A labeling method for high concentration colloidal gold

Abstract

The present invention discloses a method for labeling high-concentration colloidal gold. Buffer solutions of different pH values ​​are taken to dilute high-concentration colloidal gold solutions respectively to form colloidal gold solutions of different pH values. Proteins to be labeled are added to each colloidal gold solution respectively, and then the optimal labeling pH is determined by a high-salt destruction test. A labeling buffer solution of the optimal labeling pH is taken to dilute the high-concentration colloidal gold solution, and then the solution is divided into several equal parts, and protein solutions to be labeled are added respectively to form a mixed solution of markers with different final protein concentrations, and then the optimal protein labeling amount is determined by a high-salt destruction test. A mixed solution of markers is prepared according to the conditions for determining the optimal labeling and the pH optimal protein labeling amount, and then a blocking reaction is performed to obtain a colloidal gold labeled protein complex. In the labeling process of the present invention, gold particles do not aggregate throughout the entire process, the reaction system has a small volume, 60% of labor is saved, protein materials are saved, and in centrifugal purification, large-scale production can be completed without investing too much large-scale equipment.

Description

Technical Field

[0001] The invention relates to an immunogold labeling method, in particular to a high-concentration colloidal gold labeling method. Background Art

[0002] At present, there are few high-concentration colloidal gold products on the market, and there are few studies on the corresponding methods of labeling proteins. Usually, the experimenters still dilute the high-concentration colloidal gold to the conventional concentration, such as diluting 100OD colloidal gold to 1OD, and then adjust the pH before coupling with the protein. However, the conventional labeling process cannot maximize the advantages of high-concentration gold, and will increase the working time and reduce the working efficiency to a certain extent.

[0003] 1. High concentration of colloidal gold is more sensitive to salt concentration. In the process of labeling proteins, due to the high concentration of colloidal gold, conventional pH test paper cannot correctly detect the appropriate pH. In addition, the adsorption of colloidal gold can easily damage the probe of the pH reagent. Conventional labeling processes are difficult to adjust the appropriate labeling buffer.

[0004] 2. As far as conventional technology is concerned, the labeling process of low-concentration colloidal gold will increase the workload. For example, if 1L of 100OD colloidal gold is diluted to a conventional concentration, the volume will be enlarged to 100L. For production companies, the processing of 100L colloidal gold requires supporting large-scale centrifugal equipment, which requires at least 3 to 5 staff members to cooperate in the operation. Accordingly, energy loss, labor cost, material cost, equipment depreciation, and delivery cycle will all be affected.

[0005] 3. Conventional labeling processes for a large number of antibodies often involve determining a process to label antibodies or antigens from different manufacturers, sources, and batches. However, this often results in a large workload, and even the most suitable antibody cannot be screened out due to limitations in labeling conditions, resulting in low work efficiency. Summary of the invention

[0006] The purpose of the present invention is to provide a high-concentration colloidal gold labeling method, aiming to create a new process, adapt to the characteristics of high-concentration colloidal gold, develop the advantages of high-concentration colloidal gold, meet the needs of low-energy consumption and low-cost mass production, and at the same time be more flexible and simple, meet market demand, and be suitable for promotion.

[0007] The present invention is achieved through the following technical solution: A high-concentration colloidal gold labeling method comprises the following steps:

[0008] (1) Determining the optimal labeling pH: taking buffer solutions of different pH values ​​to dilute colloidal gold solutions of concentration not less than 100 OD respectively, to form colloidal gold solutions of different pH values, respectively adding the protein to be labeled to each colloidal gold solution, mixing, and reacting to obtain a labeling mixed solution A of buffer solutions of different pH values, diluting each labeling mixed solution A with pure water to a final colloidal gold concentration of 1 OD, and then adding sodium chloride solution to a final sodium chloride concentration of 1%. After the salt destruction reaction is completed, the color of the colloidal gold does not change, or the color changes the least, which is the optimal labeling pH, and the corresponding buffer solution is the optimal labeling buffer solution;

[0009] (2) Determine the optimal protein labeling amount: dilute the colloidal gold solution with a concentration of not less than 100 OD using the determined optimal labeling buffer, then divide the solution into several equal parts, add the protein solution to be labeled respectively, to form a labeling mixed solution B with different final protein concentrations, dilute each labeling mixed solution B with pure water to a final colloidal gold concentration of 1 OD, then add sodium chloride solution to a final sodium chloride concentration of 1%. After the salt destruction reaction is completed, the protein concentration with no change in the color of the colloidal gold or the smallest change is the optimal protein labeling amount;

[0010] (3) To determine the optimal labeling pH and the optimal antibody labeling amount, take the most appropriate labeling buffer and protein to prepare the protein diluent, then add it to a colloidal gold solution with a concentration of not less than 100 OD, react to obtain a labeling mixture C, and then add a blocking agent to perform a blocking reaction to obtain a colloidal gold-labeled protein complex.

[0011] In the present invention, the protein to be labeled can be an antigen, an antibody or other protein molecules.

[0012] In the present invention, the buffer is citric acid-trisodium citrate buffer, phosphate buffer (PB), boric acid-borax buffer or carbonate buffer, etc. In step (1), the buffer can be the same or different, and can be selected according to actual conditions. The buffer concentration is 0.01M.

[0013] In step (1), when the colloidal gold concentration is between 200 and 100 OD, the concentration of the colloidal gold solution at different pH values ​​is within the range of 10-60 OD, otherwise the buffer volume ratio is low, resulting in a decrease in buffer capacity.

[0014] In step (1), the marker mixed solution A is shaken at room temperature for 15-120 minutes, and then diluted with pure water. The salt destruction reaction time is 1-30 minutes.

[0015] In step (2), the optimal labeling buffer dilutes the colloidal gold solution having a concentration of not less than 100 OD to 10-60 OD.

[0016] In step (2), the marker mixed solution B is shaken and reacted at room temperature for 15-120 minutes, and then diluted with pure water. The salt destruction reaction time is 1-30 minutes.

[0017] In step (3), the marker mixed solution C is shaken at room temperature for 15-120 minutes, and the blocking reaction time is 15-60 minutes.

[0018] In step (3), the blocking agent is a macromolecular substance, such as bovine serum albumin (BSA), casein, polypropylene glycol (PEG), polyvinyl pyrrolidone (PVP), etc.

[0019] The present invention also includes step (4), namely purifying the colloidal gold labeled protein complex, and in the present invention, centrifugal purification is mainly performed using a centrifuge.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The method provided by the present invention can quickly label proteins such as antigens and antibodies on the surface of high-concentration colloidal gold. First, the buffer solution with the most suitable labeling pH is determined by a high-salt destruction test, and then the colloidal gold labeled protein is configured using the buffer solution with the most suitable labeling pH. After the high-salt destruction reaction, the most suitable labeling protein concentration is determined, and then the final labeled protein preparation is performed based on the buffer solution with the most suitable labeling pH and the most suitable labeling protein concentration. Through the method provided by the present invention, the gold particles do not aggregate throughout the high-concentration colloidal gold labeling process, the reaction system is small in volume, 60% of labor is saved, and up to 80% of protein materials can be saved. Even in the centrifugal purification process, large-scale production can be completed without investing too much large-scale equipment.

[0022] 2. The method provided by the present invention can complete the labeling of colloidal gold and protein under the condition of high concentration of colloidal gold, fully develops and utilizes the advantages of high concentration of colloidal gold, reduces the labeling time and the amount of labeled protein, does not require the investment of large-scale centrifugal equipment, reduces energy loss, and can save production cycle. At the same time, the performance of colloidal gold labeled protein is increased, and the method is simple to operate and convenient to promote.

[0023] 3. The method provided by the present invention can achieve accurate optimal pH determination without using a pH meter probe, while also improving the sensitivity of antigen-antibody reaction detection.

[0024] 4. The screening conditions of the method provided by the present invention are flexible and fast, and are more conducive to high-throughput and high-precision determination of the most suitable antigen or antibody in the field of lateral chromatography.

[0025] 5. The method provided by the present invention has a relatively simple operation process. Compared with the conventional low-concentration labeling method, it does not increase the complexity of the operation. In addition, during the protein labeling process, it can more accurately locate the optimal labeling pH, facilitate subsequent expansion of production, and reduce the debugging process.

[0026] 6. The present invention optimizes the flexibility of the labeling method. It can flexibly change the labeling buffer or labeling amount for antigens or antibodies from different batches from different manufacturers or even from different batches from the same manufacturer, so as to determine whether the living material is the most suitable in real time under the best conditions and reduce the omission of the most suitable living material due to mismatch of labeling conditions.

[0027] 7. The method provided by the present invention uses high-concentration colloidal gold for labeling, which shortens the delivery cycle by 30% during the production process, and has a small batch difference, can reduce the investment in large-scale centrifugal equipment, and reduce energy consumption. It is especially worthy of promotion for large-scale production.

[0028] The method of the present invention has shown significant advantages in the actual batch production process. For example, the production of 2 million doses only requires 1L of 100OD high-concentration colloidal gold. With the labeling method of the present invention, only one R&D staff and a 50ml desktop centrifuge per well are needed to complete the labeling work in 3 hours. Compared with conventional labeling work, the efficiency is greatly improved:

[0029]

[0030] 8. The method provided by the present invention improves the labeling efficiency and production efficiency, and at the same time, the performance is improved due to the use of high-concentration colloidal gold labeling sensitivity and specificity liquid. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] In the present invention, the concentration of colloidal gold is expressed in OD, and a colloidal gold concentration of 100 OD contains 1% chloroauric acid.

[0033] Example 1 High concentration colloidal gold and antibody labeling method

[0034] 1. Determine the Optimal pH Buffer for Labeling

[0035] (1) Select 0.01 M PB buffer and prepare PB buffers with three different pH values: pH 6.2, pH 7.4, and pH 8.0.

[0036] (2) Take three 40ul tubes of 100OD high-concentration colloidal gold and dilute them to 10-60OD with the above buffer respectively, and oscillate and mix at room temperature.

[0037] (3) Add the antibody to be labeled to a final concentration of 20 to 200 μg / ml and react at room temperature for 20 to 120 minutes.

[0038] (4) Take 25ul of each colloidal gold-antibody mixture in the above different pH buffers, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the colloidal gold-antibody mixture dilution after 1 to 60 minutes. The color does not change, or the change is minimal, which is the most suitable labeling pH buffer, buffer A.

[0039] 2. Determination of the optimal antibody labeling amount

[0040] (1) Take 400ul 100OD high concentration colloidal gold solution, add buffer A, dilute the colloidal gold solution to 10OD~60OD, divide it into at least 6 equal parts, add different volumes of antibody solution respectively, the final concentration of the antibody shows a certain regularity, which can be 100ug / ml, 80ug / ml, 60ug / ml, 40ug / ml, 20ug / ml, 10ug / ml, etc., shake and react at room temperature for 20min~120min, and obtain antibody-colloidal gold mixture solutions of different concentrations, that is, marker mixture B of different concentrations.

[0041] (2) Take 25ul of each of the above antibody-colloidal gold mixture solutions of different concentrations, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the colloidal gold-antibody mixture dilution after 1 to 60 minutes. The antibody concentration that does not change color or changes the least is the optimal antibody labeling amount.

[0042] 3. Blocking of Colloidal Gold-Antibody Complex

[0043] Based on the determined optimal labeling pH and optimal antibody labeling conditions, prepare a protein diluent with buffer A and antibody, then add it to a colloidal gold solution with a concentration of not less than 100 OD, react for 15 to 120 minutes to obtain a labeling mixture C, and then add a BSA solution with a final concentration of 0.2% to 5%, react for 15 minutes to 60 minutes to complete the blocking reaction.

[0044] 4. Purification of Colloidal Gold-Antibody Complex

[0045] (1) The reaction system is sealed and purified by centrifugation. Centrifugal force: 4660 x g, time: 5 min to 60 min.

[0046] (2) After centrifugation, remove the supernatant and replace it with colloidal gold re-solution to resuspend the colloidal gold-antibody labeled complex.

[0047] The colloidal gold complex solution should contain a buffer, macromolecular protein, a sugar surfactant, a preservative, and the like.

[0048] Example 2 High-concentration colloidal gold and antibody labeling method

[0049] The difference from the first embodiment is that:

[0050] 1. Determination of the optimal labeling pH

[0051] (1) Select 0.01 M BB buffer and prepare three buffers of pH 8.0, pH 8.5, and pH 9.0.

[0052] (2) Take three 40ul tubes of 100OD high-concentration colloidal gold and dilute them to 10-60OD with the above buffer respectively, and oscillate and mix at room temperature.

[0053] 3. Blocking of Colloidal Gold-Antibody Complex

[0054] Add Casein solution with a final concentration of 0.2% to 5% to the marker mixture C and react for 15 minutes to 60 minutes to complete the blocking reaction.

[0055] Example 3 High concentration colloidal gold and antibody labeling method

[0056] The difference from the second embodiment is that:

[0057] 1. Determination of the optimal labeling pH

[0058] (1) Select 0.01M pH 8.5 BB buffer, 0.01M pH 7.6 PB buffer, or 0.01M pH 6.0 citric acid-trisodium citrate buffer.

[0059] Example 4 High concentration colloidal gold and antigen labeling method

[0060] 1. Determine the Optimal pH Buffer for Labeling

[0061] (1) Select 0.01M pH 8.5 BB buffer, 0.01M pH 7.6 PB buffer, or 0.01M pH 6.0 citric acid-trisodium citrate buffer.

[0062] (2) Take three 400ul tubes of 100OD high concentration colloidal gold and dilute them to 10-60OD respectively with the above buffer.

[0063] (3) Add different volumes of the antigen solution to be labeled, with a final concentration of 10 ug / ml to 400 ug / ml, and shake the reaction at room temperature for 20 to 120 min.

[0064] (4) Take 25ul of each colloidal gold-antibody mixture in the above different pH buffers, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the colloidal gold-antibody mixture dilution after 1 to 60 minutes. The color does not change, or the change is minimal, which is the most suitable labeling pH buffer, buffer A.

[0065] 2. Determination of the optimal antibody labeling amount

[0066] (1) Take 400ul 100OD high concentration colloidal gold solution, add buffer A, dilute the colloidal gold solution to 10OD~60OD, divide it into several equal parts according to the needs of the experiment, add different volumes of antigen solution respectively, the final concentration of antigen is 10ug / ml~400ug / ml, the specific final concentration can be designed according to the needs of the experiment, specifically refer to Example 1, shake and react at room temperature for 20min~120min, and obtain antigen-colloidal gold mixture solutions of different concentrations, that is, marker mixture B of different concentrations.

[0067] (2) Take 25ul of each of the above different concentrations of antigen-colloidal gold mixture solutions, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the antigen-colloidal gold mixture dilution after 1 to 60 minutes. The antigen concentration that does not change color or changes the least is the optimal antigen labeling amount.

[0068] 3. Blocking of Antigen-Colloidal Gold Complex

[0069] Based on the determined optimal labeling pH and optimal antibody labeling conditions, prepare a protein diluent with buffer A and antibody, then add it to a colloidal gold solution with a concentration of not less than 100 OD, react for 15 to 120 minutes to obtain a labeling mixture C, and then add a BSA solution with a final concentration of 0.2% to 5%, react for 15 minutes to 60 minutes to complete the blocking reaction.

[0070] Example 5 Method for labeling procalcitonin antibody with high concentration colloidal gold

[0071] 1. Determination of the optimal labeling pH

[0072] (1) Select 0.01 M PB buffer and prepare three buffers of pH 6.2, pH 7.4, and pH 8.0.

[0073] (2) Take three 40ul vials of 100 OD high-concentration colloidal gold and dilute them to 40 OD with the above buffer respectively. Mix by shaking at room temperature.

[0074] (3) Add the procalcitonin antibody to be labeled, the final concentration can be 60ug / ml, and react at room temperature for 40 minutes to obtain colloidal gold-antibody mixtures in different pH buffers.

[0075] (4) Take 25ul of colloidal gold-antibody mixture in different pH buffer solutions, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the colloidal gold-antibody mixture dilution after 10 minutes.

[0076] (5) The color hardly changes, or changes minimally, which is the optimal labeling pH. In this experiment, the optimal labeling pH is 0.01M pH7.4 PB buffer.

[0077] 2. Determination of the Optimal Labeling Amount

[0078] (1) Take 40ul of 100 OD high-concentration colloidal gold from three tubes and dilute them to 40OD with 0.01M pH 7.4 PB buffer. Mix by shaking at room temperature.

[0079] (2) Add different concentrations of PCT (procalcitonin) antibody, with final concentrations of 40ug / ml, 50ug / ml, and 60ug / ml, respectively, and react at room temperature for 40 minutes.

[0080] (3) Take 25ul of colloidal gold-antibody mixture with different antibody concentrations, dilute to 1OD with 975ul purified water, add 100ul 10% NaCl solution, shake and mix, and observe the color of the colloidal gold-antibody mixture dilution after 10 minutes.

[0081] (2) When the color hardly changes or changes minimally, it is the optimal antibody amount. In this experiment, the optimal labeled antibody amount is 50ug / ml.

[0082] (4) According to the optimal labeling pH, 400ul of 100OD high concentration colloidal gold was diluted to 1ml, and further according to the optimal labeling amount, an appropriate amount of PCT (procalcitonin) antibody was added. After 40 minutes of antibody labeling, 50ul of 10% BSA aqueous solution was added. Furthermore, the final concentration of BSA was 0.5%, and the reaction was shaken at room temperature for 20 minutes.

[0083] (5) Centrifuge at a centrifugal force of 4660 x g for 10 min, remove the supernatant, and add 1 ml of colloidal gold reconstitution solution to reconstitute the colloidal gold labeled complex. The formula of the colloidal gold reconstitution solution contains: Tris buffer, BSA, sugar, surfactant and preservative.

[0084] (6) Using a gold-labeled three-dimensional gold dot film spraying instrument and setting the parameters to 4ul / cm, the 11 mixed labeling compounds were sprayed onto polyester fiber DL42 and cut into 7mm*300mm pieces for later use, which are called labeling compound pads.

[0085] (7) Using a gold-labeled three-dimensional gold dot membrane sprayer, another procalcitonin monoclonal antibody and goat anti-mouse IgG were coated on a nitrocellulose membrane at a parameter of 1 μl / cm and dried at 37°C for 16 hours to serve as a reaction membrane.

[0086] (8) The glass fiber was soaked in a 0.1 M PBS solution containing 0.5% Tween-20, and then dried at 37°C for 16 hours. The glass fiber was cut into pieces of 17 mm × 300 mm as a sample pad.

[0087] (9) The sample pad, fluorescent nanosphere labeled composite pad, reaction membrane and absorbent paper (22 mm × 300 mm) were adhered and assembled on a PVC base plate in a layered manner, and cut into strips to serve as procalcitonin assay test strips.

[0088] (10) Detection: Place the test strip into a suitable cartridge to make a complete test strip. Add 40-100ul of the sample to be tested into the sample well and react for 15 minutes. If the C line shows a normal red band, it is a valid test. Observe the color band of the T line. If there is no color band on the T line, the test result is negative. If there is a band, it is judged as weak positive, strong positive, etc. according to the strength of the band.

[0089] Interpretation results:

[0090]

[0091] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for labeling high-concentration colloidal gold, characterized in that: The following steps are involved: (1) Determining the optimal labeling pH: taking buffer solutions of different pH values ​​to dilute colloidal gold solutions of concentration not less than 100 OD respectively, forming colloidal gold solutions of different pH values ​​in the range of 10-60 OD, adding the protein to be labeled to each colloidal gold solution, mixing, and reacting to obtain a mixed labeling solution A of buffer solutions of different pH values, diluting each mixed labeling solution A with pure water to a final colloidal gold concentration of 1 OD, and then adding sodium chloride solution to a final sodium chloride concentration of 1%. After the salt destruction reaction is completed, the color of the colloidal gold does not change, or changes minimally, which is the optimal labeling pH, and the corresponding buffer solution is the optimal labeling buffer solution; (2) Determine the optimal protein labeling amount: dilute the colloidal gold solution with a concentration of not less than 100 OD to 10-60 OD using the determined optimal labeling buffer, then divide the solution into several equal parts, add the protein solution to be labeled respectively, to form a labeling mixed solution B with different final protein concentrations, dilute each labeling mixed solution B with pure water to a final colloidal gold concentration of 1 OD, then add sodium chloride solution to a final sodium chloride concentration of 1%. After the salt destruction reaction is completed, the protein concentration with no change in the color of the colloidal gold or the smallest change is the optimal protein labeling amount; (3) To determine the optimal labeling pH and the optimal protein labeling amount, take the most appropriate labeling buffer and protein to prepare the protein diluent, then add it to a colloidal gold solution with a concentration of not less than 100 OD, react to obtain a labeling mixture C, and then add a blocking agent to perform a blocking reaction to obtain a colloidal gold-labeled protein complex.

2. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: The protein to be labeled is an antigen, an antibody or other protein molecules.

3. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: The buffer is BB buffer, citric acid-trisodium citrate buffer, phosphate buffer, boric acid-borax buffer or carbonate buffer.

4. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: In the step (1), the marker mixed solution A is oscillated at room temperature for 15-120 minutes, and then diluted with pure water; the salt destruction reaction time is 1-30 minutes.

5. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: In the step (2), the marker mixed solution B is oscillated at room temperature for 15-120 minutes, and then diluted with pure water; the salt destruction reaction time is 1-30 minutes.

6. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: In the step (3), the marker mixed solution C is shaken at room temperature for 15-120 minutes, and the blocking reaction time is 15-60 minutes.

7. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: In the step (3), the blocking agent is bovine serum albumin, casein, polypropylene glycol or polyvinyl pyrrolidone.

8. The method for labeling high concentration colloidal gold according to claim 1, characterized in that: The method further comprises a step (4), namely, purifying the colloidal gold labeled protein complex.

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