Method for improving sensitivity and specificity of anti-CA724 antibody and application

Abstract

The invention belongs to the technical field of biology, and provides a method for improving the sensitivity and specificity of an anti-CA724 antibody and application, and the method for improving the sensitivity and specificity of the anti-CA724 antibody specifically comprises the following steps: carrying out enzyme digestion on the anti-CA724 antibody by adopting sialylase to obtain a sialylated anti-CA724 antibody, thereby improving the sensitivity and specificity of the anti-CA724 antibody. A common anti-CA724 antibody is modified through an enzyme digestion process, so that the sensitivity and the specificity of the anti-CA724 antibody are improved, and meanwhile, the detection cost can be effectively reduced.

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 anti-CA724 antibodies. Background Art

[0002] Carbohydrate antigen CA724 is a specific tumor marker and one of the laboratory markers for detecting gastric cancer and various digestive tract cancers. The content in normal human serum is <6 U / mL. Abnormal elevation occurs in various digestive tract tumors, ovarian cancer and other diseases, and it has relatively high specificity for the detection of gastric cancer. The antigen is identified as a mucinous glycoprotein with a size of 220-400 kDa. Two different monoclonal antibodies, CC49 and B72.3, can respectively recognize its glycan epitopes Galβ(1-3)sialyl-Tn and sialyl-Tn antigens. During the expression of antibodies derived from mammals, due to the influence of culture conditions during expression, the obtained anti-CA724 antibodies often have many glycosylation modifications. Glycosylation modifications have an adverse impact on both the sensitivity and specificity of anti-CA724 antibodies. Currently, the sensitivity and specificity of anti-CA724 antibodies are defective. In view of this, there is a need to provide anti-CA724 antibodies with better sensitivity and specificity. 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 problem, in the first aspect of the present invention, a method for improving the sensitivity and specificity of anti-CA724 antibodies is provided. The method includes the following steps: treating anti-CA724 antibodies with sialidase to obtain desialylated anti-CA724 antibodies, so as to improve the sensitivity and specificity of anti-CA724 antibodies.

[0005] In the production of antibodies, due to the complex biochemical structure and production process, anti-CA724 antibodies are extremely prone to a large amount of modification or degradation during production and storage. Among them, sialic acid modification is one of the main glycosylation modifications that affect the specificity and sensitivity of anti-CA724 antibodies, which will cause a mismatch between anti-CA724 antibodies and sialic acid modification, and then lead to weak aggregation between antibodies, reducing the sensitivity and specificity of anti-CA724 antibodies. By adopting the technical solution provided by the present invention and treating anti-CA724 antibodies with sialidase (neuraminidase), the sialic acid modification level of anti-CA724 antibodies can be effectively reduced, thereby effectively reducing the aggregation between anti-CA724 antibodies, further improving the specificity of anti-CA724 antibodies, and at the same time reducing the background signal interference in reagent application, and further 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] According to the molar ratio of the sialidase and the anti - CA724 antibody provided by the present invention, the enzymatic digestion efficiency can be maximized while avoiding side reactions or cost waste caused by excessive enzymes, thus improving the process economy.

[0008] Preferably, the reaction temperature for digesting the anti - CA724 antibody with sialidase is 35 - 37.5 °C.

[0009] The present invention uses a reaction temperature range of 35 - 37.5 °C to simulate the physiological environment. While maintaining the optimal activity of the sialidase, it can protect the structural integrity of the antibody and avoid problems such as high - temperature denaturation or insufficient reaction rate at low temperatures.

[0010] Preferably, the anti - CA724 antibody is an anti - CA724 antibody with sialylation modification.

[0011] The present invention aims at the anti - CA724 antibody, which can directly solve the problem of performance defects of the anti - CA724 antibody caused by glycosylation modification during the production process due to host cells, and effectively improve the sensitivity and specificity of the anti - CA724 antibody.

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

[0013] Preferably, it further includes the following step: after the enzymatic digestion is completed, the anti - CA724 antibody after the reaction is separated and purified 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 digestion product. This step ensures the purity of the final antibody and avoids interference of residues in subsequent detections.

[0015] More preferably, the anti - CA724 antibody after the reaction is separated and purified by any one or more of protein G, anion chromatography, metal ion chelating chromatography, chromatographic 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 described in the first aspect.

[0017] Furthermore, the third aspect of the present invention provides an antibody conjugate, and the antibody conjugate includes the desialylated anti - CA724 antibody described in the second aspect.

[0018] After coupling markers such as fluorescent dyes, enzymes, and radioisotopes to the digested anti-CA724 antibody provided by the present invention, the antibody can still maintain the optimized performance, effectively improving the detection signal intensity and signal-to-noise ratio, and is applicable to various detection platforms such as ELISA and immunohistochemistry.

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

[0020] Furthermore, the fourth aspect of the present invention provides a CA724 detection product, which includes the desialylated anti-CA724 antibody described in the second aspect.

[0021] Through the CA724 detection product provided by the present invention, accurate quantitative or qualitative analysis of CA724 can be effectively achieved, and in particular, the incidence of false positives or false negatives in various cancers such as digestive tract tumors and ovarian cancer can be effectively reduced.

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

[0023] 1. By modifying the ordinary anti-CA724 antibody through an enzymatic digestion process, the present invention improves the sensitivity and specificity of the anti-CA724 antibody, making its performance superior to existing imported products, being a substitute for imported products, and effectively reducing the detection cost.

[0024] 2. The method provided by the present invention for improving the sensitivity and specificity of the anti-CA724 antibody is simple to operate, has mild reaction conditions, can be seamlessly connected with the existing traditional antibody production line, and the modified antibody can be adapted to mainstream diagnostic platforms, which can accelerate industrial application.

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

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

[0027] Figure 1 It is the SDS-PAGE electrophoresis result in Example 1 of the present invention;

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

[0029] Figure 3This is the HPLC detection result in Example 3 of the present invention. Detailed implementation mode

[0030] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of specific embodiments of the present invention is provided. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the protection scope of the claims of the present invention.

[0031] It should be noted that the endpoints and any values within the disclosed ranges in this article are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0032] As described in the background art, the sensitivity and specificity of existing commercial anti-CA724 antibodies are relatively low. Based on this, the specific implementation mode of the present invention provides a method for improving the sensitivity and specificity of anti-CA724 antibodies. This method uses sialidase to cleave anti-CA724 antibodies, thereby improving the sensitivity and specificity of 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 using sialidase to cleave anti-CA724 antibodies is 35-37.5°C.

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

[0036] In some embodiments, after the cleavage is completed, the anti-CA724 antibody after the reaction 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 obtain the antibody solution through column chromatography for crude purification;

[0039] S2: Mix the antibody solution with the NRH enzyme mixture and react overnight at 37°C to obtain the digestion reaction solution;

[0040] S3: Subject the digestion reaction solution in step S2 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 anti-CA724 antibody provided by the specific embodiments of the present invention has the advantages of high sensitivity and high specificity. Compared with the existing anti-CA724 antibodies, the binding effect between desialylated anti-CA724 antibodies is significantly reduced, and aggregation is not likely to occur. The CA724 detection product prepared with the desialylated anti-CA724 antibody provided by the present invention can effectively reduce the incidence of false positives and false negatives in the detection results, providing a new detection antibody for the detection of CA724.

[0042] The technical solutions of the present invention will be further described below through specific examples. Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with commonly understood meanings are defined herein for purposes of clarification or convenience of citation, and such definitions herein should not be construed as indicating a significant difference from the conventional understanding 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 specified, the use of commercially available kits, reagents and instruments follows the protocols and parameters given by the manufacturers.

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

[0044] In the following examples, the Maccura MS-i3080 luminescence analyzer is from Maccura 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 CO₂ shaker at a rotation speed of 110 rpm / min, a temperature of 37 °C, and a CO₂ concentration of 5%. When the cell density reaches (1.5 - 3.0) × 10 6 cells / ml, perform cell passage with an inoculation density of (2.0 - 5.0) × 10 5 cells / ml. After several passages, make the cell viability reach more than 95%, 8 × 10 5Inoculate into a shake flask at [[[number]]] cells / mL and perform batch cell culture. During the culture, add the supplementary feed matching the medium to maintain the sugar content at 4 g / L. When the cell viability drops below 80%, centrifuge to collect the cell supernatant.

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

[0049] Mix the NRH enzyme solution and the antibody solution at a mass ratio of 2:1, place them in a metal bath at 37 °C and incubate overnight to allow the enzyme to fully exert its activity to cleave the sialic acid chains of the anti-CA724 antibody, forming a mixture of the cleaved anti-CA724 antibody and the NRH enzyme. The mixture was purified again using Protein G packing material, the eluate was collected, and it was dialyzed into a 20 mM PB buffer solution (pH 8.0) four times to obtain a solution of the cleaved anti-CA724 antibody.

[0050] The above-mentioned solution of the cleaved anti-CA724 antibody was further purified using DEAE packing material, gradient elution was performed using a salt solution, the eluate was collected, and after dialysis four times in a 20 mM PBS buffer solution (pH = 8.0), the antibody was concentrated to 2 mg / mL to obtain a highly purified desialylated anti-CA724 antibody. The purification effect of the antibody was evaluated by SDS-PAGE electrophoresis. The specific results are as Figure 1 shown. A clear band of the anti-CA724 antibody can be seen in the SDS-PAGE electrophoresis. Among them, lane 1 is the band of the highly purified desialylated anti-CA724 antibody (reductive), the heavy chain size is about 50 KD, and the light chain size is about 25 KD; lane 2 is the band of the highly purified desialylated anti-CA724 antibody (non-reductive); lane 3 is the Maker band. It can be Figure 1 seen that no other protein impurity bands were found in the antibody prepared by the method provided by the present invention.

[0051] Example 2

[0052] Detection of the morphological form of the antibody

[0053] Quantify the crude pure anti-CA724 antibody solution in Example 1 and the desialylated anti-CA724 antibody to the same concentration (1 mg / mL). Rinse the quantitative cup of the ZATA potentiometer repeatedly with pure water. Take the buffer used to store the antibody and place it in the ZATA potentiometer for particle size scanning, and set it as the blank background. Then, take 1 mL of the crude pure anti-CA724 antibody and the purified desialylated anti-CA724 antibody for detection respectively. The specific results are as Figure 2 shown, Figure 2 Figure 5 is the particle size scanning display diagram of the two anti-CA724 antibodies in the ZATA potentiometer. Among them, Figure 2 A in Figure 5 is the particle size scanning result of the crude pure anti-CA724 antibody, Figure 2 B in Figure 5 is the particle size scanning result of the purified desialylated anti-CA724 antibody. It can be seen from Figure 2 Figure 5 that the particle size of the normal antibody monomer is about 10 nm. The particle size scanning of the crude pure anti-CA724 antibody shows scanning peaks in the ranges of 10 nm, 100 nm, and 1000 nm, indicating that the crude pure anti-CA724 antibody is in the states of monomer, aggregate, and large particle aggregate in the storage buffer. After treatment with NRH enzyme and purification, the particle size scanning of the antibody shows only a peak in the 10 nm range, indicating that there is no aggregation phenomenon of the antibody in the storage buffer. Thus, it can be seen that the quality of the finally obtained purified desialylated anti-CA724 antibody in Example 1 is significantly better than that of the normal antibody.

[0054] Example 3

[0055] Glycosylation detection

[0056] Take 100 μg of the purified desialylated anti-CA724 antibody in Example 1, dilute it with pure water to 1 μg / μL, add 10 μg of PNGase F, and incubate overnight at 37 °C. The digested antibody is placed in a vacuum centrifugal concentrator for concentration and drying, and stored at -20 °C for later use.

[0057] Prepare the glycosylation staining solution according to the Ludger 2-AB kit instructions. Take 20 μL of the staining solution and mix it with the concentrated and dried glycosylation. Incubate at 65 °C for 3 h. Add 500 μL of 100% acetonitrile solution and mix well. Centrifuge to remove the supernatant, and air-dry the precipitate at room temperature. Add 15 μL of pure water to dissolve the precipitate, and then add 35 μL of 100% acetonitrile solution. Store at -20 °C for later use.

[0058] Use HPLC to detect the glycosylation level of the sample by loading it onto the machine. The specific results are as Figure 3 shown, and the peak area distribution table is shown in Table 1. The chromatographic conditions are as follows:

[0059] Chromatographic column: TSKgel Amide-80, 4.6 mm I.D., 25 cm, 5 μm; Detection wavelength: 428 nm; Injection volume: 10 μL; Mobile phase A: 50 mM ammonium formate solution (pH = 4.4); Mobile phase B: 100% acetonitrile solution, Flow rate: 0.8 mL / min; Column temperature: 60 °C (Auto-injection: 15 °C), Excitation wavelength: 250 nm.

[0060] It should be noted that in Table 1, all glycosylation names and the reference standards used for their determination are from Ludger Ltd., UK.

[0061] Table 1

[0062]

[0063] From Figure 3 and the results in Table 1, it can be seen that in glycosylation modification, F(6)A2 accounts for the majority (about 74%) of the antibody glycosylation modification, which is consistent with the results reported in other literatures. Compared with ordinary antibodies, the sialic acid modification of the purified desialylated anti-CA724 antibody prepared in Example 1 accounts for a relatively small proportion (about 0.56%) of the total sugar level, which is consistent with the purpose of the present invention.

[0064] Example 4

[0065] Product performance comparison

[0066] Use the crude pure anti-CA724 antibody solution in Example 1 as the control antibody. Take the control antibody and the purified desialylated anti-CA724 antibody provided in Example 1 as the labeled antibody, and combine it with the Roche CA724 coated antibody to detect the effect of the purified desialylated anti-CA724 antibody of the present invention in reagent application. Take out the pre-prepared antigen calibrator and restore it to room temperature, and mark the experimental layout. Mix 200 ug of the antibody with the acridinium ester solution in a ratio of 1:10, and mix in the dark for 2 h. Subsequently, use a desalting column for desalting, recover the centrifuged supernatant, determine the concentration using a microquantifier, and then dilute it to 100 ug / mL with PBS (pH = 7.4) buffer solution. The labeled antibody is diluted to the working concentration with a diluent (containing PBS (pH = 7.2), 4-aminoantipyrine at 0.7 g / L, paracetamol at 0.2 g / L, Triton X-100 at 1.0 g / L, SDS at 0.4 g / L, soy peptone at 10 g / L, fish gelatin protein at 5 g / L, and gentamicin sulfate at 0.4 g / L), and measure the luminescence value using a Meikang MS-i3080 model luminescence analyzer. The test results are shown in Table 2. Table 2 shows the test results of the antigen calibrator (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 75.9 U / mL; 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] It can be seen from the detection results in Table 2 that, compared with the control antibody, the activity of the purified desialylated anti-CA724 antibody prepared in the present invention is relatively increased, and the low-value background is relatively decreased, which can improve the application effect.

[0070] It can be seen from the experimental results of Examples 1 to 4 that the anti-CA724 antibody prepared by the method for improving the sensitivity and specificity of the anti-CA724 antibody provided by the present invention has higher sensitivity, stronger specificity, better antibody activity and lower reagent background compared with the existing anti-CA724 antibody, and can be used for the preparation of 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 all fall within the protection scope of the present invention.

Claims

1. A method for improving the sensitivity and specificity of anti-CA724 antibody, characterized in that, Comprising the following steps: Using sialidase to digest anti - CA724 antibody to obtain desialylated anti - CA724 antibody, so as to improve the sensitivity and specificity of the anti - CA724 antibody.

2. The method for improving the sensitivity and specificity of the anti-CA724 antibody according to claim 1, wherein The molar ratio of the sialidase to the anti - CA724 antibody is 1:(1 - 3).

3. The method for improving the sensitivity and specificity of the anti-CA724 antibody according to claim 1, wherein The reaction temperature for using sialidase to digest anti - CA724 antibody is 35 - 37.5 °C.

4. The method for improving the sensitivity and specificity of the anti-CA724 antibody according to claim 1, wherein The anti - CA724 antibody is an anti - CA724 antibody with sialylation modification.

5. The method for improving the sensitivity and specificity of anti-CA724 antibody according to claim 1, characterized in that, It further comprises the following steps: After the digestion is completed, the reacted anti - CA724 antibody is separated and purified by column chromatography and / or dialysis.

6. A desialylated anti-CA724 antibody, characterized in that, Prepared by using the method for improving the sensitivity and specificity of anti - CA724 antibody according to any one of claims 1 - 5.

7. An antibody conjugate, characterized in that, Comprising the desialylated anti - CA724 antibody according to claim 6.

8. The antibody conjugate according to claim 7, wherein, It further comprises a chemical label or a biological label conjugated with the anti - CA724 antibody.

9. A CA724 detection product, characterized in that, Comprising the desialylated anti - CA724 antibody according to claim 6.

Images