A method and application for improving the sensitivity and specificity of anti-CA724 antibodies

Abstract

The present invention belongs to the field of biotechnology and provides a method and application for improving the sensitivity and specificity of anti-CA724 antibodies. The method of improving the sensitivity and specificity of anti-CA724 antibodies of the present invention specifically comprises: enzymatically cleaving the anti-CA724 antibodies with sialylase to obtain desialylated anti-CA724 antibodies, thereby improving the sensitivity and specificity of the anti-CA724 antibodies. The present invention transforms conventional anti-CA724 antibodies through an enzymatic cleavage process, thereby improving the sensitivity and specificity of the anti-CA724 antibodies and effectively reducing detection costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method and application for improving the sensitivity and specificity of an anti-CA724 antibody. Background Art

[0002] The carbohydrate antigen CA724 (CA724) is a specific tumor marker and a laboratory marker for the detection of gastric cancer and various digestive tract cancers. Its normal serum concentration is <6 U / mL, but elevated levels are observed in various digestive tract tumors, ovarian cancer, and other diseases. It has high specificity for the detection of gastric cancer. The antigen has been identified as a mucinous glycoprotein of 220–400 kDa. Two monoclonal antibodies, CC49 and B72.3, recognize its glycan epitope, Galβ(1-3)sialyl-Tn, and sialyl-Tn antigen, respectively. During the expression of mammalian antibodies, host cell culture conditions often affect the expression, resulting in numerous glycosylation modifications in the resulting anti-CA724 antibodies. These modifications negatively impact the sensitivity and specificity of anti-CA724 antibodies. Currently, anti-CA724 antibodies exhibit limited sensitivity and specificity, leading to a need for more sensitive and specific anti-CA724 antibodies. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: how to improve the sensitivity and specificity of anti-CA724 antibodies.

[0004] To solve the above technical problems, the first aspect of the present invention provides a method for improving the sensitivity and specificity of anti-CA724 antibodies, the method comprising the following steps: using sialidase to cleave the anti-CA724 antibody to obtain a desialylated anti-CA724 antibody, thereby improving the sensitivity and specificity of the anti-CA724 antibody.

[0005] In antibody production, due to its complex biochemical structure and production process, anti-CA724 antibodies are prone to a large number of modifications or degradation during production and storage. Among them, sialic acid modification is one of the main glycosylation modifications that affects the specificity and sensitivity of anti-CA724 antibodies. It can cause mismatches between anti-CA724 antibodies and sialic acid modifications, thereby leading to weak aggregation between antibodies and reducing the sensitivity and specificity of anti-CA724 antibodies. The technical solution provided by the present invention, using sialidase (neuraminidase) to treat anti-CA724 antibodies can effectively reduce the level of sialic acid modification of anti-CA724 antibodies, thereby effectively reducing aggregation between anti-CA724 antibodies, thereby improving the specificity of anti-CA724 antibodies, and at the same time reducing background signal interference in reagent application, thereby improving the sensitivity of anti-CA724 antibody detection.

[0006] Preferably, the molar ratio of the sialidase to the anti-CA724 antibody is 1:(1-3).

[0007] The molar ratio of sialidase to anti-CA724 antibody provided by the present invention can ensure maximum enzymatic cleavage efficiency while avoiding side reactions or cost waste caused by excessive enzyme, thereby improving process economy.

[0008] Preferably, the reaction temperature for enzymatic cleavage of the anti-CA724 antibody using sialidase is 35-37.5°C.

[0009] The present invention adopts a reaction temperature range of 35-37.5°C to simulate the physiological environment, which can protect the structural integrity of the antibody while maintaining the optimal activity of sialylase and avoid the problems of high temperature denaturation or insufficient reaction rate at low temperature.

[0010] Preferably, the anti-CA724 antibody is a sialylated anti-CA724 antibody.

[0011] The present invention is directed to anti-CA724 antibodies and can directly solve the problem of performance defects of anti-CA724 antibodies caused by glycosylation modification caused by host cells during the production process of anti-CA724 antibodies, thereby effectively improving the sensitivity and specificity of anti-CA724 antibodies.

[0012] More preferably, the anti-CA724 antibody is derived from an anti-CA724 antibody expressed by cells cultured in vitro.

[0013] Preferably, the method further comprises the following steps: after the enzyme digestion is completed, separating and purifying the anti-CA724 antibodies after the reaction by column chromatography and / or dialysis.

[0014] Column chromatography and dialysis can effectively separate the enzyme, sialic acid fragments, and unreacted impurities in the enzymatic cleavage product. This step ensures the purity of the final antibody and avoids residual interference with subsequent detection.

[0015] More preferably, the anti-CA724 antibody after the reaction is separated and purified by any one or more methods selected from protein G, anion chromatography, metal ion chelation chromatography, chromatography, and dialysis.

[0016] Furthermore, the second aspect of the present invention provides a desialylated anti-CA724 antibody, which is prepared by the method for improving the sensitivity and specificity of the anti-CA724 antibody according to the first aspect.

[0017] Furthermore, the third aspect of the present invention provides an antibody conjugate, wherein the antibody conjugate comprises the desialylated anti-CA724 antibody according to the second aspect.

[0018] After coupling labels such as fluorescent dyes, enzymes, and radioactive isotopes to the enzymatically cleaved anti-CA724 antibody provided by the present invention, the antibody can still maintain optimized performance, effectively improve the signal intensity and signal-to-noise ratio of the detection, and is suitable for various detection platforms such as ELISA and immunohistochemistry.

[0019] Preferably, the antibody conjugate further comprises a chemical marker or a biological marker coupled to the anti-CA724 antibody.

[0020] Furthermore, the fourth aspect of the present invention provides a CA724 detection product, which comprises the desialylated anti-CA724 antibody according to the second aspect.

[0021] The CA724 detection product provided by the present invention can effectively achieve accurate quantitative or qualitative analysis of CA724, and in particular can effectively reduce the incidence of false positives or false negatives in various cancers such as gastrointestinal tumors and ovarian cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention transforms common anti-CA724 antibodies through an enzymatic cleavage process, improving their sensitivity and specificity, making their performance surpass that of existing imported products. It is a substitute for imported products and can effectively reduce detection costs.

[0024] 2. The method provided by the present invention for improving the sensitivity and specificity of anti-CA724 antibodies is simple to operate, requires mild reaction conditions, and can be seamlessly integrated with existing traditional antibody production lines. The modified antibodies are compatible with mainstream diagnostic platforms, accelerating industrial application.

[0025] 3. The anti-CA724 antibodies produced by the method provided by the present invention for improving the sensitivity and specificity of anti-CA724 antibodies can be used as high-precision CA724 detection products, providing a more reliable tool for tumor marker detection and promoting the development of personalized medicine.

[0026] 4. The method for improving the sensitivity and specificity of anti-CA724 antibodies provided by the present invention can be further extended to the preparation of other glycosylation-sensitive antibodies (such as CEA and CA919), and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The SDS-PAGE electrophoresis results in Example 1 of the present invention are shown below:

[0028] Figure 2 This is the particle size scanning result in Example 2 of the present invention;

[0029] Figure 3The HPLC test results in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0030] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only intended to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.

[0031] It should be noted that the endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0032] As described in the background art, existing commercial anti-CA724 antibodies have low sensitivity and specificity. Based on this, a specific embodiment of the present invention provides a method for improving the sensitivity and specificity of anti-CA724 antibodies. The method uses sialidase to enzymatically cleave the anti-CA724 antibodies, thereby improving the sensitivity and specificity of the anti-CA724 antibodies.

[0033] In some embodiments, the molar ratio of sialidase to anti-CA724 antibody is 1:(1-3).

[0034] In some embodiments, the reaction temperature for enzymatic cleavage of the anti-CA724 antibody using sialidase is 35-37.5°C.

[0035] In some embodiments, the anti-CA724 antibody is an anti-CA724 antibody with a sialylation modification.

[0036] In some embodiments, after the enzymatic cleavage is completed, the anti-CA724 antibody is separated and purified by column chromatography and / or dialysis.

[0037] More specifically, the desialylated anti-CA724 antibody provided by the present invention can be prepared by the following method:

[0038] S1: Batch culture the cell line stably expressing anti-CA724 antibody, collect the cell supernatant and perform column chromatography to obtain a crude antibody solution;

[0039] S2: The antibody solution and NRH enzyme mixture were reacted at 37°C overnight to obtain the enzyme digestion reaction solution;

[0040] S3: The enzyme digestion reaction solution in step S2 is subjected to column chromatography and dialysis to obtain desialylated anti-CA724 antibody.

[0041] The desialylated anti-CA724 antibody prepared by the method for improving the sensitivity and specificity of an anti-CA724 antibody provided by a specific embodiment of the present invention has the advantages of high sensitivity and high specificity. Compared with existing anti-CA724 antibodies, the binding interaction between the desialylated anti-CA724 antibodies is significantly reduced and aggregation is less likely to occur. The CA724 detection product prepared using the desialylated anti-CA724 antibody provided by the present invention can effectively reduce the incidence of false positives and false negatives in the test results, providing a new detection antibody for the detection of CA724.

[0042] The technical solutions of the present invention are further illustrated below by specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventional meanings are defined herein for the purpose of illustration or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters provided by the manufacturers.

[0043] In the following examples, the Roche CA724 coating antibody was derived from Roche REF09005692190, and the antigen calibrator was derived from Roche REF09175130190.

[0044] In the following examples, the Mecon MS-i3080 luminometer is from Mecon Biotechnology Co., Ltd.

[0045] Example 1

[0046] Preparation of desialylated anti-CA724 antibody

[0047] Resuscitate the cell line stably expressing anti-CA724 antibody and culture it in a shaker at 110 rpm / min, 37°C, and 5% CO2. When the cell density reaches (1.5-3.0)×10 6 cells / ml, cell passage was performed and the seeding density was (2.0~5.0)×10 5 cells / ml. After several passages, the cell viability reached above 95%, 8×10 5Cells were inoculated into shake flasks at a concentration of 10 cells / mL for batch culture. During the culture period, supplement the culture medium with supplementary feed to maintain a sugar content of 4 g / L. When cell viability dropped below 80%, the supernatant was collected by centrifugation.

[0048] The Protein G affinity medium was equilibrated with 10 column volumes of 10mM PBS (pH=7.4), after which the flow rate was reduced to allow the cell supernatant to pass through the affinity medium. After re-equilibration, the medium was eluted with 30mM citric acid solution (pH=2.7). The eluate was collected and dialyzed four times against 50mM sodium acetate solution (pH=6.0). The antibody was concentrated to 2mg / mL to obtain a crude anti-CA724 antibody solution.

[0049] The NRH enzyme solution and antibody solution were mixed in a 2:1 mass ratio and incubated overnight in a 37°C metal bath to allow the enzyme to fully activate the sialic acid chains of the anti-CA724 antibody and cleave them, forming a mixture of cleaved anti-CA724 antibody and NRH enzyme. The mixture was further purified using Protein G media, and the eluate was collected and dialyzed into 20 mM PB buffer (pH 8.0) four times to obtain the cleaved anti-CA724 antibody solution.

[0050] The above enzymatically cleaved anti-CA724 antibody solution was further purified using DEAE filler, gradient eluted with saline solution, and the eluate was collected and dialyzed four times in 20mM PBS buffer (pH=8.0). The antibody was concentrated to 2mg / mL to obtain a pure desialylated anti-CA724 antibody. The antibody purification effect was evaluated by SDS-PAGE electrophoresis. The specific results are as follows: Figure 1 As shown, clear anti-CA724 antibody bands can be seen in SDS-PAGE electrophoresis, where lane 1 is a pure desialylated anti-CA724 antibody band (reducing), with a heavy chain size of about 50KD and a light chain size of about 25KD; lane 2 is a pure desialylated anti-CA724 antibody band (non-reducing); lane 3 is a Maker band. Figure 1 It can be seen that the antibody prepared by the method provided by the present invention has no other protein bands.

[0051] Example 2

[0052] Antibody presence morphology detection

[0053] The crude anti-CA724 antibody solution and the desialylated anti-CA724 antibody in Example 1 were quantified to the same concentration (1 mg / mL). The quantitative cup of the ZATA potentiometer was repeatedly rinsed with pure water, and the buffer solution used to store the antibody was placed in the ZATA potentiometer for particle size scanning, and set to a blank background. Subsequently, 1 mL of crude anti-CA724 antibody and pure desialylated anti-CA724 antibody were each taken for detection. The specific results are shown in FIG. Figure 2 As shown, Figure 2 The figure shows the particle size scanning of two anti-CA724 antibodies in the ZATA potentiometer, among which: Figure 2 A in the figure is the particle size scanning result of the crude anti-CA724 antibody. Figure 2 B in the figure is the particle size scan result of the purified desialylated anti-CA724 antibody. Figure 2 As can be seen, while normal antibody monomer particle sizes are approximately 10 nm, the particle size scan of the crudely purified anti-CA724 antibody revealed peaks in the 10 nm, 100 nm, and 1000 nm ranges, indicating that the crudely purified anti-CA724 antibody exists in a variety of states: monomer, aggregate, and large aggregate. After NRH enzyme treatment and polishing, the antibody particle size scan revealed only a peak in the 10 nm range, indicating no aggregation of the antibody in the storage buffer. This demonstrates that the quality of the purified desialylated anti-CA724 antibody obtained in Example 1 is significantly superior to that of normal antibodies.

[0054] Example 3

[0055] Glycosylation detection

[0056] 100 μg of the purified desialylated anti-CA724 antibody from Example 1 was diluted to 1 μg / μL with purified water. 10 μg of PNGase F was added and digested overnight at 37°C. The digested antibody was concentrated to dryness in a vacuum centrifugal concentrator and stored at -20°C until ready for use.

[0057] Prepare the glycan staining solution according to the Ludger 2-AB kit instructions. Add 20 μL of the staining solution to the concentrated, dried glycans and mix thoroughly. Incubate at 65°C for 3 h. Add 500 μL of 100% acetonitrile solution and mix thoroughly. Centrifuge and remove the supernatant. Air-dry the precipitate at room temperature. Dissolve the precipitate in 15 μL of pure water, then add 35 μL of 100% acetonitrile solution. Store at -20°C until needed.

[0058] The samples were tested for glycosylation levels using HPLC. The specific results are as follows: Figure 3 The peak area distribution table is shown in Table 1, where the chromatographic conditions are as follows:

[0059] Chromatographic column: TSKgel Amide-80 4.6mm ID 25 cm, 5um; detection wavelength: 428nm; injection volume: 10uL; mobile phase A: 50mM ammonium formate solution (pH=4.4); mobile phase B: 100% acetonitrile solution, flow rate: 0.8mL / min; column temperature: 60℃ (automatic injection: 15℃) excitation wavelength: 250nm.

[0060] Note: In Table 1, all glycosylation names and the standards used for their determination are from Ludger Company, UK.

[0061] Table 1

[0062]

[0063] Depend on Figure 3 As shown in Table 1, F(6)A2 accounts for the majority of the glycosylation modifications (approximately 74%), which is consistent with the results reported in other literature. Compared with conventional antibodies, the purified desialylated anti-CA724 antibody prepared in Example 1 has a lower sialic acid content (approximately 0.56%) of the total sugar content, which is consistent with the purpose of the present invention.

[0064] Example 4

[0065] Product Performance Comparison

[0066] The crude anti-CA724 antibody solution in Example 1 was used as a control antibody. The control antibody and the purified desialylated anti-CA724 antibody provided in Example 1 were used as labeled antibodies, and paired with the Roche CA724 coated antibody to detect the effect of the purified desialylated anti-CA724 antibody of the present invention in reagent application. The antigen calibrator prepared in advance was taken out and restored to room temperature, and the experimental layout was marked. 200 μg of antibody was mixed with the acridinium ester solution in a ratio of 1:10 and mixed in the dark for 2 hours. Subsequently, a desalting column was used for desalting, and the centrifugal supernatant was recovered. After the concentration was determined using a microquantitator, the volume was adjusted to 100 μg / mL using PBS (pH = 7.4) buffer. The labeled antibody was diluted to the working concentration using diluent (containing PBS (pH = 7.2), 0.7 g / L 4-aminoantipyrine, 0.2 g / L paracetamol, 1.0 g / L Triton X-100, 0.4 g / L SDS, 10 g / L soy peptone, 5 g / L fish gelatin protein and 0.4 g / L gentamicin sulfate), and the luminescence value was measured using a Mecon MS-i3080 luminometer. The test results are shown in Table 2, which shows the test results of the antigen calibrators (S1-S10). Among them, the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S1 is 0 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S2 is 2.6 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S3 is 17.26 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S4 is 38.33 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S5 is 10. The concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S6 is 121.59 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S7 is 140.05 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S8 is 193.15 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S9 is 228.38 U / mL; the concentration of the crude pure anti-CA724 antibody or the purified desialylated anti-CA724 antibody in S10 is 250.17 U / mL.

[0067] Table 2

[0068]

[0069] The test results in Table 2 show that, compared with the control antibody, the activity of the purified desialylated anti-CA724 antibody prepared in the present invention is relatively improved, the low-value background is relatively reduced, and the application effect can be improved.

[0070] The experimental results of Examples 1 to 4 show that the anti-CA724 antibodies prepared by the method for improving the sensitivity and specificity of anti-CA724 antibodies provided by the present invention have higher sensitivity, stronger specificity, better antibody activity, and lower reagent background compared to existing anti-CA724 antibodies, and can be used to prepare CA724 detection products.

[0071] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for improving the sensitivity of anti-CA724 antibodies, characterized in that: The following steps are involved: The anti-CA724 antibody with sialylation modification is cleaved with sialylase to obtain a desialylated anti-CA724 antibody, thereby improving the sensitivity of the anti-CA724 antibody, wherein the molar ratio of the sialylase to the anti-CA724 antibody is 1:(1-3).

2. The method for improving the sensitivity of anti-CA724 antibodies according to claim 1, characterized in that: The reaction temperature for enzymatic cleavage of anti-CA724 antibody using sialylase is 35-37.5°C.

3. The method for improving the sensitivity of anti-CA724 antibodies according to claim 1, characterized in that: The following steps are also included: After the enzyme digestion is completed, the anti-CA724 antibody after the reaction is separated and purified by column chromatography and / or dialysis.

4. A desialylated anti-CA724 antibody, characterized in that: The method for improving the sensitivity of anti-CA724 antibodies according to any one of claims 1 to 3 is used to prepare the anti-CA724 antibody.

5. An antibody conjugate, characterized in that The antibody conjugate is composed of the desialylated anti-CA724 antibody according to claim 4, and The invention also comprises a chemical label or a biological marker coupled to the desialylated anti-CA724 antibody.

6. A CA724 detection product, characterized in that: The invention also comprises the desialylated anti-CA724 antibody according to claim 4.

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