KR20220018753A - Antibody specific to Fagales plant pollen or antigen-binding fragment thereof, composition or kit including the same, and detection method using the same - Google Patents

Abstract

Provided are an antibody specifically binding to oak tree pollen allergen or antigen-binding fragment thereof, kit for detecting oak tree pollen allergen using the antibody, and a quantitatively detecting method of oak tree pollen allergen, and provided is hybridoma cell lines. Accordingly, quantification of major pollen allergen is usefully used for improving a diagnostic method for allergic diseases and developing immunotherapeutic drugs.

Description

Antibody specific to Fagales plant pollen or antigen-binding fragment thereof, composition or kit including the same, and detection method using the same}

The present invention relates to an antibody or antigen-binding fragment thereof specific for pollen of an oak plant, a composition comprising the same, a kit, and a detection method using the same.

For the treatment of allergic diseases, drug treatment, surgical treatment, or immunotherapy are mainly used. Among them, immunotherapy is a method of treating allergic diseases by inducing desensitization and returning the immunological mechanism, and is spotlighted as the only fundamental treatment for allergic diseases. The fact that the dose of allergen administered at this time and the clinical effect of immunotherapy are directly proportional is known as the principle of immunotherapy. In particular, a maintenance dose of 5 to 20 ug of a major allergen is associated with clinical improvement after immunotherapy, and natural allergen products are partially formulated based on the content of specific allergens. Nevertheless, one of the major problems in immunotherapy is that there is currently no method for standardizing the content and titer of allergens.

In order to overcome the problems related to the standardization of content and titer, it is known that a method of measuring the concentration of a major allergen and allowing doctors to select an appropriate reagent based on this is known to be useful. The concentration of the major allergen can be measured by using the enzyme immunization method using a monoclonal antibody using the major allergen produced by the genetic recombination method as a standard reagent (MD Chapman et al., Journal of Allergy and Clinical Immunology 122 (2008)) 882-889.e2.)

Under this technical background, research has been continuously conducted to solve problems related to the diagnosis and treatment of pollen allergy (Korean Patent Registration No. 10-1919575), but it is still insufficient to solve this problem.

One aspect is to provide an antibody or antigen-binding fragment thereof that specifically binds to an oak pollen allergen.

Another aspect is to provide a kit for detecting oak pollen allergens comprising the antibody or antigen-binding fragment thereof.

Another aspect is to provide a method for quantitative analysis of oak pollen allergens using the antibody or antigen-binding fragment thereof.

Another aspect is to provide a hybridoma cell line (Accession No.: KCLRF-BP-00484) that produces an antibody that specifically binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

Another aspect is to provide a hybridoma cell line (Accession No.: KCLRF-BP-00485) that produces an antibody that specifically binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

One aspect provides an antibody or antigen-binding fragment thereof that specifically binds to an oak pollen allergen.

The oak pollen allergen may be Que ac 1. The oak pollen allergen may be Que ac 1.0101. The Que ac 1.0101 may be an isoform of Que ac 1, and may be a recombinant one. The Que ac 1.0101 may be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1. The Que ac 1.0101 (SEQ ID NO: 1) has been deposited with GenBank under the accession number MN201198. The amino acid sequence represented by SEQ ID NO: 1 may be the following sequence.

The Que ac 1 may be derived from a plant of the order Fagales. The oak tree plant may include plants of the family Fagaceae or the family Betulaceae. Quercus, for example, oak ( Quercus accutisima ), oak ( Q. aliena ), oak ( Q. dentata ), Japanese oak ( Q. mongolica , Mongolian oak), Japanese oak ( Q. serrata ), oyster oak ( Q. variabilis ), etc., beech ( Fagus ), for example, beech ( F. engleriana ), American beech ( F. grandifolia ), etc.), or chestnut ( Castanea ) , For example, chestnut ( C. crenata ), chestnut tree ( C. bungeana ) and the like. Birch plants include alder ( Alnus ), for example, alder ( A. japonica ), alder ( A. sibilica ), etc., birch ( Betula ), for example, birch ( B. platyphylla var. japonica ). ), Manchurian birch ( B. platyphylla ), birch tree ( B. schmidtii ), water birch ( B. davurica ), etc., hazel ( Corylus ), for example, clover hazel ( C. hallaisanensis ), hazel ( C. heterophylla ), hornbeam ( C. sieboldiana var. manshurica ), or the like, or hornbeam ( Carpinus ) plants, such as hornbeam ( C. laxiflora ), blackcurrant ( C. cordata ), hornbeam ( C. turczaninowii ) and the like. The oak tree plant may be an oak tree plant native to Korea.

The "allergen" may mean an antigen that induces an allergy or an antigen that causes an allergic disease. When antibodies are produced in vivo due to ingestion or contact with any kind of substance including an allergen substance, an antigen-antibody reaction may occur when re-intake or re-contact of the same substance occurs.

As used herein, "polypeptide", "peptide" and "protein" are used interchangeably and include reference to a polymer of amino acid residues. The term applies not only to natural amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acid. The terms also apply to those polymers containing conservative amino acid substitutions such that the protein remains functional.

The Que ac 1 is about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more similar sequence to pathogenesis related protein (PR)-10. It may have a structure and activity.

The "PR" is a protein expressed when plants are subjected to biological and abiotic stress, and is a signal that induces a defense response, and was classified into 11 types by Van Loon (1994), but the types are gradually increasing in recent years. The PR may mean a protein whose concentration is rapidly increased when attacked by a pathogen, and has antibacterial activity, stress resistance activity, RNase activity, and the like. Among them, PR-10 is known to be expressed in parsley, peas, potatoes, soybeans, asparagus or pepper, etc., Bet v 1-related proteins homologues, ribonucleases, etc. It belongs to this category.

The "antibody" refers to a protein molecule serving as a receptor for specifically recognizing an antigen, including an immunoglobulin molecule having immunological reactivity with a specific antigen. Antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, whole antibodies, or antigen-binding fragments thereof. The antibody also includes chimeric antibodies, bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies, and tetrabodies. In addition, single-chain antibodies having a binding function to FcRn (neonatal Fcreceptor), scaps, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds are included. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and the IgG includes IgG1, IgG2, IgG3 and IgG4.

The antibody may be an antibody derived from a first hybridoma cell (Accession No.: KCLRF-BP-00485) or an antibody derived from a second hybridoma cell (Accession No.: KCLRF-BP-00484) have.

The antigen-binding fragment refers to a fragment having an antigen-binding function, and includes Fc, Fd, Fab, Fab', Fv and F(ab') ₂ , and the like. The Fc refers to a tail portion of an antibody that interacts with a cell surface receptor called an Fc receptor. The Fd refers to the heavy chain portion included in the Fab fragment, and F(ab′) ₂ refers to a fragment including Fd, Fab, Fab′ and Fv. The Fab has a structure having a light chain and heavy chain variable regions, a light chain constant region and a heavy chain first constant region (CH1 domain), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region including one or more cysteine residues at the C terminus of the heavy chain CH1 domain. The F(ab') ₂ antibody is produced by forming a disulfide bond with a cysteine residue in the hinge region of Fab'. Fv (variable fragment) refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Of these, disulfide Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond, and single chain Fv (scFv) generally has a heavy chain variable region and a light chain variable region connected by a covalent bond through a peptide linker. . Such antibody fragments can be obtained using proteolytic enzymes (for example, by restriction digestion of the whole antibody with papain to obtain Fab, and by digestion with pepsin to obtain F(ab') ₂ fragments), preferably It can be produced through genetic recombination technology.

In general, an immunoglobulin has a heavy chain and a light chain, each heavy and light chain comprising a constant region and a variable region. The variable regions of the light and heavy chains include three complementarity-determining regions (CDRs) and four framework regions (FRs) called complementarity determining regions. The CDRs of each chain mainly play a role in binding to an epitope of an antigen, and are typically named CDR1, CDR2, and CDR3 sequentially, starting from the N-terminus. In addition, the FR of each chain may be sequentially named FR1, FR2, FR3, FR4 starting from the N-terminus.

The amino acid sequence is construed to include a sequence showing substantial identity to the sequence described in the sequence listing, if the mutation having a biologically equivalent activity is considered. The term "substantial identity" means that the sequence of the present invention and any other sequence are aligned to the maximum extent, and when the aligned sequence is analyzed using an algorithm commonly used in the art, at least 80% refers to a sequence showing homology, most specifically 90% homology.

Therefore, if it exhibits biologically equivalent activity, an amino acid sequence having high homology with the amino acid sequence represented by SEQ ID NO: 1, for example, the homology is 80% or more, more specifically, high homology of 90% or more It should also be construed as having an amino acid sequence within the scope of the present invention.

Another aspect provides a kit for detecting oak pollen allergens comprising the antibody or antigen-binding fragment thereof.

Among the terms or elements mentioned in the description of the antibody, the same as those already mentioned are the same as described above.

The oak pollen allergen may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

The kit may be an enzyme-linked immunosorbent assay (ELISA) kit, a 2-site ELISA kit, an RT-PCR kit, a DNA chip kit, or a protein chip kit. In one embodiment, the kit for detecting an oak pollen allergen may be a kit including essential elements necessary for performing an ELISA.

In one embodiment, the essential element comprises a labeling substance. The labeling material may be Q dot (Quantum dot), an enzyme, colloidal gold (Coloid gold), a fluorescent material (Fluorescein), a radioactive material or a dye (Dye).

The enzyme is Horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, luciferase, beta-D-galactosidase, malic acid It may include, but is not limited to, dehydrogenase (MDH: malate dehydrogenase), acetylcholinesterase (acetylcholinesterase), or an analog having a similar activity thereto. In the case of using such an enzyme or analog, binding of the labeling material can be checked by binding the enzyme or analog to the secondary antibody and then reacting by adding a substrate capable of exhibiting a color reaction by the enzyme or analog. .

The fluorescent material is fluoresceine isothiocyanate, phycobiliproteins, 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein , tetrachloro-6-carboxyfluorescein (tetrachloro-6-carboxyfluorescein), FAM (5-carboxyfluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichloroflorescein ), Cy3 (cyanine-3), Cy5 (cyanine-5), 6-carboxytetramethyl-rhodamine (6-carboxytetramethyl-rhodamine) or TAMRA (5-Carboxytetramethylrhodamine), BHQ3 (black hole quencher3) may be used, However, the present invention is not limited thereto. When the fluorescent material is used, binding of the labeling material can be confirmed by binding the fluorescent material to the secondary antibody and detecting the fluorescent material using a fluorescence microscope or the like.

The radioactive material may be ¹²⁵ I, ¹³¹ I, ¹⁴ C or ³ H, but is not limited thereto. In the case of using the radioactive material, binding of the labeling material can be checked by binding the radioactive material to the secondary antibody and detecting the radioactive material with an X-ray film or the like.

The kit may include an antibody derived from a first hybridoma cell (Accession No.: KCLRF-BP-00485) or an antibody derived from a second hybridoma cell (Accession No.: KCLRF-BP-00484). have. The antibody derived from the first hybridoma cell (Accession No.: KCLRF-BP-00485) may be a capture antibody. The antibody derived from the second hybridoma cell (Accession No.: KCLRF-BP-00484) may be a detection antibody.

Another aspect is the step of contacting the test sample with the antibody or antigen-binding fragment thereof according to claim 1; And it provides a quantitative analysis method of oak pollen allergen comprising the step of measuring the amount of the antibody to the oak pollen allergen in the contacted sample.

Among the terms or elements mentioned in the description of the antibody or kit, the same as those already mentioned are the same as described above.

As the quantitative analysis method, various known quantitative analysis methods may be used. In one embodiment, the quantitative analysis method is Western blotting (western blotting), ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA: radioimmunoassay), radioimmunodiffusion (radial immunodiffusion), Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography ( immunochromatography), FACS analysis (fluorescenceactivated cell sorter analysis), and protein chip technology assay (protein chip technology assay) may be any one selected from the group consisting of. The ELISA may be a 2-site ELISA. When using the ELISA method, the analysis may be performed on a microplate.

The ELISA was performed using an antibody derived from a hybridoma cell (Accession No.: KCLRF-BP-00485) as a capture antibody and an antibody derived from a hybridoma cell (Accession No.: KCLRF-BP-00484) as a detection antibody. can be carried out.

In one embodiment, before the contacting step, the quantitative analysis method may further include the step of dissociating oak pollen allergen in the sample. The "dissociation" may refer to physical or chemical treatment of the sample so that the oak pollen allergen in the sample has an appropriate structure or state that can be recognized by the antibody.

In one embodiment, the quantified oak pollen allergen may be used for immunotherapy. In the immunotherapy, the drug may be administered using various routes in the body. Specifically, subcutaneous immunotherapy (SCIT) in which the drug is injected subcutaneously or sublingual immunotherapy (SLIT) administered sublingually may be used, but is not limited thereto. In particular, lingual immunotherapy is non-invasive compared to subcutaneous injection, can be self-administered, and has a low frequency and severity of side effects, so it can be usefully used.

Another aspect provides a hybridoma cell line (Accession No.: KCLRF-BP-00484) that produces an antibody that specifically binds to a polypeptide represented by the amino acid sequence of SEQ ID NO: 1.

Among the terms or elements mentioned in the description of the antibody, kit, or quantitative analysis method, the same as those already mentioned are the same as described above.

Another aspect provides a hybridoma cell line (Accession No.: KCLRF-BP-00485) that produces an antibody that specifically binds to the polypeptide represented by the amino acid sequence of SEQ ID NO: 1.

Among the terms or elements mentioned in the description of the antibody, kit, or quantitative analysis method, the same as those already mentioned are the same as described above.

An antibody or antigen-binding fragment thereof, or a kit according to an aspect may be used to quantify a pollen allergen of an oak extract. In particular, since the allergen is a major allergen of oak tree, which is one of the major causes of pollen allergy in Koreans, the quantification can be usefully used for diagnosing pollen allergy in Koreans. In addition, the quantification can be utilized in the development of an effective immunotherapeutic agent for pollen allergy.

1 is a photograph comparing IgE-reactive components between oak (S) and commercial oak mix (O) (Red, Virginia live and white oak) by separating protein (20 μg) by 15% SDS-PAGE under reducing conditions. to be.
2 is a photograph comparing IgE reactive components between oak and commercial oak mixes (Red, Virginia live and white oak), which were used as serum pooled from oak allergy patients.
Figure 3 is a graph showing the cross-reactivity (Cross-reactivity) of oak and commercial oak mix.
4 is a photograph showing the detection of native and recombinant Que ac 1 by Western blot by developing an oak pollen extract (20 μg) and recombinant Que ac 1 (5 μg) under 15% SDS-PAGE.
5 is a photograph of western blot detection of Que ac 1 and recombinant Que ac 1 in the extract using a monoclonal antibody.
6 is a graph showing a 2-Site ELISA titration curve for an oak pollen extract.
7 is a graph showing a 2-Site ELISA titration curve for recombinant Que ac 1.
8 is a photograph showing whether PR-10 protein is recognized in various oak pollen extracts by the monoclonal antibody generated against recombinant Que ac 1.
9 is a photograph showing whether PR-10 protein is recognized in various oak pollen extracts by the monoclonal antibody generated against recombinant Que ac 1.
10 is a photograph showing whether PR-10 protein is recognized in various oak pollen extracts by the monoclonal antibody generated against recombinant Que ac 1.
11 is a photograph showing the detection of a subtype of Que ac 1 in a pollen extract separated by two-dimensional gel electrophoresis.
12 is a photograph of detecting a subtype of Que ac 1 with the monoclonal antibody 3E6.
13 is a photograph of detecting a subtype of Que ac 1 with the monoclonal antibody 11G5.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Experimental Preparation

Example 1-1. Allergen extract preparation

Pollen from Mongolian oak was collected by a botanical taxonomist, and pollen from Quercus alba (White oak) and birch ( Betula verrucosa ; common silver birch) was purchased from Allergon (Angelholm, Sweden). Pollen extracts were prepared as previously described (Jeong KY et al., Yonsei Med J2016; 57 (2): 399-406). Specifically, pollen was collected from a field in Seoul, and the collected pollen was degreased three times with ethyl ether (1:5 w/v) and then allergen in phosphate buffered saline (PBS) at pH 7.4 at 4 °C for 48 hours. was extracted. The extract was centrifuged at 13,000 g for 15 min at 4°C. The supernatant was then dialyzed against distilled water (cutoff of 3.5 kDa, Spectrum, USA). The dialyzed sample was filtered (0.22 μm, Millipore, USA) and insoluble matter was sterilized and removed. After measuring the protein concentration by Bradford assay (Bio-Rad, USA), the extract was lyophilized and stored at -80°C until used.

Oak pollen extract prepared in the same manner as described above was obtained from Prolagen (Seoul, Korea). An oak mix, a mixture of pollen extracts from three different oak trees (red oak, virginia live oak and white oak), was purchased from HollisterStier Laboratories LLC (Spokane, USA). Allergy units were given at 1:20 w/v (20,000 PNU/mL).

Example 1-2. target selection

This study was approved by the institutional ethics committee. Consent was obtained from the patient for skin testing and blood collection. Fifteen (18 to 55 years old; average 30 years old) visitors to the Allergy-Asthma Center at Severance Hospital in Seoul, Korea were enrolled in an in vivo standardized test of Japanese hop extract (Table 1). In the spring, an intradermal skin test (IDT) was performed on a patient suffering from various allergic symptoms due to pollen. The clinical characteristics of registered oak allergy patients are shown in Table 1 below.

gender age ED50 BAU sensitization profile
(ImmunoCAP or SPT)* sIgE for t7
(class) Total IgE M 19 13.8 80,274.16 t3, t7, g6, w6, w22, d2, 69.3 (5) 968 M 34 13.0 33,333.33 t1, t2, t3, t7, t8, t10, t11, t12, t14, t15, t17, t19, t70, t205,, t213, g2, g3, g5, g6, g8, w1, w5, w10, w9, w22 , e1, e5, 3.29 (2) 48.2 M 30 12.9 29,865.28 t2, t3, t7, t8, t10, t17, d1, d2, e1 M 29 15.6 579,954.61 t1, t2, t3, t7, t10, t11, t12, t17, t70, t205, g2, g3, g6, g8, w1, w5, w10, w9, w22, w19, d1, d2, e4, e71 F 57 14.7 215,766.93 t2, t3, t7, t10, t11, t12, t14, t70, t205, g2, g8, w8, w9, w22, d1, d2, e1, e4, e5, e6, e73, e84, i6, 13.9 (3) F 24 13.4 51,728.19 t3, t7, w1, w6, w22, d2, 24.4 (4) 186 F 55 13.0 33,333.33 t1, t2, t3, t7, t8, t10, t11, t12, t14, t15, t19, t70, t205, g8, g2, g3, g5, g6, w1, w5, w6, w8, w10, w9, w22, w19, d1, d2, d72, e1, m1, 3.92 (3) M 19 12.9 29,865.28 t2, t3, t7, w8, w22, d1, d2 14.4 (3) M 35 11.1 4,133.79 t1, t2, t3, t7, t8, t15, t19, w1, w5, 3.30 (2) M 18 10.8 2,973.12 t3, t7, t19, w1, w6, w12, w22, d1, d2, g2, g5, g6, e1, e5, >100 (6) 540 M 35 12.4 17,242.73 t2, t3, t7, t10, t70, t15, g8, g5, w22, m1 13.4 (3) F 25 10.4 1,915.86 t2, t3, t7, t10, t17, g8, w22, d1, d2, e1, e3, e5, i6, 8.19 (3) M 24 10.3 1,716.53 d1, d2, t1, t2, t3, t7, t9, t10, t12, t14, t15, t19, t70, g2, g3, g5, g6, g8, w1, w9, w12, e1 30.2 (4) M 35 12.5 19,245.01 d1, d2, t1, t2, t3, t7, t8, t10, t15, t70, w1, w6, e1, i6, i206 M 24 13.5 57,735.03 t7 Mean 30.8 12.64 83,238.70 BAU/mL 22,406.86

*t1, quadrupedal maple (box elder); t2, gray alder; t3, birch (common silver birch); t7, oak; t8, olive; t9, elm (elm); t10, walnut; t11, European maple (maple leaf sycamore); t12, willow; t14, poplar; t15, Japanese cedar; t17, American ash (white ash); t19, acacia; t70, mulberry; t205, alder; t213, pine; g2, Bermuda grass; g3, cocksfoot; g5, rye-grass; g6, Timothy grass; g8, white bran (redtop); w1, ragweed; w5, wormwood; w6, mugwort; w9, plantain; w12, seaweed (goldenrod); w22, ginseng vine (Japanese hop); d1, Vertical dust mite ( Dermatophagoides pteronyssinus ); d2, Great-legged dust mite ( D. farinae ); d72, Tyrophagus putrescentiae ; e1, cat dander; e3, horse dander; e4, cow dander; e5, dog dander; e6, guinea pig epithelium; e71, mouse epithelium; e73, rat epithelium, e84, hamster epithelium; i6, German cockroach; i206, dysentery (American cockroach); m1, Penicillium chrysogenum .

Example 2. SDS-PAGE and IgE immunoblotting analysis

For analysis of the obtained protein, the protein was run on a 15% sodium dodecyl sulfate-polyacrylamide gel. The isolated proteins were stained with Coomassie blue or electroblotted on PVDF membranes. Membranes were blocked with 3% skim milk in PBS containing 0.05% Tween 20 (PBST) and then incubated overnight with pooled serum diluted 1:4. Then incubated with alkaline phosphate-conjugated monoclonal anti-human IgE (1,000 fold dilution) and nitro blue tetrazolium (NBT)-3-bromo-4-chloro-5-indolylphosphate (BCIP) (Promega , USA) was used as a substrate for visualization.

As a result, a clear protein band of about 15 kDa appeared on SDS-PAGE in both oak (S) and oak mix (O) (Fig. 1). In addition, IgE immunoblotting showed strong IgE reactivity to these components ( FIG. 2 ). In particular, the 23 kDa component exhibited significant IgE reactivity.

Example 3. Evaluation of Allergen Potency by Quantitative Intradermal Skin Test

To determine the potency (Bioequivalent Allergy Unit: BAU) of the extract (Turkeltaub et al., J Allergy Clin Immunol 1982;70:343-352 and Jeong et al., Yonsei Med J2016; 57 (2): 399-406) , An intradermal test using pollen extract was performed on 15 patients with very severe allergy to oak.

Specifically, 50 μL of serial 3-fold dilutions of the extract, 0.9% NaCl, 0.4% phenol, and 0.03% human serum albumin solution were injected onto the skin. After 15 min, the sum of the longest and midpoint orthogonal diameters (ΣE) of the erythema in millimeters was recorded. To analyze the titer of the extract, the best fit linear regression line was calculated for the mean of ΣE versus log3 dose in triplicate for each patient tested. The extraction concentration with the sum of 50 mm erythema of the 14th 3-fold dilution (ED ₅₀ ) was randomly assigned to 100,000 BAU/mL.

As a result, it was found that the sum of erythema of 50 mm by the skin intradermal test was the 13.05th dilution. Therefore, it was found that the allergic titer of oak pollen extract was 35,391.8 BAU/mL. Commercial oak mix inhibited oak extract by up to 96.4%, while self-inhibition reached 98.6%. Also, as shown in Figure 3, 1:20 w/v (20,000 PNU/mL) of oak mix corresponds to 28,508.1 BAU/mL (ie, 0.701 BAU/PNU) for 60.5 ng of oak, and 73.5 ng was found to be necessary for 50% inhibition of IgE reactivity.

Example 4. Preparation of monoclonal antibody against recombinant Que ac 1

The major isoform, recombinant Que ac 1.0101 (SEQ ID NO: 1), was produced in E. coli and purified using Ni-affinity column chromatography available from Qiagen. Mouse monoclonal antibodies were produced by fusion of splenocytes from BALB/c mice immunized with recombinant Que ac 1 and myeloma cells (Sp 2.0-Ag 14). Three immunizations were performed with an interval of 2 weeks using Freund's adjuvant. Hybridomas that produced antibodies to Que ac 1 were screened by ELISA and Western blot. Five selected hybridomas were expanded by limiting dilution and monoclonal antibodies were purified using protein G affinity chromatography (Sigma-Aldrich). The subtype of the purified antibody was determined with an isotyping reagent (Pierce, ThermoFisher scientific, USA).

As a result, as shown in FIG. 4 , a monoclonal antibody against Que ac 1 was produced by immunization with recombinant Que ac 1.0101, which is a major subtype of Que ac 1. As shown in FIG. 5 , native Que Five hybridoma-producing antibodies 3E6 (Accession No.: KCLRF-BP-00484), 8G7, 9E11, 10G10 and 11G5 (Accession No.: KCLRF-BP-00485) recognizing ac 1 were selected by ELISA and immunoblotting. . Among them, monoclonal antibodies 3E6 and 9E11 were in the form of IgG1, and 8G7, 10G10 and 11G5 were in the form of IgG2b.

Example 5. Identification of the optimal combination of antibodies for 2-site ELISA

We tried to identify the optimal combination of antibodies for 2-site ELISA using 5 different antibodies.

Specifically, the purified monoclonal antibody was biotinylated with EZ-link174 ^® sulfo-NHS-LC-biotin (ThermoFisher Scientific, USA). That is, 2 mg/mL of antibody was incubated with NHS-LC-biotin on ice for 4 hours, and extensive dialysis against PBS was performed to remove unreacted NHS-LC-biotin.

Capture antibody (11G5 at 10 μg/mL) was diluted with carbonate buffer (pH 9.6) and coated at 4° C. overnight. After blocking with 3% skim milk, serially diluted antigens (pollen extract, native Que ac 1 or recombinant Que ac 1) were added to the wells and incubated for 1 hour at room temperature. Detection antibody (biotinylated 3E6 diluted 1:1,000) was added and incubated for 1 hour at room temperature. Then, 1 mg/mL of streptavidin-conjugated peroxidase (Sigma-Aldrich) diluted 1:1,000 was added to the wells. The microtiter plates were washed at least 3 times with PBST between each step. Color was enhanced with TMB Microwell Peroxidase Substrate (SerCare Life Science). After adding 0.5 MH ₂ SO ₄ , the absorbance at 450 nm was measured. All samples were tested twice. The optimal combination of antibodies was confirmed by comparing titration curves of recombinant Que ac 1 using 5 different antibodies.

As a result, the combination of biotinylated 3E6 (1:1,000 dilution) as detection antibody and 11G5 (10 μg/mL) as capture antibody was found to be the optimal combination for 2-site ELISA. As shown in FIGS. 6 and 7 , it was found that the detection limits were approximately 0.1 ng/mL for recombinant Que ac 1 and 1 μg/mL for oak pollen extract.

Example 6. Recognition of Que ac 1 homologs by monoclonal antibodies

By testing cross-recognition of homologous allergens, we tried to confirm that the newly developed ELISA can quantify major allergens in oak species.

Immunoblotting was performed to confirm that the monoclonal antibodies could recognize homologous molecules from different oak and birch pollen extracts. Various pollen extracts were separated on 15% SDS-PAGE and stained with Coomassie blue or electroblotted on PVDF membranes. Membranes were then blocked with 3% skim milk in PBST and incubated with hybridoma culture supernatants (3E6 and 11G5) for 1 h. It was then incubated with alkali phosphate-conjugated goat anti-mouse IgG (F _ab -specific, Sigma-Aldrich) (diluted 1:1,000) and visualized using NBT and BCIP.

As a result, the monoclonal antibody could not detect Bet v 1 ( FIG. 8 ). In addition, it was not easy to identify Que m 1, which is a major allergen of Japanese oak, by immunoblotting. On the other hand, as shown in Figures 9 and 10, 3E6 was found to be more specific for Que ac 1 compared to 11G5.

Example 7. Two-dimensional gel electrophoresis and immunoblotting analysis of native Que ac 1

To confirm the detection of the Que ac 1 subtype by the monoclonal antibody, two-dimensional gel electrophoresis was performed. Anion exchange using DEAE, hydrophobic interaction using phenyl sepharose, and gel filtration using Sephadex G75 similar to that previously disclosed for native Que ac 1 (Korean Application No. 10-2019-0115485) was purified with Isoelectric focusing was performed on 11 IPG strips (ReadyStrip IPG strips, Protean IEF system; Bio-Rad) using an immobilized gradient of pH 4-7. Then, 15% SDS-polyacrylamide gel electrophoresis was performed. Proteins were stained with Coomassie Blue or electroblotted on PVDF membranes. Que ac 1 subtype recognized by the monoclonal antibody was detected by NBT and BCIP as described above.

As a result, as shown in Fig. 11, a number of subtypes were separated by 2D gel. As shown in Figures 12 and 13, 3E6 showed a more specific reactivity to a specific subtype compared to 11G5. More spots detected by 11G5 may indicate the presence of small amounts of various subtypes.

Example 7. Allergen analysis for various extracts

Quantification of the native Que ac 1 homologue allergen was performed using the developed 2-site ELISA. The results are shown in Table 2 below.

allergen 2-site ELISA
(μg/mL) correction constant
(Correction factor) actual concentration
(μg/mL) titer BAU/PR-10 Natural Que ac 1 0.543 1.839 1.00 - - Oak Tree Extract 0.924 1.839 1.70 35,391.8 BAU/mL 38,302.8 (20,828.1, corrected) Japanese oak extract 0.389 - - - - American Oak Extract 0.929 - - - - Oak Mix (red, Virginia live, and white oak) 0.634 - - 28,508.1 BAU/mL (1:20 w/v, 20,000 PNU/mL) 44,965.3 Birch Extract Below detection limit - - - -

As shown in Table 2, Que ac 1 was found to be 0.924 μg/mL in the oak extract when measured by 2-site ELISA. However, Que ac 1 2-site ELISA was found to be able to detect 54.3% of native Que ac 1. Therefore, it was found that the actual Que ac 1 concentration in the standardized pollen extract was 1.7 μg/mL. The above results indicate that the method according to one embodiment can effectively standardize antigens. The concentration of Que ac 1 homologue (PR-10 molecule) measured with the 2-site ELISA was found in Japanese oak, American oak and Estimated to be 389 ng/mL, 929 ng/mL and 634 ng/mL in the oak mix, respectively. 1 μg of Que ac 1 can be calculated as 20,818 BAU, and 1 mg of oak pollen extract showed 35,391 BAU/mL by ΣED50.

Claims (16)

An antibody or antigen-binding fragment thereof that specifically binds to an oak pollen allergen. The antibody or antigen-binding fragment thereof according to claim 1, wherein the oak pollen allergen is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is derived from a first hybridoma cell (Accession No.: KCLRF-BP-00485). The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is derived from a second hybridoma cell (Accession No.: KCLRF-BP-00484). A kit for detecting oak pollen allergens comprising the antibody according to claim 1 or an antigen-binding fragment thereof. The kit for detecting oak pollen allergens according to claim 5, wherein the oak pollen allergen is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. The method according to claim 6, wherein the antibody is an antibody derived from a first hybridoma cell (Accession No.: KCLRF-BP-00485) or an antibody derived from a second hybridoma cell (Accession No.: KCLRF-BP-00484) A detection kit for detecting oak pollen allergens. The method according to claim 7, wherein the antibody produced from the first hybridoma cell (Accession No.: KCLRF-BP-00485) is used as a capture antibody, and the second hybridoma cell (Accession No.: KCLRF-BP-00484) The antibody produced from the kit for detection of oak pollen allergen is used as a detection antibody. The kit for detecting an oak pollen allergen according to claim 8, wherein the detection kit uses a 2-site ELISA. contacting the test sample with the antibody or antigen-binding fragment thereof according to claim 1; and
A method for quantitative analysis of oak pollen allergens, comprising measuring the amount of an antibody against oak pollen allergen in the contacted sample. The method according to claim 11, wherein measuring the amount of the antibody Q dot (Quantum dot), enzyme, colloidal gold (Coloid gold), fluorescent material (Fluorescein), at least one selected from the group consisting of radioactive material and dye (Dye) A quantitative analysis method of oak pollen allergens using a marker. The method for quantitative analysis of oak pollen allergens according to claim 10, further comprising dissociating the oak pollen allergen in the sample before the contacting step. The method according to claim 10, wherein the quantitative analysis method is western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complement fixation assay, immunofluorescence, immunochromatography ( Immunochromatography), FACS analysis (fluorescenceactivated cell sorter analysis), and protein chip technology assay (protein chip technology assay) is any one selected from the group consisting of a quantitative analysis method of oak pollen allergen. The method for quantitative analysis of oak pollen allergens according to claim 13, wherein the ELISA is a 2-site ELISA. A hybridoma cell line producing an antibody that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 (Accession No.: KCLRF-BP-00484). A hybridoma cell line producing an antibody that specifically binds to a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1 (Accession No.: KCLRF-BP-00485).

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