Anti-KL-6 bispecific double antibody detection kit and application thereof

Abstract

The invention discloses an anti-KL-6 bispecific diantibody detection kit, which is characterized in that the anti-KL-6 bispecific diantibody detection kit comprises an anti-KL-6 bispecific diantibody, and the anti-KL-6 bispecific diantibody is a diantibody of an anti-KL-6 specific PTS domain heavy chain variable region and an anti-KL-6 specific SEA domain heavy chain variable region. The invention also discloses application of the kit. The anti-KL-6 specific double antibody disclosed by the invention can simultaneously and specifically recognize and combine a PTS domain and an SEA domain on KL-6. The ELISA kit can be used for quantitative detection of the KL-6 protein. Compared with the existing detection technology, the ELISA can more sensitively detect the KL-6 protein in the serum of the patient, and is simple and convenient to operate, low in cost and easy to popularize and apply, so that the clinical treatment effect, clinical typing or outcome, especially the clinical typing of the connective tissue disease patient with interstitial pulmonary fibrosis, can be predicted.

Description

Technical Field

[0001] The present invention relates to the field of antibody kit manufacturing, and in particular to an anti-KL-6 bispecific double antibody detection kit and uses thereof. Background Art

[0002] The clinical significance of sialolyzed sugar chain antigen, also known as Krebs Von den Lungen-6 (KL-6), is mainly reflected in the diagnosis and evaluation of pulmonary fibrosis. As a sensitive biological marker, KL-6 can reflect the degree of damage to alveolar epithelial cells. During the process of pulmonary fibrosis, due to factors such as inflammation and oxidative stress, alveolar epithelial cells will be damaged, leading to increased KL-6 levels. Therefore, increased KL-6 levels can be used as an indirect indicator of alveolar damage. KL-6 can sensitively reflect the degree of damage to the alveolar epithelium and interstitium, as well as the regeneration of type II alveolar cells. This is of great significance for evaluating lung conditions and formulating treatment plans.

[0003] In addition, the increase in KL-6 levels is closely related to the activity of pulmonary fibrosis. During the active stage of pulmonary fibrosis, due to factors such as inflammation and immune response, the lung tissue will undergo continuous damage and repair process, resulting in a continuous increase in KL-6 levels. Changes in KL-6 levels can predict the disease progression and prognosis of pulmonary fibrosis. During the treatment of pulmonary fibrosis, monitoring changes in KL-6 levels can also evaluate the treatment effect. If the KL-6 level gradually decreases as the treatment progresses, it usually indicates that the treatment is effective; conversely, if the KL-6 level continues to rise, it may mean that the treatment is not effective or the condition is worsening.

[0004] Currently, there are few detection methods for KL-6 protein, and the cost is high. The present invention finds out the amino acid sequence of the anti-KL-6 specific PTS domain heavy chain variable region / SEA domain heavy chain variable region double antibody, prepares the anti-KL-6 bispecific double antibody, and at the same time, establishes an ELISA kit for KL-6 protein detection for quantitative detection of KL-6 protein. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the object of the present invention is to provide an anti-KL-6 bispecific dual antibody detection kit and its use.

[0006] The quantitative detection of KL-6 protein is achieved by manufacturing an ELISA kit for KL-6 protein detection.

[0007] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0008] An anti-KL-6 bispecific double antibody detection kit, comprising:

[0009] An anti-KL-6 bispecific diabody, wherein the anti-KL-6 bispecific diabody is a diabody of an anti-KL-6 specific PTS domain heavy chain variable region and a SEA domain heavy chain variable region, and the antibody amino acid sequence of the anti-KL-6 specific PTS domain heavy chain variable region is:

[0010]

[0011] The antibody amino acid sequence of the SEA domain heavy chain variable region is:

[0012]

[0013]

[0014] In a preferred embodiment of the present invention, the anti-KL-6 bispecific double antibody detection kit further comprises a 96-well ELISA plate and an enzyme-labeled secondary antibody, wherein the enzyme-labeled secondary antibody is an HRP antibody, a SEA antibody or a VH antibody.

[0015] A use of an anti-KL-6 bispecific dual antibody detection kit, the use being to evaluate the KL-6 protein level in an individual's serum sample.

[0016] In a preferred embodiment of the present invention, the evaluation is based on the detection of the KL-6 protein content in the serum sample of the test individual.

[0017] The beneficial effects of the present invention are:

[0018] The anti-KL-6 specific double antibody of the present invention can simultaneously specifically recognize and bind to the PTS domain and the SEA domain on KL-6. The ELISA kit can be used for the quantitative detection of KL-6 protein. Compared with the existing detection technology, ELISA can more sensitively detect the KL-6 protein in the patient's serum, is simple to operate, low in cost, and easy to promote and apply, so as to predict the clinical treatment effect, clinical typing or prognosis, especially the clinical typing of connective tissue disease patients with interstitial pulmonary fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the structural model of the anti-KL-6 specific double antibody of the present invention that specifically binds to the PTS domain and SEA domain of the KL-6 protein at the same time.

[0020] Figure 2 Schematic diagram of KL-6 expression in patients with connective tissue disease and interstitial pulmonary fibrosis.

[0021] Figure 3 Schematic diagram of the diagnostic efficacy of KL-6 in patients with connective tissue disease and interstitial pulmonary fibrosis. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following structure, the description of the known structure and technology is omitted to avoid unnecessary confusion of the concept of the present invention.

[0023] The present invention is specifically described below in conjunction with embodiments:

[0024] First, the acquisition of anti-KL-6 specific double antibody:

[0025] 1. Immunized animals are obtained:

[0026] 0.5 ml of 2 mg / ml human KL-6 antigen (Abcam) was mixed with an equal volume of Freund's complete adjuvant and injected subcutaneously into 10 6-8 week old BALB / C mice at multiple points in the abdomen. After the above one week, the above steps were repeated every week. Three days after the fourth injection, 15 μl of blood was collected from the eyeball, left to stand at room temperature for 2 hours, centrifuged at 25°C and 1 2000 rpm for 15 minutes, and the supernatant was taken and the titer was tested by ELISA. The plasma was diluted with PBS at 1:2, 1:4, 1:8 and 1:10, and 100 μl / well was added to the plate coated with antigen (5 μg / ml), and each sample was made in parallel. PBS was used as a negative control well, covered and incubated at 37°C for 2 hours. Washed 3 times with PBST washing solution. Rabbit anti-mouse HRP-IgG (Abcam, 1:10000 dilution) was added, and 100 μl / well was incubated at 37°C for 2 hours. Washed 3 times with PBST washing solution. After adding 100 μl / well TMB solution for color development for 15 minutes, 50 μl / well dilute sulfuric acid solution was used to terminate the reaction. The titer was considered acceptable if a positive reaction was observed at a dilution of 1:10.

[0027] If titer testing is available, whole blood is collected from the eye, allowed to stand at room temperature for 2 hours, centrifuged at 25°C and 12,000 pm for 10 minutes, the supernatant is collected, and stored at -80°C.

[0028] 2. Hybridoma preparation: The mice were killed by dislocation and disinfected with 75% alcohol. The spleen cells of the mice were aseptically obtained and prepared into a cell suspension. 1 × 108 spleen cells were fused with 4 × 107 myeloma (SP2 / 0) cells using 50% PEG as a fusion agent. After 7 days of HAT selection culture, the cells were switched to HT culture and cultured at 37°C, 5% CO2 to obtain monoclonal hybridoma cells.

[0030] 3. Positive clone screening:

[0031] Using the limiting dilution method, the hybridoma cells are diluted multiple times and inoculated into multi-well cell culture plates so that there is no more than one cell in each well, and then cultured to allow them to proliferate.

[0032] Then, the specificity of the antibodies secreted by the cells in the supernatant of each well is detected. The culture wells whose supernatants can bind to specific antigens are positive wells. The preferred clones that can secrete antibodies that can specifically bind to PTS domain and SEA domain antigens in the monoclonal hybridoma cells are screened by ELISA reaction.

[0033] 4. Design of anti-KL-6 specific diabody:

[0034] The structural model of the anti-KL-6 specific double antibody that can specifically bind to the PTS domain and SEA domain of the KL-6 protein at the same time is shown in the attached figure. Figure 1 shown.

[0035] An anti-KL-6 specific diabody consisting of a heavy chain variable region of an anti-PTS specific antibody (anti-PTS VH) and a heavy chain of an anti-SEA specific antibody (anti-SEAVH).

[0036] Among them, the anti-KL-6 bispecific diabody, the anti-KL-6 bispecific diabody is a diabody of the anti-KL-6 specific PTS domain heavy chain variable region and the SEA domain heavy chain variable region, and the antibody amino acid sequence of the anti-KL-6 specific PTS domain heavy chain variable region is:

[0037]

[0038] The antibody amino acid sequence of the SEA domain heavy chain variable region is:

[0039]

[0040] 5. Construction of vector for anti-KL-6 specific double antibody

[0041] The complete heavy chain (anti-PTS VH - anti-SEA VH) coding gene was connected to pEE6.4 through the Hind III / EcoRI cloning site to obtain pEE6.4-PTS VH antibody and pEE6.4-PTS SEA VH antibody, respectively; finally, the pEE6.4-PTS VH antibody and pEE6.4-SEA VH antibody were double-digested with BamH I / Not I to obtain the vector expressing the anti-KL-6 specific double antibody.

[0042] 6. Preparation of anti-KL-6 specific diabody

[0043] The density of HEK293F host cells was adjusted to 2X 106 / ml, and the shake flask was placed in a 5% CO2 constant temperature shaker. The experiment was started after constant temperature shaking and culture at 37 degrees Celsius and 120rpm for 15 minutes. Prepare two 50ml sterile centrifuge tubes, add 10ml KPM and 200μg anti-KL-6 specific double antibody vector to one of them, and gently blow and mix; take another centrifuge tube, add 10ml KPM and 1ml TA-293 transfection reagent, and gently blow and mix; transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and gently blow and mix; let it stand at room temperature for 15 minutes to prepare the plasmid-vector complex; remove the cells from the constant temperature shaker, add the prepared plasmid-vector complex while shaking, and return to the CO2 constant temperature shaker for shaking culture. The product expression levels were measured on the 6th day.

[0044] 7. Antibody Purification

[0045] After centrifugation at 5000 rpm, the supernatant was collected and filtered through a 0.22 μm filter.

[0046] The antibody was purified by protein A affinity chromatography column, and after the concentration was determined, it was packaged and stored to obtain the anti-KL-6 specific double antibody.

[0047] Second, the development and detection method of an ELISA kit for KL-6 determination:

[0048] 1. The concentration of PTS VH antibody is 0.25ug / ml, 55ul is coated on a 96-well plate, 100ul is added to each reaction well, and incubated at 4 degrees overnight;

[0049] 2. Washing: After coating, discard the coating solution and wash with PBST, repeat 3-5 times;

[0050] 3. Blocking: 5% BSA, 400ul / well, incubate at room temperature for 2h and then wash, same as 3.2;

[0051] 4. Add standard and test serum, 100ul per well, and incubate at room temperature for 1 hour;

[0052] 5. Dilute standard (known positive serum):

[0053] Standard dilution: blank, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560; Blank: 100ul PBS, serum to be tested was diluted 1:100, and 100ul was added respectively;

[0054] 6. Washing, same as 3.2;

[0055] 7. Add secondary antibody at 1:2000 (HRP-SEA VH antibody), incubate at room temperature for 30 minutes, and wash as in 2.

[0056] 8. Add substrate solution and color for 15 minutes;

[0057] 9. Terminate the reaction and read the result with a 450 / 630 nm microplate reader.

[0058] Clinical relevance analysis:

[0059] Clinical data of the above test samples were collected, the correlation between KL-6 and clinical parameters and disease severity was analyzed, and the changes in KL-6 levels before and after treatment were evaluated. The predictive value of KL-6 for patients was analyzed based on the patients' clinical manifestations, imaging abnormalities, serological indicators, etc.

[0060] exist Figure 2 Among them, compared with the normal control group and patients with connective tissue disease without interstitial pulmonary fibrosis, the serum KL-6 level of patients with connective tissue disease combined with interstitial pulmonary fibrosis was significantly increased.

[0061] HRCT score and IL-18 score can reflect the degree of interstitial pulmonary fibrosis to a certain extent. Figure 3 It can be seen that KL-6 has the highest diagnostic efficacy for the degree of interstitial pulmonary fibrosis compared with HRCT score and IL-18 score (AUC=0.8894).

[0062] The basic principles and main features of the invention and the advantages of the invention are shown and described above.

[0063] Those skilled in the art should understand that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An anti-KL-6 bispecific double antibody detection kit, characterized in that: include: An anti-KL-6 bispecific diabody, wherein the anti-KL-6 bispecific diabody is a diabody of an anti-KL-6 specific PTS domain heavy chain variable region and a SEA domain heavy chain variable region, and the antibody amino acid sequence of the anti-KL-6 specific PTS domain heavy chain variable region is: The antibody amino acid sequence of the SEA domain heavy chain variable region is:

2. An anti-KL-6 bispecific double antibody detection kit according to claim 1, characterized in that: The anti-KL-6 bispecific double antibody detection kit also includes a 96-well ELISA plate and an enzyme-labeled secondary antibody, wherein the enzyme-labeled secondary antibody is an HRP antibody, a SEA antibody or a VH antibody.

3. Use of an anti-KL-6 bispecific double antibody detection kit according to claim 1, characterized in that: The use is to assess the level of KL-6 protein in a serum sample of an individual.

4. Use of an anti-KL-6 bispecific double antibody detection kit according to claim 1, characterized in that: The assessment is based on the detection of the KL-6 protein content in the serum sample of the test individual.

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