CN118879641A - Monoclonal antibody pair, hybridoma cell and application of Der p1, the main allergen of house dust mite - Google Patents

Abstract

The invention relates to a pair of monoclonal antibodies of Der p1, a hybridoma cell and application thereof, in particular to a pair of monoclonal antibodies for detecting Der p1, a hybridoma cell strain secreting the pair of monoclonal antibodies and application thereof. The invention provides hybridoma cells for preparing a Der p1 monoclonal antibody of a main allergen of house dust mites, which have the preservation number of: CGMCC No.45843, cgmccno.45844. The invention prepares the anti-Der p1 monoclonal antibody pair by using the two hybridoma cells, can specifically identify and combine Der p1 protein, has high specificity and strong sensitivity, and has higher detection sensitivity when the obtained monoclonal antibody is used for detecting house dust mite allergen Der p 1.

Description

Monoclonal antibody pair, hybridoma cell and application of Der p1, the main allergen of house dust mite

Technical Field

The invention belongs to the technical field, and in particular relates to a pair of monoclonal antibodies to a main allergen Der p1 of a house dust mite, a hybridoma cell and an application thereof.

Background Art

House dust mite (Dermatophagoides pteronyssinus) is the main allergen that causes allergic diseases. Among them, the main allergen Der p 1, which exists in the intestinal contents and midgut tissues of mites, is a cysteine protease with a molecular weight of 25,000 and belongs to the papain family. At present, about 95% of the serum of patients allergic to house dust mites reacts with Der p 1.

It may induce allergic reactions by cutting off the IL-2 receptor on the surface of peripheral blood T cells through cysteine protease activity, inhibiting T cell proliferation and IFN-γ secretion, or by cutting off CD23 on the surface of B cells, eliminating the inhibitory signal of IgE synthesis and stimulating the production pathway of IgE.

At present, standardized house dust mite allergens are often used in clinical practice to specifically diagnose house dust mite allergies and perform desensitization treatment. As the main allergen of house dust mites, the content of Der p1 in desensitization drugs has become an important indicator for evaluating the effectiveness of drugs. Therefore, the preparation of a monoclonal antibody that can detect Der P1 antigen is of great significance for the quality control of allergen extracts and the standardization and mutual comparison of allergen products.

Several anti-Der p 1 monoclonal antibodies are currently on the market, but they all have varying degrees of cross-reactivity with the main allergen of dust mites, Der f 1, which affects the specificity of detection. Der p 1 and Der f 1 belong to the papain family of cysteine proteases, and their amino acid sequences have 82% sequence similarity. However, their amino acid residues are unevenly distributed on the surface of the protein spatial structure, and their allergen epitopes do not completely overlap.

The invention uses semi-solid culture technology to successfully prepare a specific anti-Der p 1 monoclonal antibody that does not cross-react with Der f 1.

Summary of the invention

The present invention aims to provide a hybridoma cell for preparing a monoclonal antibody against Der p 1, a major allergen of house dust mites. The antibody prepared by the cell line has good specificity and detection sensitivity for Der p 1, a major allergen of house dust mites, and can be used to establish an immunological detection method for Der p 1, a major allergen of house dust mites.

Another object of the present invention is to provide an antibody pair for detecting the content of Der p 1, a major allergen of house dust mites, and the use of the antibody pair in the quantitative detection of allergen products.

To achieve the above object, the present invention adopts the following technical solutions:

The hybridoma cells used to prepare monoclonal antibodies against Der p 1, the major allergen of house dust mites, were deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on April 17, 2024, and their deposit numbers are: CGMCC No.45843 and CGMCC No.45844.

The invention utilizes purified natural Der p 1 protein to immunize BALB/c mice, obtains spleen cells of the immunized mice and fuses them with SP2/0 myeloma cells, screens hybridoma cells on a methylcellulose semisolid culture medium, and uses an Elisa method to screen positive hybridoma cells to obtain a hybridoma cell line that specifically secretes anti-Der p 1 protein monoclonal antibodies.

The antibody pair used for detecting the content of Der p 1, a major allergen of house dust mites, is prepared by intraperitoneally injecting the hybridoma cells obtained above into parous BALB/c female mice, collecting ascites, purifying monoclonal antibodies, and obtaining anti-Der p 1 monoclonal antibodies DP1.10 and DF1.7.

Use of the antibody pair for detecting the content of the major allergen Der p 1 of house dust mites as described above in the preparation of a preparation for detecting the major Der p 1 antigen of house dust mites.

A detection kit for detecting the main Der p1 antigen of house dust mites comprises a monoclonal antibody prepared by mouse hybridoma cells with a deposit number of CGMCC No. 45843 as a capture antibody, and a monoclonal antibody labeled with HRP prepared by mouse hybridoma cells with a deposit number of CGMCC No. 45844 as a detection antibody.

A method for detecting the main allergen Der p 1 of house dust mites comprises the following steps:

S1. The antibody prepared by hybridoma cell CGMCC No.45844 is used as capture antibody to coat the ELISA coated plate;

S2, after washing, block with blocking solution and then wash;

S3, adding the test solution to the ELISA coated plate, incubating, and washing;

S4, adding HRP-conjugated antibodies prepared by hybridoma cell CGMCC No.45845, incubating, and washing;

S5. After adding TMB, the reaction was terminated by adding stop solution and the absorbance at a wavelength of 450 nm was detected by an ELISA reader.

In the above method, preferably, standard samples with different Der p 1 protein concentrations are prepared at the same time. After detection according to the above method, a dose-response standard curve is drawn, and then the absorbance value of the test solution is substituted into the standard curve to obtain the concentration of Der p 1 protein in the test solution.

The beneficial effects of the present invention are:

The hybridoma cell for preparing monoclonal antibody against Der p 1, a major allergen of house dust mites, provided by the present invention can prepare monoclonal antibody against Der p 1, which can specifically recognize and bind to Der p 1 protein, has high specificity and strong sensitivity. Under the same conditions, the monoclonal antibody against Der p 1 provided by the present invention can be used in ELISA experimental technology to detect Der p 1, a major allergen of house dust mites, with high sensitivity and rapidity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of a dose-response standard curve.

FIG2 shows the absorbance values of specific reaction detection.

DETAILED DESCRIPTION

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the present invention all belong to the scope of the present invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are of analytical grade or above.

Example 1 Immunization of BLAB/c mice

(I) Preparation of Antigen

Take 40g of house dust mite raw material (purchased from Beijing Xinhualian Xiehe Pharmaceutical Co., Ltd.), add 800ml NH ₄ HCO ₃ , and extract overnight at 4°C. Centrifuge twice at 12,000g for 15min, take the supernatant, filter with filter paper, 0.22μm, about 600ml after filtration, slowly add 234g (NH ₄ ) ₂ SO ₄ , and let stand overnight at 4°C. Centrifuge twice at 12,000g for 15min, keep the supernatant, and dissolve the precipitate with 0.05M Tris-HCl, pH8.0 (about 37ml). Dialyze with 0.05M Tris-HCl, pH8.0 overnight. After dialysis, filter through 0.22μm PVDF membrane, use DEAE DEAE-cellulose anion exchange column: column balance (0.05M Tris-HCl, PH 8.0), elution with 500ml 0.05M Tris-HCL solution containing 0.3M NaCl, PH 7.4, under this condition, acidic protein Der p 1 (PI<PH, negatively charged) is eluted in the elution stage. Ultrafiltration concentration to 40ml, and dialyzed into 0.05M sodium borate, containing 2M sodium chloride, pH 8.0 buffer. Continue to purify using Phenyl column (0.05M sodium borate, containing 2M sodium chloride, pH 8.0 balance, then eluted with 0.05M sodium borate solution containing 50% ethylene glycol, pH 8.0). Collect the eluate, ultrafiltration concentration to 20ml, and then purify using Superdex 75 column, collect the target peak, and ultrafiltration concentration to 5ml. The purified Der p 1 protein was dialyzed into 0.05 M Tris-acetate buffer (containing 0.5 M sodium chloride, pH 7.5), and then purified using a Zinc column (0.05 M Tris-acetate buffer, pH 7.5 equilibrium, 0.05 M Tris-acetate buffer, pH 7.5-5.0 gradient elution, collecting the peak eluted in the pH 5.7-5.2 interval). The purified Der p 1 protein was finally obtained and dialyzed into PBS for storage.

(II) Immunization of mice

Five healthy female BALB/c mice aged 6 weeks (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were taken, and 30 μg of purified Der p 1 protein (per mouse) was fully mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points in the abdomen. After the first immunization, each immunization was separated by two weeks, and 30 μg of Der p 1 protein was fully mixed with an equal volume of Freund's incomplete adjuvant and then boosted. One week after the fifth immunization, blood was collected from the orbital vein, and serum was collected. The mouse serum titer was determined using the indirect ELISA method to evaluate the immune effect. If the serum titer was qualified, 35 μg of Der p 1 protein (per mouse) was injected once through the tail vein for booster immunization three days before fusion.

Example 2 Preparation of anti-Der p 1 monoclonal antibody

(I) Preparation of myeloma cells

One week before fusion, SP2/0 myeloma cells (purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences) were revived and expanded to ensure that the cells were in the logarithmic growth phase and in the best condition at the time of fusion; on the day of fusion, the cells were gently blown off with a pipette, collected in a 50 mL centrifuge tube, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in IMDM medium and counted for later use.

(II) Preparation of feeder cells

Prepare feeder cells on the day of fusion as follows:

1. Take a healthy female BALB/c mouse of 3 weeks old, remove the eyeball of the mouse and bleed it, and disinfect the body surface with 75% ethanol;

2. Place the mouse on the operating table with the abdomen facing upwards; cut the chest cavity below the sternum of the mouse, and the white transparent thymus can be seen;

3. Remove the thymus and soak it in IMDM medium;

4. Place the thymus in a 200-mesh sieve and grind it gently with a tissue grinder until there are no obvious lumps;

5. Rinse the sieve with 10 mL of IMDM medium and collect the rinse in a 15 mL centrifuge tube. Centrifuge at 300 g for 5 min and discard the supernatant.

6. Resuspend the thymocytes in 10 ml of fetal bovine serum.

(III) Preparation of spleen cells from immunized mice

Prepare mouse spleen cells on the day of fusion as follows:

1. Take one mouse immunized in Example 1, remove its eyeballs and bleed it, and use 75% ethanol to disinfect its body surface;

2. Place the mouse on the operating table with the left ventral side facing up; cut the skin in the middle of the left ventral side of the mouse, separate the skin and peritoneum, fully expose the abdominal wall, and the red long spleen can be seen;

3. Lift the peritoneum on the lower side of the spleen, cut it open and flip it up to expose the spleen. Lift the spleen with forceps, separate the connective tissue under the spleen with scissors, and take out the spleen; put it into a sterile EP tube containing 5 ml IMDM culture medium;

4. Place the spleen in a 200-mesh sieve and grind it gently with a tissue grinder until there are no obvious lumps;

5. Rinse the sieve with 10 mL of IMDM medium and collect the rinse in a 15 mL centrifuge tube. Centrifuge at 300 g for 5 min and discard the supernatant.

6. Resuspend mouse spleen cells in IMDM.

(IV) Cell fusion

1. Take 1×10 ⁸ immune mouse spleen cells and 1×10 ⁷ SP2/0 myeloma cells and add them to a 50 mL centrifuge tube, add IMDM to a final volume of 10 ml, and centrifuge at 300 g for 5 min;

2. Discard the supernatant and remove the remaining liquid as much as possible. Tap the bottom of the fusion tube with your palm to loosen and evenly distribute the precipitated cells.

3. Place the centrifuge tube in 37℃ sterile water, slowly add 1ml of 50% PEG 1500 fusion agent into the centrifuge tube, add it within about 1 minute, and let it stand for 1 minute;

4. Slowly add 2 ml of IMDM medium along the wall of the centrifuge tube for about 2 minutes.

5. Then slowly add 8 ml of IMDM medium within 2 minutes to terminate the effect of PEG 1500 fusion agent;

6. Centrifuge the fused cells at 300g for 5 minutes, discard the supernatant, mix the precipitate with thymocytes, add 2 ml of HAT selection medium, and add IMDM medium to make the final volume 15 ml.

7. Pour 25 ml of 2% methylcellulose IMDM semi-solid culture medium and mix thoroughly. Inoculate into a sterile plastic dish with a diameter of 35 mm, 2 ml/dish. Place the plastic dish in a humidified box and culture it in a 37°C, 5% CO ₂ incubator.

(V) Clonal transfer

On the sixth day after fusion, a healthy female BALB/c mouse aged 3 weeks was taken, and feeder cells were prepared according to (II) 1-5. An appropriate amount of DMEM medium containing 20% fetal bovine serum was added to the precipitate to make the cell density 10 ⁵ cells/mL. Under a dissecting microscope, the clones in the dish were transferred to a 96-well plate with a pipette for further culture.

(VI) Screening of hybridoma cells

Two days after clone transfer, positive clones were screened by indirect enzyme-linked immunosorbent assay.

The purified Der p 1 protein obtained in Example 1 was diluted to 25ug/mL with coating solution (0.05M Tris-HCl), and 100μL was coated in each well, and the cells were kept overnight at 4℃; the cells were blocked at room temperature for 2h; 100μL of cell culture supernatant was added to each well, and the cells were incubated at 37℃ for 30min; after washing, enzyme-labeled secondary antibody (1:5000) was added, 100μL/well, and the cells were incubated at 37℃ for 30min; after washing, substrate solution (TMB) was added, 100uL/well, and the cells were protected from light and developed for 10min; 50uL 2M H ₂ SO ₄ was added to each well to terminate the reaction and observe the results. The positive clones screened out were retested the next day, and the recombinant His-alt a 1 protein was used as an antigen to exclude clones targeting the His tag. Six positive clones were screened out, and the labeled numbers were DP1.1, DP1.6, DP1.10, DP1.11, DP1.77, and DF1.7, respectively. The positive clones screened were amplified and stored in liquid nitrogen.

(VII) Large-scale preparation of monoclonal antibodies

Ascites was prepared using the in vivo ascites induction method. The specific steps are as follows:

1. Select multiparous female BLAB/c mice and intraperitoneally inject 0.5 mL of sterilized liquid paraffin oil per mouse;

2. Resuscitate the above-mentioned 6 frozen positive clones of hybridoma cells and continue to culture. Select hybridoma cells in the logarithmic growth phase, gently blow them off, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend them with DMEM, count and set aside;

3. Inject 5×10 ⁵ -1×10 ⁶ hybridoma cells into each mouse intraperitoneally;

4. After 7-10 days, the abdomen of the mouse will be significantly enlarged, and ascites will be collected;

5. Centrifuge the mouse ascites at 3000 rpm for 10 min, collect the supernatant, and store it at -20°C for later use.

(VIII) Purification of Ascites

1. Dilute the ascites with 4 volumes of acetate buffer (60 mM pH 4.0, pH 4.5);

2. Add 25 μl of caprylic acid per ml of diluted sample, stirring for 30 min, and centrifuge at 10,000 g at 16°C for 30 min.

3. Filter the supernatant with filter paper to remove fine residues, add 10-fold concentrated PBS (9 samples plus 1 part PBS), and adjust the pH to 7.4 with 5M NaOH;

4. Cool the supernatant to 4°C, add 45% saturated ammonium sulfate to precipitate the supernatant (0.277 g/ml), and let stand at 4°C for 30 min;

5. Centrifuge at 10,000 g for 10 min at 4°C, take a small amount of PBS to resuspend the precipitate, dialyze against PBS at 4°C overnight to obtain 6 purified antibody solutions (the corresponding antibodies are denoted as antibodies DP1.1, DP1.6, DP1.10, DP1.11, DP1.77, and DF1.7).

Example 3 Identification and pairing of anti-Der p 1 monoclonal antibodies

(I) Pairing test

Take 5 mg of horseradish peroxidase (HRP), dissolve it in 0.5 mL of 0.2 M sodium acetate buffer (pH 5.6), mix it with 0.25 mL of 0.1 M sodium periodate solution, and place it at 4 ° C for 30 min. Add 0.5 mL of 2.5% ethylene glycol solution, mix it and place it in a 4 ° C refrigerator for 30 min. Take the purified antibody obtained in Example 2 and prepare it into 5 mg/mL 1 mL, adjust the pH to 9.0 with 1 M sodium carbonate buffer (pH 9.5). Mix the purified antibody solution and HRP solution, adjust the pH to 9.0 with 1 M sodium carbonate buffer (pH 9.5), and place it in a 4 ° C refrigerator overnight. Add 0.1 mL of 0.1 M sodium borohydride solution, place it at 4 ° C for 2 hours to terminate the reaction, and obtain HRP-bound antibody. Add the HRP-bound antibody to a dialysis bag, place it in 0.01 M PBS solution (pH 7.4), and dialyze it at 4 ° C overnight. Collect HRP-conjugated antibodies, add 0.5% BSA and store in a 4°C refrigerator. Use the double antibody sandwich method to pair antibodies, unlabeled purified antibodies as capture antibodies, HRP-conjugated antibodies as binding antibodies, and screen a series of paired antibody pairs. The specific experimental operation is as follows:

1. Take capture antibodies (DP1.1, DP1.6, DP1.10, DP1.11, DP1.77, DF1.7) and add coating buffer (0.05M Tris-HCl) to a final concentration of 3.5μg/mL. Add 100uL to each well and incubate at 4℃ overnight.

2. Discard the solution and add 300 μL TBST to each well and wash three times.

3. Add 200 μL of blocking solution (0.5% BSA by mass) to each well and leave at room temperature for 2 hours.

4. Discard the blocking solution and make a Der p 1 capture coated plate after drying.

5. Take 100 ng/mL of Der p 1 antigen, 100 μL/well, and add it into the wells of the coated plate configured above;

6. Cover with sealing film and incubate at 37°C for 30 minutes;

7. Take out, wash the plate 3 times with washing solution (TBST), and pat dry;

8. Dilute the HRP-conjugated antibody prepared above with 1:1000 blocking solution, 100 μL/well, and add to the wells of the coated plate prepared above;

9. Cover with sealing film and incubate at 37°C for 30 minutes;

10. Take out, wash the plate 3 times with washing solution (TBST), and pat dry;

11. Add 100 μL TMB to each well and react for 10 minutes;

12. Add 50 μL of stop solution (2M H ₂ SO ₄ ) to each well to terminate the reaction;

13. Use an enzyme-labeled instrument to detect the absorbance value at a wavelength of 450 nm. The results are shown in Table 1.

Table 1 ELISA detection absorbance values

The results showed that the absorbance value of the antibody DF1.7-DP1.10 HRP group was the highest, indicating that the antibodies of these two strains had the strongest ability to specifically bind to the Der p 1 antigen. This pair of antibodies was selected for antibody type identification, titer determination and cross-reactivity determination.

The obtained hybridoma cells DP1.10 and DF1.7 were sent to the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) for patent preservation on April 17, 2024, and their preservation numbers are: CGMCC No.45843 and CGMCC No.45844, respectively, and they are classified and named as mouse hybridoma cells.

(II) Identification of antibody types

Agar was prepared into 100 mL of 2% agarose solution with barbiturate buffer at pH 6.0 and boiled in water. When the temperature of the molten agar was about 50°C, 0.01 g of sodium azide was added and 16 mL of agarose solution was transferred to a transparent pre-coated glass plate (10×8.5 cm) to make an agar plate. After the agar solidified, a central hole and six peripheral holes were punched in the agar, and the agar in the holes was removed with a needle. The antibodies p1.10 and f1.7 prepared by the present invention were placed in the central hole, and IgG1, IgG2a, and IgG2b (purchased from Southern Biotech) solutions were placed in the peripheral holes. The agar plate with the sample added was placed in a wet box and placed in a 37°C incubator for 24 hours to observe the results. The precipitation line formed between the central hole and the IgG1 hole indicated that the antibody DP1.10 and DF1.7 cell line subtype prepared by the present invention was IgG1.

(III) Determination of antibody titer

Coat the purified Der p 1 protein, 25 μg per plate, overnight at 4°C; block at room temperature for 2 hours; dilute the purified monoclonal antibody (8 mg/mL) with blocking solution (0.5% BSA) (starting from 1:500 dilution), 100 μL/well, and add blank (PBST) or negative control solution, incubate at 37°C for 30 minutes; after washing, add goat anti-mouse enzyme-labeled secondary antibody (1:5000), 100 μL/well, incubate at 37°C for 30 minutes; after washing, add substrate solution (TMB), 100 μL/well, color development in the dark for 10 minutes; add 50 μL2MH ₂ SO ₄ to each well to terminate the reaction, and measure the OD450 value of each well with an enzyme-labeled instrument. If the OD value of the well to be tested is greater than 0.1 and greater than 2.1 times that of the negative control well, it is judged as positive, and the highest dilution multiple judged as positive is the antibody titer. The titer of the monoclonal antibodies DP1.10 and DF1.7 prepared by the present invention is 1:10 ⁷ .

It is shown that the mouse hybridoma cells DP1.10 and DF1.7 obtained in the present invention can be used to prepare monoclonal antibodies against major allergens of house dust mites, and can be used to prepare reagents for detecting Der p 1 protein or house dust mites.

Example 4 Application of Antibody Pairs in ELISA Kits

According to the existing chemiluminescence diagnostic reagent production process, the DF1.7 antibody obtained in Example 2 was used as a capture antibody to be coated and configured into a Der p 1 coated plate, the antibody labeled HRP obtained in Example 3, namely DP1.10-HRP, was used as a binding antibody to be configured into an enzyme conjugate of the Der p 1 antibody, and the Der p 1 antigen was configured into a series of calibrators (1 ng/ml, 2 ng/ml, 4 ng/ml, 8 ng/ml, 16 ng/ml, 32 ng/ml, 64 ng/ml) to form a complete kit, and a series of verifications were performed on it.

(I) Performance verification indicators

Refer to the technical requirements of existing Der p 1 detection kits on the market and formulate performance verification indicators for detection reagents.

1. Linearity of dose-response curve

In the linear range of 1ng/ml-64ng/ml, the linear correlation coefficient (r) of the dose-response curve should be no less than 0.9900.

2. Detection limit

Should not be higher than 1ng/ml.

3. Limit of Quantitation

Should not be higher than 2ng/ml.

4. Specificity

The established method was used to detect the concentrations of gradient diluted antigens Der p 1, Der p 2, Der f 1, and Der f 2 to verify the specificity of the method.

5. Accuracy

The recovery rate of spiked samples was tested with a test kit and the result should be within the range of 85%-115%.

6. Precision

The precision (CV) should not be greater than 15%.

(II) Experimental configuration

1. Der p 1 capture antibody coated plate configuration

Use antibody DF 1.7 as capture antibody, add coating buffer (0.05M Tris-HCl), and the final concentration of capture antibody coating is 3.8μg/mL. Add 100μl to each well and incubate at 4℃ overnight. Take out and wash 3 times with 300μL TBST per well. Add 200μl blocking solution (0.5% BSA) to each well and leave at room temperature for 2h. Discard the blocking solution and make Der p 1 capture antibody coated plate after drying.

2. Der p 1 enzyme conjugate configuration

The DP1.10-HRP conjugated antibody obtained in Example 3 was added to the enzyme diluent (0.1 M Tris + 0.5% BSA) at a ratio of 1:2000.

3. Calibration product configuration

The Der p 1 antigen was diluted with an antigen diluent (0.5% BSA-PBS) into seven gradients (1 ng/ml, 2 ng/ml, 4 ng/ml, 8 ng/ml, 16 ng/ml, 32 ng/ml, 64 ng/ml).

4. Recovery rate standard solution configuration

The Der p 1 antigen was diluted with antigen diluent (0.5% BSA-PBS) as the standard solution, and 1.38 ng/mL dust mite extract (purchased from Beijing Union New Hualian Pharmaceutical Co., Ltd.) was selected as the low value sample.

5. Quality control product configuration

Der p 1 antigen was diluted with antigen diluent (0.5% BSA-PBS) to obtain QC1 (2 ng/ml±10%), QC2 (7 ng/ml±10%), and QC3 (20 ng/ml±10%).

(III) Sample addition operation

1. Take 50 μl of the above-prepared calibrator, recovery standard solution and quality control product respectively and add them into the wells of the above-prepared coated plate, then take 50 μl of the above-prepared DP1.10-HRP conjugated antibody and add it into the wells of the above-prepared coated plate, and shake on an oscillator for 30 seconds to mix them thoroughly;

2. Cover with sealing film and incubate at 37℃ for 30 minutes;

3. Take out and wash the plate 3 times with washing solution (TBST);

4. Add 100 μl TMB to each well and react for 5 minutes;

5. Add 50 μl of stop solution (2M H ₂ SO ₄ ) to each well to terminate the reaction;

6. Use an enzyme-labeled instrument to detect the absorbance value at a wavelength of 450 nm.

(IV) Performance Index Assessment

1. Linearity of dose-response curve

The calibrators of the duplicate assay kit (concentrations were 1 ng/ml, 2 ng/ml, 4 ng/ml, 8 ng/ml, 16 ng/ml, 32 ng/ml, and 64 ng/ml, respectively) were measured three times in parallel, and the dose-response curve of the kit was fitted using the four-parameter method. The dose-response curve is shown in Figure 1.

As shown in Figure 1, the linear correlation coefficient ^R2 of the Der p 1 antigen curve in the concentration range of 1-64ng/ml is greater than 0.9950, which meets the indicators.

2. Detection limit

Using sample diluent (0.5% BSA-PBS) as the sample, repeat the measurement 24 times, obtain the OD450 value of the 24 measurement results, calculate the mean (M) and standard deviation (SD), obtain the A450 value corresponding to M+2SD, substitute it into the standard curve equation of the standard substance, and find the corresponding concentration value, which is the detection limit. The results are shown in Table 2, and the detection limit is 0.52ng/mL.

Table 2

3. Limit of Quantitation

After the standard was diluted to 0.5, 1, and 1.5 ng/ml, the determination was repeated 10 times, and the average and SD of the 10 determination results were calculated to obtain the coefficient of variation (CV). The lowest concentration with CV ≤ 15% was selected as the limit of quantification. The results are shown in Table 3, and the limit of quantification was 1 ng/mL.

Table 3

Theoretical concentration (ng/mL) Detection concentration (ng/mL) SD CV 0.5 0.8 0.246 30.71% 1 1.12 0.144 12.89% 1.5 1.52 0.131 8.60%

4. Specificity

The established method was used to detect a variety of dust mite allergens. Except for Der p 1, the OD 450 values of different concentrations of Der p 2, Der f 1, and Der f 2 (purchased from Indoor Biotechnologies) were all lower than the blank, indicating that the double antibody sandwich Elisa kit prepared by the present invention can specifically detect Der p 1, and has no cross reaction to Der p 2, Der f 1, and Der f 2. The results are shown in Figure 2.

5. Accuracy

The Der p 1 antigen prepared in Example 1 was prepared into a standard solution (100, 10 ng/mL) and added to a low-value sample of known concentration at a volume ratio of 1:9. The test was repeated 3 times using the kit, and the recovery rate R was calculated according to the formula below. The results are shown in Table 4.

Where:

R—Recovery rate

V—Volume of standard solution added

V ₀ — low value sample volume

C—Measured concentration of sample after spike

C ₀ — low value sample concentration

_CS —Concentration of standard solution

Table 4

6. Precision

Ten wells of each of the quality control products QC1, QC2 and QC3 were measured in parallel, and the CV was calculated. The results are shown in Table 5.

Table 5

M (ng/mL) SD CV QC1 2.24 0.13 5.80% QC2 7.75 0.23 2.97% QC3 20.07 1.79 8.92%

The results indicate that the antibodies prepared by the hybridoma cells DP1.10 and DF1.7 for preparing monoclonal antibodies against Der p 1, a major allergen of house dust mites, can obtain monoclonal antibodies against Der p 1 with good specificity. The antibodies have strong specificity and can be used as coated antibodies in ELISA to detect Der p 1 antigen.

Comparative Example

The Der p 1 detection kit (Indoor Biotechnologies) available on the market was used to detect Der p 1 by double antibody sandwich method, and the detection limit obtained was 0.78 ng/mL, and the total incubation time of the sample and the detection antibody was 2 hours.

This indicates that the reagent for detecting Der p 1 provided by the present invention has a high sensitivity.

Claims (6)

1. A hybridoma cell for the preparation of a monoclonal antibody to Der p 1, a major allergen of the house dust mite, characterized in that the hybridoma cell has a deposit number of: CGMCC No.45843 and cgmccno.45844.

2. The antibody pair for detecting the content of main allergen Der p1 of house dust mites is characterized in that hybridoma cells CGMCC No.45843 and CGMCC No.45844 are respectively injected into BALB/c female mice intraperitoneally, ascites are collected, monoclonal antibody purification is carried out, and monoclonal antibodies DP1.10 and DF1.7 of the Der p1 are obtained.

3. The use of an antibody for detecting the allergen Der p1 content of house dust mites according to claim 2 for the preparation of a preparation for detecting the main Der p1 antigen of house dust mites.

4. A detection kit for detecting Der p1, a main allergen of house dust mite, comprises a monoclonal antibody prepared from a mouse hybridoma cell with a preservation number of CGMCC No.45843 as a capture antibody and a monoclonal antibody labeled HRP prepared from a mouse hybridoma cell with a preservation number of CGMCC No.45844 as a detection antibody.

5. A method for detecting the main allergen Der p1 of house dust mites, characterized in that it comprises the following steps:

S1, taking an antibody prepared from hybridoma cell CGMCC No.45844 as a capture antibody coating ELISA coating plate;

s2, after washing, sealing by using sealing liquid, and washing;

S3, adding the liquid to be detected into an ELISA coating plate, incubating and washing;

s4, adding an HRP-bound antibody prepared from hybridoma cell CGMCC No.45845, incubating and washing;

S5, adding TMB for reaction, adding a stop solution for stopping, and detecting the absorbance value at the wavelength of 450nm by using an enzyme-labeled instrument.

6. The method of claim 5, wherein the Der p 1 protein concentration in the test solution is obtained by preparing standards of different Der p 1 protein concentrations, measuring the test solution according to the method, drawing a dose-response standard curve, and substituting the absorbance value of the test solution into the standard curve.