Rapid protein labeling kit

Abstract

The invention discloses a rapid protein labeling kit which comprises a freeze-drying agent, an initiator and a terminator, and the freeze-drying agent is obtained by dissolving a micromolecular to-be-labeled substance in a buffer solution and freeze-drying; the starter comprises the following components: an alkaline buffer solution with the pH value of 8.5-9.5 and the concentration of 0.2-0.5 M; 10%-20% of an organic solvent; 0.05% to 0.2% of a preservative; the terminator comprises the following components: 0.1 to 0.5 M of amino acid with free amino groups; 1%-5% of bovine serum albumin; and 0.05%-0.2% of a preservative. The kit is used for protein marking, the operation is simple, the marking efficiency is high, the stability of a marker is good, and the kit can be stored for a long time.

Description

Technical Field

[0001] The invention belongs to the technical field of medical in vitro diagnosis, and in particular relates to a protein rapid labeling kit. Background Art

[0002] In the in vitro diagnostic (IVD) industry, protein labeling technology, as one of the core key technologies, plays a vital role in improving the sensitivity, specificity, stability and reliability of diagnostic reagents. With the continuous advancement of medical diagnostic technology and the growing clinical needs, the requirements for protein labeling technology are becoming increasingly stringent and diverse.

[0003] First, from the perspective of the market demand for in vitro diagnostic reagents, rapid and accurate diagnostic results are an important basis for clinical decision-making. Traditional protein labeling methods, such as radioisotope labeling, although highly sensitive, have disadvantages such as radioactive contamination, complex operation, and high cost, which limit their widespread application. Although fluorescent labeling technology is intuitive and easy to detect, it is easily interfered by background fluorescence in high-sensitivity requirements and complex biological samples, affecting the accuracy of the test results.

[0004] As an emerging and highly promising protein labeling method, chemiluminescent labeling technology has gradually attracted the attention of the industry due to its unique advantages. Chemiluminescent labeling technology uses the covalent binding between small molecule luminescent substances (such as acridinium esters, isoluminol, etc.) and proteins to generate light signals through chemical reactions under specific conditions (such as alkaline environments), thereby achieving protein labeling and detection. This technology has the advantages of high sensitivity, low background interference, and easy automated detection. It is particularly suitable for the detection and quantitative analysis of low-concentration proteins.

[0005] However, the existing chemiluminescent labeling technology still faces many challenges in the development of in vitro diagnostic reagents. On the one hand, low labeling efficiency is one of the key factors restricting its application. Traditional labeling methods often require long reaction times and complex operating steps, resulting in low labeling efficiency and difficulty in meeting the needs of rapid detection. Complicated operating steps are also a risk point that causes batch-to-batch differences in labeling. On the other hand, nonspecific adsorption and stability of labeled products are also technical problems that need to be solved urgently. Nonspecific adsorption can lead to the generation of false positive signals, affecting the accuracy of the test results; and the poor stability of the labeled product will lead to signal attenuation and decreased detection sensitivity, affecting the storage and service life of the reagents. Summary of the invention

[0006] The present invention realizes rapid, efficient and stable labeling of proteins by optimizing the freeze-drying process of small molecule luminescent materials, designing reasonable formulations of initiators, stop solutions and stabilizers. Specifically, the present invention uses freeze-drying technology to process small molecule luminescent materials, effectively improving their stability and resolubility; at the same time, by optimizing the formulations of initiators and stop solutions, the labeling reaction time is shortened, and the entire operation does not require purification processes such as centrifugation and dialysis, which optimizes the process flow and reduces nonspecific adsorption; finally, the storage stability of the labeled product is further improved by adding stabilizers. These improvement measures not only simplify the operating steps and improve experimental efficiency, but also significantly improve the performance and stability of the labeled products, providing strong technical support for the development of key components of in vitro diagnostic reagents. Therefore, the first object of the present invention is to provide a protein rapid labeling kit. The second object of the present invention is to provide a method for using the protein rapid labeling kit.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] As a first embodiment of the present invention, a protein rapid labeling kit comprises a lyophilized agent, an initiator and a terminator.

[0009] The lyophilized agent is obtained by dissolving the small molecule to be labeled in a buffer solution, then filling the solution into a lyophilization bottle and lyophilizing the solution.

[0010] The starter comprises the following components:

[0011] Alkaline buffer, pH 8.5-9.5, 0.2M-0.5M;

[0012] Organic solvent, 10%-20%;

[0013] Preservatives, 0.05%-0.2%;

[0014] The terminator comprises the following components:

[0015] Amino acids with free amino groups, 0.1-0.5M;

[0016] Bovine serum albumin, 1%-5%;

[0017] Preservatives, 0.05%-0.2%.

[0018] According to the present invention, the protein rapid labeling kit further comprises a stabilizer, and the stabilizer comprises the following components:

[0019] Acidic buffer with pH 2.0-4.0, 0.2M-0.5M;

[0020] Glycerol, 80% (volume ratio);

[0021] EDTA, 1%-2%;

[0022] Organic solvent, 1%-5%;

[0023] Preservatives, 0.05%-0.2%.

[0024] According to the present invention, the small molecule to be labeled in the lyophilized agent is acridinium ester, acridinium sulfonamide, isoluminol or fluorescein.

[0025] According to the present invention, the buffer in the lyophilized agent comprises the following components:

[0026] Acidic buffer 0.05M;

[0027] Organic solvent, 50% (volume ratio);

[0028] Lyoprotectant, 0.1%-0.5%.

[0029] Furthermore, the acidic buffer solution is acetic acid, morphosulfonic acid, etc.

[0030] Furthermore, the organic solvent is dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and the like.

[0031] Furthermore, the lyophilization protective agent is sugars such as sucrose and trehalose.

[0032] According to the present invention, the alkaline buffer in the starter is a carbonate alkaline buffer or a borate buffer.

[0033] Furthermore, the organic solvent in the starter is dimethyl sulfoxide-DMSO or N,N-dimethylformamide-DMF or the like.

[0034] Furthermore, the preservative in the starter is P300, BRO (Bropol preservative) or PC950, etc.

[0035] According to the present invention, the amino acid with a free amino group in the terminator is arginine, lysine, and the like.

[0036] Furthermore, the preservative in the terminator is P300, BRO or PC950, etc.

[0037] According to the present invention, the acidic buffer in the stabilizer is acetic acid or morphosulfonic acid.

[0038] Furthermore, the organic solvent in the stabilizer is dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and the like.

[0039] Furthermore, the preservative in the stabilizer is P300, BRO or PC950.

[0040] As a second aspect of the present invention, a method for using the above-mentioned protein rapid labeling kit comprises the following steps:

[0041] Step 1: dilute the protein to be labeled with the initiator to 0.1-1 mg / ml, add it to the lyophilized agent, and react at room temperature for 15 minutes;

[0042] Step 2, take 100-300 μL of terminator and terminate the reaction for 5 minutes;

[0043] Step 3: Add a stabilizer equal to the total volume of the labeled protein, initiator and terminator. The mixture can be stored at -20°C for a long time.

[0044] The protein rapid labeling kit of the present invention has the beneficial effects of innovatively adopting a composite blocking process (acidic conditions, amino acids, bovine serum albumin), reducing background noise (S0) and improving sensitivity.

[0045] The protein rapid labeling kit of the present invention is used for labeling, and its beneficial effects are:

[0046] (1) Simple operation and short time: Protein labeling with this kit can generally be completed within 30 minutes. This kit does not require purification after labeling, so there is no need to perform tedious steps such as desalting and centrifugation. Due to the simple operation steps, there are fewer uncontrollable factors between batches, which greatly reduces the risk of batch differences.

[0047] (2) High labeling efficiency: Different reaction concentrations can be selected according to the characteristics of the protein to be labeled, and the luminescent substance can be added to the reaction system in solid form to increase the reaction molar concentration, improve the labeling efficiency, and achieve a high reactivity of the labeled product.

[0048] (3) Stability of markers: The reaction system contains dual stabilizers of protein and luminescent substance, which can be stored for a long time at low temperature. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0050] Example 1

[0051] 1. Lyophilized agent: prepare an acidic buffer solution with a pH of 2.0 and a concentration of 0.05M acetic acid, add an equal volume of dimethyl sulfoxide-DMSO and a mass ratio of 0.1% trehalose, mix well and set aside; use the lyophilized liquid to prepare 0.5mg / mL acridinium ester with succinimide functional group, dispense into lyophilized bottles, 0.02ml per bottle; freeze-dry in a lyophilizer and set aside;

[0052] 2. Starter: Prepare a borate buffer with a pH of 8.5 and a concentration of 0.2 M. Then, add dimethyl sulfoxide (DMSO) to a final concentration of 10% and P300 (E1A binding protein p300) to a final concentration of 0.05%. Dispense into bottles for later use;

[0053] 3. Terminator: prepare 0.1M lysine, 1% bovine serum albumin (BSA), 0.05% P300 aqueous solution, mix well and divide into portions for use;

[0054] 4. Stabilizer: prepare acetic acid buffer with a pH of 2.0 and a concentration of 0.2 M, add glycerol to a final concentration of 80% (volume ratio), 1% ethylenediaminetetraacetic acid (EDTA), 1% dimethyl sulfoxide-DMSO, 0.05% P300, mix well and divide into portions for use.

[0055] 5. Assembly: Take one bottle of lyophilized agent, one bottle of initiator, one bottle of terminator, one bottle of stabilizer, and one bottle of brown glass reaction bottle, put them into the packaging box, attach the instruction manual, and the protein rapid labeling kit is assembled.

[0056] Example 2

[0057] 1. Lyophilized agent: prepare pH 4.0, 0.05M morphosulfonic acid buffer, add equal volume of dimethyl sulfoxide-DMSO, 0.5% sucrose and mix thoroughly for later use; prepare 0.5mg / mL acridinium ester with succinimide functional group with lyophilized liquid, dispense into lyophilized bottles, 0.02ml per bottle; freeze-dry in a lyophilizer and set aside;

[0058] 2. Starter: Prepare pH 9.5, 0.2M carbonate buffer, add dimethyl sulfoxide-DMSO to a final concentration of 20% DMSO and 0.1% BRO. Dispense into bottles for later use;

[0059] 3. Terminator: prepare 0.1M arginine, 5% bovine serum albumin (BSA), and 0.1% BRO aqueous solution, mix well and divide into portions for use;

[0060] 4. Stabilizer: prepare pH 4.0, 0.5 M morphosulfonic acid buffer, add glycerol to a final concentration of 80%, 2% ethylenediaminetetraacetic acid (EDTA), 5% dimethyl sulfoxide-DMSO, 0.1% BRO, mix well and divide into portions for later use.

[0061] 5. Assembly: Take one bottle of lyophilized agent, one bottle of initiator, one bottle of terminator, one bottle of stabilizer, and one bottle of brown glass reaction bottle, put them into the packaging box, attach the instruction manual, and the protein rapid labeling kit is assembled.

[0062] Example 3

[0063] The starter in Example 1 does not contain DMSO, and the other components remain the same as those in Example 1.

[0064] Example 4

[0065] The terminator in Example 1 does not contain arginine, and other components remain the same as those in Example 1.

[0066] Example 5

[0067] In Example 1, no stabilizer is used, and other components remain the same as those in Example 1.

[0068] Example 6 Antibody labeling of the present invention

[0069] This example is to perform antibody labeling on the single-label rapid labeling kits of the above-mentioned examples 1, 2, 3, 4 and 5 to obtain CEA labeled antibodies.

[0070] 1. Take 20 μL of carcinoembryonic antigen 5 mg / ml (CEA) antibody and add it to the reaction bottle, add 180 μL of the starter, dilute the antibody to 0.5 mg / ml, mix well and set aside;

[0071] 2. Add the diluted antibody to the lyophilized agent, mix well and transfer to the reaction bottle, react at room temperature for 15 minutes;

[0072] 3. Add 100 μL of stop solution and react at room temperature for 5 minutes;

[0073] 4. Add 300 μL of stabilizer, mix thoroughly, and store at -20°C for later use.

[0074] Comparative Example 1 (Traditional Marking)

[0075] 1. Take 0.1 mg of CEA antibody, dilute to 0.5 mg / ml, and dialyze in carbonate buffer overnight.

[0076] 2. Dissolve acridinium ester in DMSO to 0.5 mg / ml, add to the antibody according to the proportion, and react for 1 hour;

[0077] 3. Add 10 μL ethanolamine to terminate the reaction for 30 min and dialyze overnight;

[0078] 4. Add an equal volume of triglyceride (total volume of steps 1, 2, and 3) and store at -20°C for later use.

[0079] Example 7 Result Detection

[0080] 1. Add the CEA labeled antibodies prepared according to the methods of Example 6 and Control Example 1 into the detection buffer at a ratio of 1:3000, mix well and set aside; wherein the formula of the detection buffer is as follows:

[0081] pH7.4 50mM PBS; 1% BSA; 0.1% Tween 20 and 0.1% P300.

[0082] 2. Use a fully automatic chemiluminescence analyzer to detect clinical sample reactivity, sensitivity, precision and other indicators;

[0083] 3. Long-term stability: The prepared labeled antibodies were tested for reactivity every 2 months to monitor long-term stability. The specific results are shown in Tables 1, 2 and 3.

[0084] Table 1 Comparison of reactivity

[0085]

[0086] The results in Table 1 show:

[0087] It can be seen that the CEA labeled antibodies labeled with Examples 1 and 2 have a 3-fold increase in low-value sensitivity compared to the (S1 / S0) of the CEA labeled antibody labeled with Example 3, and have significantly improved high-value reactivity and linear range;

[0088] The reactivity and sensitivity of the CEA-labeled antibody labeled with Example 4 were significantly inferior to those of the CEA-labeled antibodies labeled with Examples 1 and 2.

[0089] The overall reactivity of the CEA-labeled antibody labeled with Example 5 is consistent with that of the CEA-labeled antibody labeled with Examples 1 and 2, and the high-value reactivity and linear range are significantly improved, but the low-value sensitivity (S1 / S0) is significantly weaker than that of the CEA-labeled antibody labeled with Examples 1 and 2.

[0090] Conclusion: The markers prepared by the rapid labeling kits of Example 1 and Example 2 innovatively adopted a composite blocking process (acidic conditions, amino acids, bovine serum albumin), which reduced background noise (S0) and improved sensitivity.

[0091] Table 2 Precision comparison table

[0092]

[0093] The results in Table 2 show:

[0094] Compared with the traditional marking method, the rapid marking process of Example 6 also improves the precision.

[0095] Table 3 Stability comparison table

[0096]

[0097] The results show:

[0098] The long-term stability of the CEA labeled antibody labeled with Examples 1 and 2 is significantly better than that of the CEA labeled antibody with Control Group 1. Similarly, due to the lack of stabilizer in the protein rapid labeling kit of Example 5, the real-time stability of the CEA labeled antibody labeled with Example 5 is significantly worse than that of the CEA labeled antibody labeled with Examples 1 and 2.

[0099] Conclusion: 1) The rapid protein labeling kit prepared in Example 1 and Example 2 can significantly shorten the time for antibody labeling and detection from 3 days in the traditional process to 30 minutes; and can reduce the equipment and consumables required for purification and temperature control during the operation, so that protein labeling is not restricted by the site.

[0100] 2) By optimizing and adjusting the formula of freeze-drying buffer, initiator, terminator and preservative, the synergistic effect of each component can solve the problems of strong non-specific adsorption and poor real-time stability that occur in other labeling processes;

[0101] 3) The sensitivity and linear range of the markers prepared using this process are significantly better than those prepared using traditional labeling processes.

[0102] In summary, the protein rapid labeling kit of the present invention can improve the real-time stability of the labeled product through the synergistic effect of its components. When using this kit for protein labeling, there is no need to perform additional purification steps on small molecule labeled proteins, and the problem of nonspecific adsorption can be avoided. Therefore, the use of the protein rapid labeling kit of the present invention not only optimizes the process flow, but also significantly shortens the reaction time, and reduces the equipment and consumables required in the labeling process. It can be seen that the present invention shows a wide range of application prospects and important practical significance in the field of in vitro diagnostic reagent development, which not only solves many problems in the prior art, but also promotes the innovation and development of in vitro diagnostic technology.

[0103] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A protein rapid labeling kit, characterized in that: Including lyophilized agent, starter and stopper, The lyophilized agent is obtained by dissolving the small molecule to be labeled in a buffer solution and lyophilizing; The starter comprises the following components: Alkaline buffer, pH 8.5-9.5, 0.2M-0.5M; Organic solvent, 10%-20%; Preservatives, 0.05%-0.2%; The terminator comprises the following components: Amino acids with free amino groups, 0.1-0.5M; Bovine serum albumin, 1%-5%; Preservatives, 0.05%-0.2%.

2. The protein rapid labeling kit according to claim 1, characterized in that: Also included is a stabilizer, which includes the following components: Acidic buffer with pH 2.0-4.0, 0.2M-0.5M; Glycerol, 80%; EDTA, 1%-2%; Organic solvent, 1%-5%; Preservatives, 0.05%-0.2%.

3. The protein rapid labeling kit according to claim 1, characterized in that: The small molecule to be labeled in the lyophilized agent is acridinium ester, acridinium sulfonamide, isoluminol or fluorescein.

4. The protein rapid labeling kit according to claim 1 or 3, characterized in that: The buffer in the lyophilized agent comprises the following components: Acidic buffer 0.05M; Organic solvent, 50%; Lyoprotectant, 0.1%-0.5%.

5. The protein rapid labeling kit according to claim 4, characterized in that: The acidic buffer is acetic acid or morphosulfonic acid; the organic solvent is dimethyl sulfoxide or N,N-dimethylformamide.

6. The protein rapid labeling kit according to claim 4, characterized in that: The freeze-drying protective agent is sucrose or trehalose.

7. The protein rapid labeling kit according to claim 2, characterized in that: The alkaline buffer in the initiator is a carbonate alkaline buffer or a borate buffer; the acidic buffer in the stabilizer is acetic acid or morphosulfonic acid.

8. The protein rapid labeling kit according to claim 2, characterized in that: The preservatives in the initiator, terminator and stabilizer are P300, BRO or PC950.

9. The protein rapid labeling kit according to claim 1, characterized in that: The amino acid with a free amino group in the terminator is arginine or lysine.

10. The protein rapid labeling kit according to claim 2, characterized in that: The organic solvent in the starter and stabilizer is dimethyl sulfoxide or N,N-dimethylformamide.

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