Method for improving detection sensitivity of ganglioside GM1 and application

Abstract

The invention relates to a method for improving the detection sensitivity of ganglioside GM1, which comprises the following steps: S1, diluting GM1 recombinant protein by using a coating solution, spraying the diluted GM1 recombinant protein on a nitrocellulose membrane, and drying; s2, carrying out sealing treatment on the nitrocellulose membrane by adopting a sealing solution, and cutting to form a detection membrane strip; s3, carrying out sample incubation on the detection membrane strip and a sample together, cleaning, adding an enzyme conjugate for incubation, adding a substrate for incubation, and carrying out result judgment in a result judgment template; by optimizing the pH value of the coating liquid and adding the modified trehalose, carboxymethyl cellulose and beta-cyclodextrin, the stability and binding efficiency of the recombinant protein on the nitrocellulose membrane are improved. Experimental data show that the positive coincidence rate is increased to 96% from 88% of a conventional method.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for improving the detection sensitivity of ganglioside GM1 and its application. Background Art

[0002] Gangliosides are a class of glycosphingolipids containing sialic acid, which mainly exist on the outside of cell membranes and are especially abundant in the nervous system.

[0003] Ganglioside antibodies are a class of autoantibodies targeting gangliosides that commonly occur in neurological diseases. Gangliosides are important glycolipid molecules present on the surface of nerve cells and are involved in cell adhesion, signal transduction, and myelin sheath-axon interactions.

[0004] Ganglioside antibodies and their related diseases include: acute Guillain-Barré syndrome (GBS), multifocal motor neuropathy (MMN), chronic sensory ataxia neuropathy (CANOMAD), Miller-Fisher syndrome (Miller-Fisher Syndrome), brainstem encephalitis, etc.

[0005] In addition to neurological diseases, ganglioside antibodies also have potential applications in cancer treatment. For example, certain gangliosides (such as GD2) are highly expressed on the surface of tumor cells and can serve as targets for CAR-T cell therapy. Furthermore, gangliosides can be used as tumor diagnostic markers or in the development of immunotherapy drugs.

[0006] Antibodies associated with multifocal motor neuropathy (MMN) are anti-monosialoganglioside GM1 antibodies, mostly IgM antibodies, with a positive incidence of 40-70%. Furthermore, anti-monosialoganglioside GM1 antibody titers are elevated in 22-30% of patients with Guillain-Barré syndrome, and antibody titers correlate with disease activity. Antibody titers peak during the acute phase and then gradually decline.

[0007] Clinical laboratory detection of ganglioside-specific autoantibodies has important clinical significance for the diagnosis, differential diagnosis, typing, disease monitoring and prognosis of related diseases.

[0008] The existing conventional product preparation method has a slightly lower positive compliance rate of only about 88%, with a high missed detection rate and insufficient sensitivity. The main reason is that the composition and process parameters of the coating and blocking solutions are not optimized, resulting in low antigen-antibody binding efficiency. Summary of the Invention

[0009] Based on this, the present invention develops a method for improving the detection sensitivity of ganglioside GM1, which optimizes the process of coating and sealing, improves the positive compliance rate of the product, and reduces missed detections.

[0010] One object of the present invention is to provide a method for improving the sensitivity of ganglioside GM1 detection, comprising the following steps:

[0011] S1. Dilute GM1 recombinant protein with coating solution, spray onto nitrocellulose membrane, and dry.

[0012] S2. Block the nitrocellulose membrane with a blocking solution and cut it into test strips;

[0013] S3. The test membrane strip and the sample are incubated together, washed, and then the enzyme conjugate is added for incubation. The substrate is then added for incubation and placed in the result judgment template for result judgment;

[0014] The coating solution is a mixture of modified trehalose, carboxymethyl cellulose, β-cyclodextrin and Tris-HCl buffer.

[0015] Specifically, the pH of the coating solution is 7.4-9. By adjusting the pH to be controlled within the range of 7.4-9, the natural conformation of the recombinant protein can be maintained, and protein denaturation or aggregation caused by pH deviation can be avoided.

[0016] Furthermore, the blocking solution is a mixture of Tris-HCl buffer and bovine serum albumin.

[0017] Furthermore, the preparation method of the modified trehalose comprises the following steps:

[0018] Trehalose, hydroxypropyl methylcellulose, oxalic anhydride and sodium hydroxide are added into a solvent, mixed, and heated to react under the action of a catalyst to obtain modified trehalose.

[0019] Furthermore, in step S2, the molecular weight of the hydroxypropyl methylcellulose is 50-500 kDa.

[0020] Further, the pH of the Tris-HCl buffer is selected from 7.4-9;

[0021] Furthermore, the drying temperature is 35-38°C.

[0022] Furthermore, the incubation temperature is 18-25°C.

[0023] Furthermore, the substrate solution is BCIP / NBT color development solution.

[0024] Furthermore, the enzyme conjugate is goat anti-human IgG-alkaline phosphatase antibody.

[0025] The present invention also provides application of the method for improving the detection sensitivity of ganglioside GM1 in a kit.

[0026] The present invention has the following beneficial effects:

[0027] The present invention optimizes the pH of the coating solution and adds modified trehalose, carboxymethyl cellulose, and β-cyclodextrin. First, by reacting trehalose, hydroxypropyl methylcellulose, and oxalic anhydride, hydroxypropyl methylcellulose is introduced into the modified trehalose, so that the modified trehalose forms a cross-linked network structure, and enhances its compatibility with carboxymethyl cellulose, improves the hydrogen bond and electrostatic interaction between the two, and promotes the formation of a cross-linked network. It can not only more effectively adsorb recombinant proteins, but also significantly increase the effective binding site density on the surface of the nitrocellulose membrane, increase the loading capacity and directional fixation efficiency of the recombinant protein, thereby improving the stability and binding efficiency of the recombinant protein on the nitrocellulose membrane, and maintaining a high positive detection rate for the sample. Experimental data show that the positive coincidence rate is increased from 88% of the conventional method to 96%. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0029] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0030] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0031] GM1 recombinant protein was purchased from Hytest, brand 8G16-2b.

[0032] Sample diluent, washing buffer, enzyme conjugate, and color development solution were purchased from the Anti-Ganglioside Antibody Spectrum 2 (IgG) Detection Kit (European Monoblot Method), catalog number: DL 1130-1601-2G.

[0033] The sample is a patient's blood sample.

[0034] Hydroxypropyl methylcellulose, YB47286, was purchased from Hunan Yunbang Biotechnology Co., Ltd.

[0035] Example 1

[0036] A method for improving the detection sensitivity of ganglioside GM1 comprises the following steps:

[0037] S1. Dilute GM1 recombinant protein in coating solution (ensure the concentration of GM1 recombinant protein is 200 μg / mL) and evenly spray onto nitrocellulose membrane using a biodot sprayer. Dry in a 37°C oven for 120 min.

[0038] S2. Soak the nitrocellulose membrane in blocking solution for 120 minutes, remove excess liquid, dry in a 37°C oven for 120 minutes, and cut into test strips.

[0039] S3-1. Place the test strip and 1.5 mL of sample diluent in an incubation tank and incubate for 5 minutes. Aspirate the incubation tank and add 1.5 mL of diluted sample. Incubate the sample on a rocking shaker at 25°C for 120 minutes. Aspirate the tank and wash the test strip three times with 1.5 mL of wash buffer on a rocking shaker for 5 minutes each.

[0040] S3-2. Add 1.5 mL of enzyme conjugate and incubate for 60 min. Aspirate the liquid in the tank and wash the test membrane strips three times with 1.5 mL of wash buffer on a rocking shaker for 5 min each.

[0041] S3-3. Add 1.5 mL of substrate solution and incubate on a rocking shaker at 25°C in the dark for 10 min. Aspirate the liquid in the tank and rinse the membrane strips three times with distilled water for 1 min each.

[0042] S3-4. Place the test film strip in the result determination template and let it air dry before judging the result;

[0043] The coating solution is a mixture of 5% modified trehalose, 0.5% hydroxypropyl methylcellulose, 0.1% β-cyclodextrin and the remainder Tris-HCl buffer with a pH of 8.5;

[0044] The blocking solution is a mixture of 10% bovine serum albumin and the remainder of a Tris-HCl buffer solution with a pH of 8.5.

[0045] The preparation method of the modified trehalose comprises the following steps:

[0046] Trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine were mixed in anhydrous DMF as solvent, heated at 65°C with stirring for 12 hours, and the solvent was removed and recrystallized to obtain modified trehalose.

[0047] The mass ratio of trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine is 3:1:4:2.5:0.5.

[0048] Example 2

[0049] A method for improving the detection sensitivity of ganglioside GM1 comprises the following steps:

[0050] S1. Dilute GM1 recombinant protein in coating solution (ensure the concentration of GM1 recombinant protein is 200 μg / mL) and evenly spray onto nitrocellulose membrane using a biodot sprayer. Dry in a 37°C oven for 120 min.

[0051] S2. Soak the nitrocellulose membrane in blocking solution for 120 minutes, remove excess liquid, dry in a 37°C oven for 120 minutes, and cut into test strips.

[0052] S3-1. Place the test strip and 1.5 mL of sample diluent in an incubation tank and incubate for 5 minutes. Aspirate the incubation tank and add 1.5 mL of diluted sample. Incubate the sample on a rocking shaker at 25°C for 120 minutes. Aspirate the tank and wash the test strip three times with 1.5 mL of wash buffer on a rocking shaker for 5 minutes each.

[0053] S3-2. Add 1.5 mL of enzyme conjugate and incubate for 60 min. Aspirate the liquid in the tank and wash the test membrane strips three times with 1.5 mL of wash buffer on a rocking shaker for 5 min each.

[0054] S3-3. Add 1.5 mL of substrate solution and incubate on a rocking shaker at 25°C in the dark for 10 min. Aspirate the liquid in the tank and rinse the membrane strips three times with distilled water for 1 min each.

[0055] S3-4. Place the test film strip in the result determination template and let it air dry before judging the result;

[0056] The coating solution is a mixture of 7% modified trehalose, 1% hydroxypropyl methylcellulose, 0.2% β-cyclodextrin and the remainder Tris-HCl buffer with a pH of 8.5;

[0057] The blocking solution is a mixture of 10% bovine serum albumin and the remainder of a Tris-HCl buffer solution with a pH of 8.5.

[0058] The preparation method of the modified trehalose comprises the following steps:

[0059] Trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine were mixed in anhydrous DMF as solvent, heated at 65°C with stirring for 12 hours, and the solvent was removed and recrystallized to obtain modified trehalose.

[0060] The mass ratio of trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine is 3:1:4:2.5:0.5.

[0061] Example 3

[0062] A method for improving the detection sensitivity of ganglioside GM1 comprises the following steps:

[0063] S1. Dilute GM1 recombinant protein in coating solution (ensure the concentration of GM1 recombinant protein is 200 μg / mL) and evenly spray onto nitrocellulose membrane using a biodot sprayer. Dry in a 37°C oven for 120 min.

[0064] S2. Soak the nitrocellulose membrane in blocking solution for 120 minutes, remove excess liquid, dry in a 37°C oven for 120 minutes, and cut into test strips.

[0065] S3-1. Place the test strip and 1.5 mL of sample diluent in an incubation tank and incubate for 5 minutes. Aspirate the incubation tank and add 1.5 mL of diluted sample. Incubate the sample on a rocking shaker at 25°C for 120 minutes. Aspirate the tank and wash the test strip three times with 1.5 mL of wash buffer on a rocking shaker for 5 minutes each.

[0066] S3-2. Add 1.5 mL of enzyme conjugate and incubate for 60 min. Aspirate the liquid in the tank and wash the test membrane strips three times with 1.5 mL of wash buffer on a rocking shaker for 5 min each.

[0067] S3-3. Add 1.5 mL of substrate solution and incubate on a rocking shaker at 25°C in the dark for 10 min. Aspirate the liquid in the tank and rinse the membrane strips three times with distilled water for 1 min each.

[0068] S3-4. Place the test film strip in the result determination template and let it air dry before judging the result;

[0069] The coating solution is a mixture of 8% modified trehalose, 2% hydroxypropyl methylcellulose, 0.5% β-cyclodextrin and the remainder Tris-HCl buffer with a pH of 8.5;

[0070] The blocking solution is a mixture of 10% bovine serum albumin and the remainder of a Tris-HCl buffer solution with a pH of 8.5.

[0071] The preparation method of the modified trehalose comprises the following steps:

[0072] Trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine were mixed in anhydrous DMF as solvent, heated at 65°C with stirring for 12 hours, and the solvent was removed and recrystallized to obtain modified trehalose.

[0073] The mass ratio of trehalose, hydroxypropyl methylcellulose, oxalic anhydride, sodium hydroxide and 4-dimethylaminopyridine is 3:1:4:2.5:0.5.

[0074] Comparative Example 1

[0075] In this comparative example, unmodified trehalose of equal mass was used to replace the modified trehalose, and other components and preparation methods were the same as those in Example 1.

[0076] Comparative Example 2

[0077] In the coating solution of this comparative example, hydroxypropyl methylcellulose was removed, that is, (the coating solution was a mixture of 5% modified trehalose, 0.1% β-cyclodextrin and the remainder Tris-HCl buffer with a pH of 8.5), and the other components and preparation method were the same as those in Example 1.

[0078] Comparative Example 3

[0079] In the coating solution of this comparative example, hydroxypropyl methylcellulose was removed, that is, (the coating solution was a mixture of 5% modified trehalose, 0.5% hydroxypropyl methylcellulose and the remainder Tris-HCl buffer with a pH of 8.5), and the other components and preparation method were the same as those in Example 1.

[0080] Comparative Example 4

[0081] In this comparative example, the blocking solution was not used to block the nitrocellulose membrane in step S2, and the other components and preparation methods were the same as those in Example 1.

[0082] Comparative Example 5

[0083] A kit is an anti-ganglioside antibody spectrum 2 (IgG) detection kit (European monoblot method), with the product number being DL1130-1601-2G.

[0084] Test Example 1

[0085] The pH of the Tris-HCl buffer in the coating solution of Example 1 was adjusted, and the positive coincidence rate at different pH values ​​was tested. The kit of Comparative Example 5 was used as a reference, and 50 positive samples were tested. After the test, the dried test membrane strips were scanned using a flatbed scanner (EUROIMMUNAG) and the results were evaluated using EUROLineScan software. The evaluation criteria are shown in Table 1.

[0086] Table 1 EUROLineScan software evaluation criteria

[0087] Software interpretation grade determination 0-10 - Negative ≥11 + Positive

[0088] The test results are shown in Table 2.

[0089] Table 2 Test results of the detection method of Example 1 at different pH values

[0090]

[0091]

[0092] As can be seen from Table 2, buffer solutions at pH 7.8, pH 8.0, pH 8.2, pH 8.5, and pH 9 can improve the positive coincidence rate, with pH 8.2-8.5 being the best, increasing it by 4%.

[0093] Test Example 2

[0094] The positive coincidence rate of Example 1 and Comparative Examples 1-3 was tested, and the kit of Comparative Example 5 was used as a reference, and 50 positive samples were tested respectively.

[0095] The test results are shown in Table 3.

[0096] Table 3 Test results of Example 1 and Comparative Examples 1-3

[0097]

[0098]

[0099]

[0100] As shown in Table 3, adding modified trehalose, carboxymethyl cellulose, and β-cyclodextrin to a buffer solution at pH 8.5 slightly improved the results, and a combination of the three reagents resulted in a 6% improvement. Finally, optimizing the coating solution increased the positive coincidence rate by 10%.

[0101] Test Example 3

[0102] The positive detection rates of the test methods of Example 1 and Comparative Example 4 were tested, and the kit of Comparative Example 5 was used as a reference. 50 positive samples were tested respectively, and the test results are shown in Table 4.

[0103] Table 4 Positive detection rates of Example 1 and Comparative Example 4

[0104]

[0105]

[0106] As can be seen from Table 4, the blocking solution of pH 7.4 Tris-HCl buffer + 10 wt % BSA can increase the positive coincidence rate by 2%.

[0107] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0108] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for improving the sensitivity of ganglioside GM1 detection, characterized in that: The steps include: S1. Dilute GM1 recombinant protein with coating solution, spray onto nitrocellulose membrane, and dry. S2. Block the nitrocellulose membrane with a blocking solution and cut it into test strips; S3. The test membrane strip and the sample are incubated together, washed, and then the enzyme conjugate is added for incubation. The substrate is then added for incubation and placed in the result judgment template for result judgment; The coating solution is a mixture of modified trehalose, carboxymethyl cellulose, β-cyclodextrin and Tris-HCl buffer.

2. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: The blocking solution is a mixture of Tris-HCl buffer and bovine serum albumin.

3. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: The preparation method of the modified trehalose comprises the following steps: Trehalose, hydroxypropyl methylcellulose, oxalic anhydride and sodium hydroxide are added into a solvent, mixed, and heated to react under the action of a catalyst to obtain modified trehalose.

4. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: In step S2, the molecular weight of the hydroxypropyl methylcellulose is 50-500 kDa.

5. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: The pH of the Tris-HCl buffer is selected from 7.8-9.

6. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: In step S1, the drying temperature is 35-38°C.

7. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: In step S3, the incubation temperature is 18-25°C.

8. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: In step S3, the substrate solution is BCIP / NBT color development solution.

9. The method for improving the detection sensitivity of ganglioside GM1 according to claim 1, characterized in that: In step S3, the enzyme conjugate is goat anti-human IgG-alkaline phosphatase antibody.

10. Use of the method for improving the detection sensitivity of ganglioside GM1 according to any one of claims 1 to 9 in a kit.

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