Biotin labeling buffer solution
阅读说明：本技术 生物素标记缓冲液 (Biotin labeling buffer solution ) 是由 贾海英 赵转 冯坤瑞 向阳 于 2021-08-18 设计创作，主要内容包括：本发明涉及一种生物素标记缓冲液,属于生物材料技术领域。本发明的生物素标记缓冲液包括以下组分：十二水合磷酸氢二钠、二水合磷酸二氢钠、三水合磷酸氢二钾、磷酸二氢钾、氯化钠、氯化钾；制得的生物素标记缓冲液的pH值为7.2～7.6；缓冲液每1L溶解有十二水合磷酸氢二钠6～6.4g、二水合磷酸二氢钠0.6～0.9g、三水合磷酸氢二钾4～4.4g、磷酸二氢钾0.4～0.6g、氯化钠0.6～0.8g、氯化钾0.6～0.8g。本发明的生物素标记缓冲液的离子强度及缓冲环境使得抗体更加稳定,抗体与生物素更容易结合,使用更低剂量的生物素即可与抗体完成标记,效率较常规标记缓冲液更高。生物素标记缓冲液标记的重复性高于常规标记缓冲液,且具有背景低、信噪比高的优势。(The invention relates to a biotin labeling buffer solution, belonging to the technical field of biological materials. The biotin labeling buffer of the invention comprises the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride; the pH value of the prepared biotin labeling buffer solution is 7.2-7.6; 6 to 6.4g of disodium hydrogen phosphate dodecahydrate, 0.6 to 0.9g of sodium dihydrogen phosphate dihydrate, 4 to 4.4g of dipotassium hydrogen phosphate trihydrate, 0.4 to 0.6g of potassium dihydrogen phosphate, 0.6 to 0.8g of sodium chloride and 0.6 to 0.8g of potassium chloride are dissolved in 1L of the buffer solution. The ionic strength and the buffering environment of the biotin labeling buffer solution enable the antibody to be more stable, the antibody and the biotin are more easily combined, the biotin and the antibody can be labeled by using a lower dose of biotin, and the efficiency is higher than that of a conventional labeling buffer solution. The repeatability of the biotin labeling buffer solution is higher than that of the conventional labeling buffer solution, and the biotin labeling buffer solution has the advantages of low background and high signal to noise ratio.)
1. A biotin labeling buffer comprising the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride;
the pH value of the biotin labeling buffer solution is 7.2-7.6;
wherein 6 to 6.4g of disodium hydrogen phosphate dodecahydrate, 0.6 to 0.9g of sodium dihydrogen phosphate dihydrate, 4 to 4.4g of dipotassium hydrogen phosphate trihydrate, 0.4 to 0.6g of potassium dihydrogen phosphate, 0.6 to 0.8g of sodium chloride and 0.6 to 0.8g of potassium chloride are dissolved in 1L of the water solvent of the biotin labeling buffer.
2. The biotin labeling buffer of claim 1, further comprising surfactants Tritonx-100 and Tween20, wherein Tritonx-1000.3 mL and Tween 200.2 mL are dissolved in 1L of the aqueous solvent.
3. The biotin labeling buffer of claim 1, wherein the aqueous solvent is purified water for in vitro diagnostic reagents.
4. The biotin-labeling buffer of claim 1, wherein the biotin-labeling buffer uses biotin-X-NHS which is N-hydroxysuccinimide ester of biotin-6- (bioaminocaproyl) hexanoic acid.
5. The biotin labeling buffer of claim 4, wherein when the biotin labeling antibody is used, the antibody is dissolved in the biotin labeling buffer and dialyzed at 2 ℃ to 8 ℃ for 16 hours.
6. The biotin labeling buffer of claim 5, wherein the antibody is dissolved in the biotin labeling buffer at a concentration of 1.0 to 4.0 mg/mL.
7. The biotin-labeling buffer of claim 5, wherein the biotin is dissolved in the biotin-labeling buffer containing the antibody and reacted at 18 to 37 ℃ for 1 hour.
8. The biotin labeling buffer of claim 5, wherein the molar ratio of biotin to antibody is 1:5 to 1: 10.
9. The biotin labeling buffer of claim 5, wherein the solvent for biotin is an organic solvent or an aqueous solution, and the organic solvent is one or more of dimethylformamide, dimethylsulfoxide, and pyridine.
10. The biotin labeling buffer of claim 5, wherein the biotin labeled antibody is subsequently purified by dialysis against 20mmol/L phosphate buffer.
Technical Field
The invention relates to a biotin labeling buffer solution, belonging to the technical field of biological materials.
Background
The antibody can be labeled by enzyme, fluorescein or biotin, and is used for observing the immune complex of antigen and antibody. Biotin binds to proteins such as antigens and antibodies, and does not affect the immunological activity of the latter, but results are different when labeled with an enzyme. Thus, in recent years, the demand for the use of biotinylated antibodies in clinical assays and immunoassays has increased rapidly. The biotinylation of the antibody is mainly achieved by adding activated biotin, but biotin and the antibody often have the problem of low labeling efficiency in the labeling process, and a large amount of biotin is usually required for labeling the antibody to complete the labeling.
Disclosure of Invention
The present invention is directed to provide a biotin labeling buffer solution, which can improve the efficiency of labeling an antibody with biotin, i.e., less biotin is added to complete the antibody labeling.
In order to achieve the purpose, the invention provides the following technical scheme: a biotin labeling buffer comprising the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride;
the pH value of the biotin labeling buffer solution is 7.2-7.6;
wherein 6 to 6.4g of disodium hydrogen phosphate dodecahydrate, 0.6 to 0.9g of sodium dihydrogen phosphate dihydrate, 4 to 4.4g of dipotassium hydrogen phosphate trihydrate, 0.4 to 0.6g of potassium dihydrogen phosphate, 0.6 to 0.8g of sodium chloride and 0.6 to 0.8g of potassium chloride are dissolved in 1L of the water solvent of the biotin labeling buffer.
Furthermore, the biotin labeling buffer also comprises surfactant Tritonx-100 and Tween20, and Tritonx-1000.3 mL and Tween 200.2mL are dissolved in 1L of aqueous solvent of the biotin labeling buffer.
Further, the aqueous solvent is purified water for in vitro diagnostic reagents.
Furthermore, the biotin used in the biotin labeling buffer solution is biotin-X-NHS, and the biotin-X-NHS is biotin-6- (biological amino caproyl amino) hexanoic acid N-hydroxysuccinimide ester.
Further, when the biotin-labeled antibody is used, the antibody is dissolved in the biotin-labeled buffer and dialyzed at 2 ℃ to 8 ℃ for 16 hours.
Further, the concentration of the antibody dissolved in the biotin labeling buffer solution is 1.0-4.0 mg/mL.
Further, dissolving the biotin in a biotin labeling buffer solution containing the antibody, and reacting for 1h at 18-37 ℃.
Further, the molar ratio of the biotin to the antibody is 1: 5-1: 10.
Further, the solvent of the biotin is an organic solvent or an aqueous solution, and the organic solvent is one or more of dimethylformamide, dimethyl sulfoxide and pyridine.
Further, the biotin-labeled antibody is purified by dialysis in 20mmol/L phosphate buffer.
The invention has the beneficial effects that: the biotin labeling buffer solution is a buffer solution with low ion concentration, in the buffer solution, the antibody is more stable due to the ionic strength and the buffer environment, and the antibody and the biotin are more easily combined, so that the ratio of the biotin used for labeling to the antibody is lower, namely the biotin labeling of the antibody is higher by using the biotin and the antibody with lower doses. The repeatability of the biotin labeling buffer solution is higher than that of the conventional labeling buffer solution, and the biotin labeling buffer solution has the advantages of low background and high signal-to-noise ratio.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 is a graph comparing the luminescence values of samples with different concentrations detected by CA15-3 biotin-labeled antibody;
FIG. 2 is a graph comparing the luminescence values of samples with different concentrations detected by CA72-4 biotin-labeled antibody.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The invention provides a biotin labeling buffer solution which comprises the following components: disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride;
the pH value of the biotin labeling buffer solution is 7.2-7.6;
6-6.4 g of disodium hydrogen phosphate dodecahydrate, 0.6-0.9 g of sodium dihydrogen phosphate dihydrate, 4-4.4 g of dipotassium hydrogen phosphate trihydrate, 0.4-0.6 g of potassium dihydrogen phosphate, 0.6-0.8 g of sodium chloride and 0.6-0.8 g of potassium chloride are dissolved in 1L of a water solvent of the biotin labeling buffer solution, and the buffer solution with low ion concentration is prepared, so that the labeling efficiency can be improved, the used biotin can be reduced, and resources can be saved.
The biotin labeling buffer also comprises surfactant Tritonx-100 and Tween20, wherein Tritonx-1000.3 mL and Tween 200.2mL are dissolved in each 1L of aqueous solvent. Because amino exists on the antibody to be biotinylated, the amino is not easy to approach, so that the antibody biotinylation is possibly insufficient, and the problem of insufficient biotinylation of the antibody can be effectively solved by adding surfactants Tritonx-100 and Tween 20. The amount of the surfactant can be increased or decreased according to the use condition of the reagent, and other surfactants can be used, which are not listed here.
The water solvent in the biotin labeling buffer solution is purified water for the in-vitro diagnostic reagent.
The biotin used in the biotin labeling buffer solution is biotin-X-NHS, and the biotin-X-NHS is biotin-6- (biological amino caproyl amino) hexanoic acid N-hydroxysuccinimide ester. The use of biotin labeled with NHS, i.e. biotin which has been activated, which is soluble in water, allows simple and efficient biotinylation of the antibody in solution.
When the biotin-labeled antibody is used, the antibody is dissolved in a biotin-labeled buffer solution, and the concentration of the antibody after being dissolved in the biotin-labeled buffer solution is 1.0-4.0 mg/mL, because the original solution of the antibody contains sodium azide, glycerol and other substances, which can affect the subsequent biotin-labeled antibody, the antibody needs to be dialyzed for 16 hours at the temperature of 2-8 ℃ for purification to remove the substances, and then the purified antibody is obtained.
And calculating the using amount of the biotin required to carry out a labeling reaction according to the molar ratio of the biotin to the antibody added into the biotin labeling buffer solution of 1: 5-1: 10. And dissolving biotin in an organic solvent or an aqueous solution, adding the dissolved biotin into a biotin-labeled buffer solution containing an antibody, and reacting at 18-37 ℃ for 1h to perform a biotin-labeled antibody reaction. The biotin is dissolved in a biotin-labeling buffer containing an antibody, that is, the biotin is dissolved in the biotin-labeling buffer after antibody dialysis.
After the reaction was completed, the reaction mixture was purified by dialysis against 20mmol/L phosphate buffer to remove free biotin.
The molar ratio of biotin to the antibody added into the biotin labeling buffer solution is 1: 5-1: 10, and the biotinylation of the antibody can be completed by using a small dose of biotin and the antibody.
The biotin is dissolved in an organic solvent or an aqueous solution, and the organic solvent is one or more of dimethylformamide, dimethyl sulfoxide and pyridine. It should be noted that the solvent of biotin may also be other solvents, and may be selected according to actual needs, which are not described herein again.
The biotin-labeled antibody needs to be purified by dialysis in 20mmol/L phosphate buffer solution, and the dialysis time can be adjusted according to specific operation conditions. The biotin buffer solution with the dissolved antibody needs dialysis for purification, and residual biotin in the solution can be removed, and other purification methods can also be used. It should be noted that the phosphate buffer used herein is a conventional phosphate buffer, and thus, the description thereof is omitted.
The following is a detailed description of specific examples:
example 1
The following reagents were weighed:
disodium hydrogen phosphate dodecahydrate: 6g of a mixture;
sodium dihydrogen phosphate dihydrate: 0.6 g;
dipotassium hydrogen phosphate trihydrate: 4g of the total weight of the mixture;
potassium dihydrogen phosphate: 0.4 g;
sodium chloride: 0.6 g;
potassium chloride: 0.6 g;
Tritonx～100：0.3mL；
Tween20：0.2mL；
weighing a proper amount of purified water for the in vitro diagnostic reagent, sequentially adding the weighed disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride into the purified water for the in vitro diagnostic reagent, and uniformly stirring to fully dissolve and uniformly mix the materials;
tritonx-100 and Tween20 are added and stirred evenly to be fully dissolved and evenly mixed, the volume of the in vitro diagnostic reagent is determined to be 1L by purified water, and then hydrochloric acid is added to adjust the pH value to be 7.2.
Filtering with 0.45 μm filter membrane to obtain biotin-labeled buffer solution, and storing at 2-8 deg.C for further use.
Diluting 1mg of an antibody to be labeled to the concentration of 2mg/mL by using a biotin labeling buffer solution, transferring the antibody to a dialysis bag (with the molecular weight cutoff of 8000-12000D), dialyzing the antibody in the biotin labeling buffer solution for 16h, and keeping the ambient temperature at 2-8 ℃.
0.56mg (molecular weight 589) of biotin was added in 95.1. mu.L of dimethylformamide, an organic solvent, so that the biotin concentration was set to 10 mmol/L. 6.6 mu L of biotin with the concentration of 10mmol/L is added into biotin labeling buffer with the antibody content of 1mg, and the molar ratio of the antibody to the biotin is 1: reacting for 1h at the temperature of 10, 18-37 ℃.
After the labeling reaction is finished, the labeled antibody is transferred into a dialysis bag and dialyzed for 16h in phosphate buffer solution with the ion concentration of 20mmol/L, pH value of 7.2.
Example 2
The following reagents were weighed:
disodium hydrogen phosphate dodecahydrate: 6.4 g;
sodium dihydrogen phosphate dihydrate: 0.9 g;
dipotassium hydrogen phosphate trihydrate: 4.4 g;
potassium dihydrogen phosphate: 0.6 g;
sodium chloride: 0.8 g;
potassium chloride: 0.6-0.8 g;
Tritonx～100：0.3mL；
Tween20：0.2mL；
weighing a proper amount of purified water for the in vitro diagnostic reagent, sequentially adding the weighed disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride into the purified water for the in vitro diagnostic reagent, and uniformly stirring to fully dissolve and uniformly mix the materials;
tritonx-100 and Tween20 are added and stirred evenly to be fully dissolved and evenly mixed, the volume of the in vitro diagnostic reagent is determined to be 1L by purified water, and then hydrochloric acid is added to adjust the pH value to be 7.2.
Filtering with 0.45 μm filter membrane to obtain biotin-labeled buffer solution, and storing at 2-8 deg.C for further use.
Diluting 1mg of an antibody to be labeled to the concentration of 2mg/mL by using a biotin labeling buffer solution, transferring the antibody to a dialysis bag (with the molecular weight cutoff of 8000-12000D), and dialyzing in the biotin labeling buffer solution for 16h at the ambient temperature of 2-8 ℃.
0.56mg (molecular weight 589) of biotin was added to 95.1. mu.L of dimethylformamide, and the concentration was 10 mmol/L. And 6.6 mu L of biotin with the concentration of 10mmol/L is added into biotin labeling buffer solution with the antibody content of 1mg, and the molar ratio of the antibody to the biotin is 1: reacting for 1h at the temperature of 10, 18-37 ℃.
After the labeling reaction is finished, the labeled antibody is transferred into a dialysis bag and dialyzed for 16h in phosphate buffer solution with the ion concentration of 20mmol/L, pH value of 7.2.
Example 3
Disodium hydrogen phosphate dodecahydrate: 6g of a mixture;
sodium dihydrogen phosphate dihydrate: 0.6 g;
dipotassium hydrogen phosphate trihydrate: 4g of the total weight of the mixture;
potassium dihydrogen phosphate: 0.4 g;
sodium chloride: 0.6 g;
potassium chloride: 0.6 g;
Tritonx～100：0.3mL；
Tween20：0.2mL；
weighing a proper amount of purified water for the in vitro diagnostic reagent, sequentially adding the weighed disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride into the purified water for the in vitro diagnostic reagent, and uniformly stirring to fully dissolve and uniformly mix the materials;
tritonx-100 and Tween20 are added and stirred evenly to be fully dissolved and evenly mixed, the volume of the in vitro diagnostic reagent is determined to be 1L by purified water, and then hydrochloric acid is added to adjust the pH value to be 7.2.
Filtering with 0.45 μm filter membrane to obtain biotin-labeled buffer solution, and storing at 2-8 deg.C for further use.
Diluting 1mg of an antibody to be labeled to the concentration of 2mg/mL by using a biotin labeling buffer solution, transferring the antibody to a dialysis bag (with the molecular weight cutoff of 8000-12000D), and dialyzing in the biotin labeling buffer solution for 16h at the ambient temperature of 2-8 ℃.
0.56mg (molecular weight 589) of biotin was added to 95.1. mu.L of dimethylformamide, and the concentration was 10 mmol/L. Adding 3.3 mu L of biotin with the concentration of 10mmol/L into biotin labeling buffer solution with the antibody content of 1mg, wherein the molar ratio of the antibody to the biotin is 1:5, reacting for 1h at the temperature of 18-37 ℃.
After the labeling reaction is finished, the labeled antibody is transferred into a dialysis bag and dialyzed for 16h in phosphate buffer solution with the ion concentration of 20mmol/L, pH value of 7.2.
Example 4
The following reagents were weighed:
disodium hydrogen phosphate dodecahydrate: 6.4 g;
sodium dihydrogen phosphate dihydrate: 0.9 g;
dipotassium hydrogen phosphate trihydrate: 4.4 g;
potassium dihydrogen phosphate: 0.6 g;
sodium chloride: 0.8 g;
potassium chloride: 0.6-0.8 g;
Tritonx～100：0.3mL；
Tween20：0.2mL；
weighing a proper amount of purified water for the in vitro diagnostic reagent, sequentially adding the weighed disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, sodium chloride and potassium chloride into the purified water for the in vitro diagnostic reagent, and uniformly stirring to fully dissolve and uniformly mix the materials;
tritonx-100 and Tween20 are added and stirred evenly to be fully dissolved and evenly mixed, the volume of the in vitro diagnostic reagent is determined to be 1L by purified water, and then hydrochloric acid is added to adjust the pH value to be 7.2.
Filtering with 0.45 μm filter membrane to obtain biotin-labeled buffer solution, and storing at 2-8 deg.C for further use.
Diluting 1mg of an antibody to be labeled to the concentration of 2mg/mL by using a biotin labeling buffer solution, transferring the antibody to a dialysis bag (with the molecular weight cutoff of 8000-12000D), and dialyzing in the biotin labeling buffer solution for 16h at the ambient temperature of 2-8 ℃.
0.56mg (molecular weight 589) of biotin was added to 95.1. mu.L of dimethylformamide, and the concentration was 10 mmol/L. Adding 3.3 mu L of biotin with the concentration of 10mmol/L into biotin labeling buffer solution with the antibody content of 1mg, wherein the molar ratio of the antibody to the biotin is 1:5, reacting for 1h at the temperature of 18-37 ℃.
After the labeling reaction is finished, the labeled antibody is transferred into a dialysis bag and dialyzed for 16h in phosphate buffer solution with the ion concentration of 20mmol/L, pH value of 7.2.
The biotin-labeled antibodies prepared in examples 1 to 4 and those prepared in a conventional labeling buffer were used to react with the antigens of the conjugate, and the experimental data are shown in tables 1 to 6, and it should be noted that the antibodies used in examples 1 to 4 may be CA15-3 antibody, CA72-4 antibody or other antibodies.
Tables 1 to 3 show the experimental data of CA15-3 labeled antibody, and tables 4 to 6 show the experimental data of CA72-4 labeled antibody.
Diluting the biotin-labeled CA15-3 antibody obtained in examples 1 to 4 into a working solution by using a buffer solution, and detecting CA15-3 samples with the concentration of 0 and the concentration of 80U/mL respectively; CA15-3 antibody labeled with 0.1mol/L phosphate buffer solution (conventional labeling buffer solution) at pH 7.2 was used as a control, and CA15-3 samples at 0 concentration and 80U/mL concentration were also tested to calculate the signal-to-noise ratio, and the experimental results are shown in Table 1.
TABLE 1 signal-to-noise ratio of CA15-3 labeled antibody
Observing table 1, the signal-to-noise ratio of the conventional labeling buffer solution labeled antibody is obviously lower than that of the buffer solution prepared in the application, and the background noise of the buffer solution prepared in the application is obviously lower than that of the conventional labeling buffer solution.
The biotin-labeled CA15-3 antibodies obtained in examples 1 to 4 were diluted with buffers to give working solutions, and luminescence values of CA15-3 samples at concentrations of 0U/mL, 5U/mL, 20U/mL, 80U/mL, 200U/mL, and 500U/mL were measured; the luminescence measurements were also performed on CA15-3 samples at concentrations of 0U/mL, 5U/mL, 20U/mL, 80U/mL, 200U/m, and 500U/mL using 0.1mol/L of CA15-3 antibody labeled with phosphate buffer (conventional labeling buffer) at pH 7.2 as a control, the experimental data are shown in Table 2, and a comparison graph is plotted in FIG. 1.
TABLE 2 CA15-3 labeled antibody test sample data
By observing fig. 1, it can be seen that the conventional labeling buffer solution labeled antibody and the buffer solution labeled antibody configured in example 1, example 2, example 3, and example 4 detect samples with the same concentration, the sample luminescence value curve of the conventional labeling buffer solution group is significantly lower than that of the buffer solution of the present application, and the labeling efficiency of the biotin labeling buffer solution of the present application is higher. And the labeling effect of the buffers configured in the examples 1, 2, 3 and 4 is higher than that of the conventional labeling buffer.
Diluting the biotin-labeled CA15-3 antibody obtained in examples 1 to 4 into a working solution by using a buffer solution, repeating the test for 10 times on CA15-3 samples with the concentrations of 0U/mL and 80U/mL, and calculating the repeatability of the labeled antibody; as a control, CA15-3 antibody labeled with 0.1mol/L phosphate buffer (conventional labeling buffer) at pH 7.2 was used, and the statistical data and results are shown in Table 3.
TABLE 3 repeated detection of CA15-3 labeled antibody
Looking at table 3, the CV values of the conventional labeling buffers were all > 10%, while the CV values of the buffers formulated in the present application were all < 5%, and the reproducibility of the buffers formulated in the present application was significantly higher than that of the conventional labeling buffers.
Diluting the biotin-labeled CA72-4 antibody obtained in examples 1 to 4 into a working solution by using a buffer solution, and detecting CA72-4 samples with the concentrations of 0U/mL and 50U/mL respectively; CA72-4 antibody labeled with 0.1mol/L phosphate buffer solution (conventional labeling buffer solution) at pH 7.2 was used as a control, and CA72-4 samples at concentrations of 0U/mL and 50U/mL were also tested to calculate the signal-to-noise ratio, and the results are shown in Table 4.
TABLE 4 signal-to-noise ratio of CA72-4 labeled antibody
Observing table 4, the signal-to-noise ratio of the conventional labeling buffer solution labeled antibody is obviously lower than that of the buffer solution prepared in the application, and the background noise of the buffer solution prepared in the application is obviously lower than that of the conventional labeling buffer solution.
The biotin-labeled CA72-4 antibodies obtained in examples 1 to 4 were diluted with buffer solutions to working solutions, and luminescence values of CA72-4 samples at concentrations of 0U/mL, 2U/mL, 10U/mL, 50U/mL, 150U/mL, and 300U/mL were measured; the luminescence measurements were also performed on CA72-4 samples at concentrations of 0U/mL, 2U/mL, 10U/mL, 50U/mL, 150U/mL, and 300U/mL using 0.1mol/L of CA72-4 antibody labeled with phosphate buffer (conventional labeling buffer) at pH 7.2 as a control, the experimental data are shown in Table 5, and a comparison graph is plotted in FIG. 2.
TABLE 5 CA72-4 labeled antibody test sample data
By observing fig. 2, it can be seen that the conventional labeling buffer solution labeled antibody and the buffer solution labeled antibody configured in example 1, example 2, example 3, and example 4 detect samples with the same concentration, the sample luminescence value curve of the conventional labeling buffer solution group is significantly lower than that of the buffer solution of the present application, and the labeling efficiency of the biotin labeling buffer solution of the present application is higher. And the labeling effect of the buffers configured in the examples 1, 2, 3 and 4 is higher than that of the conventional labeling buffer.
Diluting the biotin-labeled CA72-4 antibodies obtained in examples 1 to 4 into working solutions by using buffer solutions, repeating the test for 10 times on CA72-4 samples with the concentrations of 0U/mL and 50U/mL, and calculating the repeatability of the labeled antibodies; as a control, CA72-4 antibody labeled with 0.1mol/L phosphate buffer (conventional labeling buffer) at pH 7.2 was used, and the statistical data and results are shown in Table 6.
TABLE 6 repeated detection of CA72-4 labeled antibody
Looking at table 6, the CV values of the conventional labeling buffers were all > 10%, while the CV values of the buffers formulated in the present application were all < 5%, and the reproducibility of the buffers formulated in the present application was significantly higher than that of the conventional labeling buffers.
In conclusion, the biotin labeling buffer solution is a buffer solution with low ion concentration, in the buffer solution, the antibody is more stable due to the ionic strength and the buffer environment, and the antibody and the biotin are more easily combined, so that the ratio of the biotin to the antibody used for labeling is lower, namely the biotin labeling of the antibody is higher by using the biotin and the antibody with lower doses. The repeatability of the biotin labeling buffer solution is higher than that of the conventional labeling buffer solution, and the biotin labeling buffer solution has the advantages of low background and high signal-to-noise ratio.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.