the target antibody in the sample to be tested; (c) a step of binding the specific antigen to the target antibody in the sample to be tested; (d) a step of binding the specific antibody to the specific antigen; (e) a step of detecting the captured antibody; and (f) a color development step; Optionally, the capture antibody is an antibody targeting the Fc fragment of the IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE; The specific antigen is mosquito salivary protein (MSP); optionally, the MSP further comprises a tag sequence, and the tag is selected from the group consisting of a His tag, a Flag tag, an HA tag and a V5 tag, preferably a V5 tag.

2. The method according to claim 1, characterized in that The specific antibody is an antibody against the tag, preferably an antibody against the V5 tag; Optionally, the specific antibody is IgG, preferably mouse IgG.

3. The method according to claim 1 or 2, characterized in that In step (e), a detection antibody is added to detect the captured antibody; the detection antibody is an antibody against the specific antibody, preferably an anti-mouse IgG antibody.

4. The method according to claim 3, characterized in that The detection antibody further comprises a tracer marker, and the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

5. The method according to claim 1, wherein The sample includes serum, plasma and / or whole blood.

6. The method according to claim 1, characterized in that The method further comprises a blocking step after step (a), and a terminating step after step (f); Preferably, a washing step is further included after at least one of steps (b) to (e).

7. The method according to any one of claims 1 to 6, characterized in that The specific antigen is obtained by using a Drosophila cell expression system.

8. A kit for detecting mosquito salivary protein-specific IgE, characterized in that: The kit comprises: a capture antibody, a specific antigen, a specific antibody and / or a detection antibody; Optionally, the capture antibody is an antibody targeting the Fc fragment of the IgE, preferably a monoclonal antibody targeting the Fc fragment of human or mouse IgE; Optionally, the specific antigen is mosquito salivary protein (MSP); optionally, the MSP further comprises a tag sequence, and the tag is selected from the group consisting of a His tag, a Flag tag, an HA tag and a V5 tag, preferably a V5 tag; The specific antibody is an antibody against the tag, preferably an antibody against the V5 tag; optionally, the specific antibody is IgG, preferably mouse IgG; The detection antibody is an antibody against a specific antibody, preferably an anti-mouse IgG antibody.

9. The kit according to claim 8, characterized in that The capture antibody further comprises a tracer marker, and the tracer marker is selected from one or more of the group consisting of luminol, alkaline phosphatase, horseradish peroxidase, acridinium ester and adamantane; preferably, the tracer marker is horseradish peroxidase.

10. The kit according to claim 8 or 9, characterized in that The kit further comprises one or more of an ELISA plate, a washing solution, a sample diluent, a negative control, a positive control, a substrate color development solution, and a stop solution.

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