A method for preparing an immunocolloidal gold calibration card suitable for POCT equipment

Abstract

The invention discloses a method for preparing an immunocolloidal gold calibration card suitable for POCT equipment, comprising the following steps: preparing a colloidal gold solution by a trisodium citrate reduction method; adding an antibody to the colloidal gold solution to obtain a colloidal gold labeled antibody; and obtaining an immunocolloidal gold calibration card by streaking the colloidal gold labeled antibody. The preparation and application of the immunocolloidal gold calibration card of the invention eliminates the difference in measured values ​​between instruments caused by components and assembly of optical path modules of various manufacturers, realizes the comparability of the detection results of the colloidal gold POCT instrument, and improves and ensures the accuracy of the instrument detection results.

Description

Technical Field

[0001] The invention belongs to the field of medical diagnosis, and in particular relates to a method for preparing an immune colloidal gold calibration card suitable for POCT equipment. Background Art

[0002] Colloidal gold is a uniform and stable gold particle polymerized by chloroauric acid (HAuCl4) in the presence of reducing agents such as white phosphorus, ascorbic acid, sodium citrate, tannic acid, etc. It has a strong protein adsorption function and carries a negative charge in a weakly alkaline environment. Colloidal gold can form a strong bond with the positively charged groups of protein molecules. Based on some physical properties of colloidal gold, such as high electron density, particle size, shape and color reaction, and the immune and biological characteristics of the conjugate, colloidal gold is widely used in immunology, histology, pathology and cell biology.

[0003] At present, the colloidal gold POCT instrument optical path calibration method commonly used by enterprises is to use the company's supporting reagents, drip them into the immune colloidal gold reagent strips, wait for the display board to be completed, measure the voltage values ​​on the standard machine and the machine to be tested respectively, calculate the calibration coefficient by automatically fitting the curve, and complete the optical path calibration. With the continuous growth of instrument production and the number of market instrument maintenance, the disadvantages exposed by this method are becoming more and more obvious. The reagent-based calibration method has a long display board time (waiting at least 10 minutes), a short service life (only maintains stability for 10 minutes), requires supporting reagents and equipment, and the calibration steps are cumbersome, which is not conducive to the calibration and maintenance of mass-produced instruments and market instruments. Summary of the invention

[0004] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for preparing an immunocolloidal gold calibration card suitable for POCT equipment, so as to solve the problems of cumbersome operation and difficult maintenance of the calibration steps in the prior art.

[0005] To solve the above problems, the present invention adopts the following solutions:

[0006] A method for preparing an immunocolloidal gold calibration card comprises the following steps:

[0007] The colloidal gold solution was prepared by trisodium citrate reduction method;

[0008] adding the antibody to the colloidal gold solution to obtain the colloidal gold labeled antibody;

[0009] The immunocolloidal gold calibration card was obtained by streaking with colloidal gold labeled antibody.

[0010] Further, the colloidal gold solution is prepared by a trisodium citrate reduction method, comprising:

[0011] The trisodium citrate solution is added to the chloroauric acid solution under heating, reflux and stirring conditions to react to obtain a colloidal gold solution.

[0012] Furthermore, the volume ratio of the trisodium citrate solution to the chloroauric acid solution is 1-2:100; the mass concentration of the chloroauric acid solution is 0.01%; and the mass concentration of the trisodium citrate solution is 1%.

[0013] Further, adding the antibody to the colloidal gold solution to obtain the colloidal gold labeled antibody comprises:

[0014] Adjust the pH of the colloidal gold solution to 6-7;

[0015] Add the antibody dropwise into the colloidal gold solution under stirring at room temperature;

[0016] A BSA stabilizer is added to the colloidal gold solution after the antibody is added to obtain a colloidal gold labeled antibody.

[0017] Furthermore, the amount of the antibody in the colloidal gold solution is 5-100 uL / mL; the concentration of the BSA stabilizer in the colloidal gold solution is 10-100 μL / mL.

[0018] Furthermore, the concentration of the BSA stabilizer is 10%.

[0019] Furthermore, the antibody is added dropwise to the colloidal gold solution, and stirred at room temperature for 10-30 min. A BSA stabilizer is added to the colloidal gold solution after the antibody is added, and stirring is continued at room temperature for 20-30 min to obtain a colloidal gold labeled antibody.

[0020] Furthermore, the antibody is diluted with double distilled water or PBS to a concentration of 1 mg / mL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The immunocolloidal gold calibration card prepared by the present invention can meet the application of optical path calibration of colloidal gold POCT instruments; high precision and repeatability ensure the stability of continuous detection between different instruments, and achieve efficient use; long storage period can achieve stability of detection in a long-term storage state, reduce production cycle, and extend market use time; small volume is convenient for carrying, and improves the convenience of calibration application; use without reagents can eliminate the influence of reagent stability on measured values, simplify calibration steps, shorten calibration time, and improve calibration efficiency; simple preparation method and common raw materials can also meet the needs of multiple companies to produce calibration cards adapted for their own instruments, which is conducive to industry norms and unified calibration standards of colloidal gold POCT instruments, and has broad application prospects. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] A method for preparing an immunocolloidal gold calibration card comprises the following steps:

[0025] (1) Colloidal gold synthesis

[0026] The invention adopts a trisodium citrate reduction method to prepare a colloidal gold solution, wherein 100 mL of a 0.01% chloroauric acid solution is added into a cleaned and dried 250 mL beaker, heated to boiling under stirring conditions of a magnetic stirrer, kept in a boiling state for 2 minutes, 1-2.0 mL of a 1% trisodium citrate solution is added, the solution is heated and boiled for 5-15 minutes, cooled to room temperature, restored to the original volume (100 mL) with distilled water, and stored at 4°C for later use.

[0027] (2) Preparation of colloidal gold-labeled antibodies

[0028] The prepared colloidal gold solution was adjusted to pH 6-7 with 0.2 mol / L K2CO3, the antibody was diluted with double distilled water or PBS to a concentration of 1 mg / mL, and then added dropwise to the colloidal gold solution under stirring at room temperature to make the amount of antibody in the colloidal gold solution 5-100 uL / mL, and stirring was maintained for 10-30 min. 10% BSA stabilizer was added to make the concentration of BSA stabilizer in the colloidal gold solution 10-100 μL / mL, and stirring was continued at room temperature for 20-30 min. Finally, centrifuged at 4°C, 4000 r / min for 30 min, and the precipitate was resuspended in 0.2 M phosphate buffer (containing 0.1%-1% Tween-80), centrifuged twice, and concentrated for later use.

[0029] (3) Calibration card marking process

[0030] The colloidal gold-labeled antibody solution was diluted 1-10 times with 0.01 mol / L PB, and the nitrocellulose membrane was streaked with gold dots using an automatic streaking instrument. The quality control line was the colloidal gold-labeled antibody solution diluted 2 times, and the detection lines were the colloidal gold-labeled antibody solutions diluted 1 times, 5 times, and 10 times, respectively. The streaked reagent strips were placed in an oven at 37°C for drying, and then cut and packaged for later use.

[0031] Example 1

[0032] (1) Synthesis of colloidal gold: Using the trisodium citrate reduction method, add 100 mL of 0.01% chloroauric acid solution into a cleaned and dried 250 mL beaker, heat to boiling under stirring with a magnetic stirrer, keep boiling for 2 min, then add 1 mL of 1% trisodium citrate solution, continue heating and boiling for 5 min, cool to room temperature, restore to the original volume with distilled water, and store at 4°C for later use.

[0033] (2) Preparation of colloidal gold-labeled antibodies: Adjust the pH of the prepared colloidal gold solution to 6 using 0.2 mol / L K2CO3, dilute the antibody to a concentration of 1 mg / mL with double distilled water or PBS, and then add it dropwise to the colloidal gold solution under stirring at room temperature to make the amount of antibody in the colloidal gold solution 5 uL / mL. Keep stirring for 10 min, add 10% BSA stabilizer to make the concentration of BSA stabilizer in the colloidal gold solution 10 μL / mL, continue stirring at room temperature for 20 min, and finally, centrifuge at 4°C, 4000 r / min for 30 min, resuspend the precipitate in 0.2 M phosphate buffer (containing 0.1% Tween-80), repeat centrifugation twice, and concentrate for later use.

[0034] (3) Calibration card marking process

[0035] The gold-labeled antibody solution was diluted 1, 2, 5, and 10 times with 0.01 mol / L pH PB, and the nitrocellulose membrane was streaked with gold using an automatic streaking instrument. The quality control line was the colloidal gold-labeled antibody solution diluted 2 times, and the detection lines were the colloidal gold-labeled antibody solution diluted 1, 5, and 10 times, respectively. The streaked reagent strips were placed in an oven at 37°C for drying, and then cut and packaged for later use.

[0036] The accuracy of the immunocolloidal gold calibration card obtained by the above method is 2.4%, the repeatability is 1.6%, and the service life is more than half a year.

[0037] Example 2

[0038] (1) Synthesis of colloidal gold: Using the trisodium citrate reduction method, add 100 mL of 0.01% chloroauric acid solution into a cleaned and dried 250 mL beaker, heat to boiling under stirring with a magnetic stirrer, keep boiling for 2 min, then add 1.5 mL of 1% trisodium citrate solution, continue heating and boiling for 10 min, cool to room temperature, restore to the original volume with distilled water, and store at 4°C for later use.

[0039] (2) Preparation of colloidal gold-labeled antibodies: Adjust the pH of the prepared colloidal gold solution to 6.5 using 0.2 mol / L K2CO3, dilute the antibody to a concentration of 1 mg / mL with double distilled water or PBS, and then add it dropwise to the colloidal gold solution under stirring at room temperature to make the amount of antibody in the colloidal gold solution 50 uL / mL. Keep stirring for 20 min, add 10% BSA stabilizer to make the concentration of BSA stabilizer in the colloidal gold solution 50 μL / mL, continue stirring at room temperature for 25 min, and finally, centrifuge at 4°C, 4000 r / min for 30 min, resuspend the precipitate in 0.2 M phosphate buffer (containing 0.5% Tween-80), repeat centrifugation twice, and concentrate for later use.

[0040] (3) The calibration card marking process is the same as that in Example 1. The obtained calibration card has an accuracy of 1.8%, a repeatability of 1.1%, and a service life of more than half a year.

[0041] Example 3

[0042] (1) Synthesis of colloidal gold: Using the trisodium citrate reduction method, add 100 mL of 0.01% chloroauric acid solution into a cleaned and dried 250 mL beaker, heat to boiling under stirring with a magnetic stirrer, keep boiling for 2 min, then add 2.0 mL of 1% trisodium citrate solution, continue heating and boiling for 15 min, cool to room temperature, restore to the original volume with distilled water, and store at 4°C for later use.

[0043] (2) Preparation of colloidal gold-labeled antibodies: Adjust the pH of the prepared colloidal gold solution to 7 using 0.2 mol / L K2CO3, dilute the antibody to a concentration of 1 mg / mL with double distilled water or PBS, and then add it dropwise to the colloidal gold solution under stirring at room temperature to make the amount of antibody in the colloidal gold solution 100 uL / mL. Keep stirring for 30 min, add 10% BSA stabilizer to make the concentration of BSA stabilizer in the colloidal gold solution 100 μL / mL, continue stirring at room temperature for 30 min, and finally, centrifuge at 4°C, 4000 r / min for 30 min, resuspend the precipitate in 0.2 M phosphate buffer (containing 1% Tween-80), repeat centrifugation twice, and concentrate for later use.

[0044] (3) The calibration card marking process is the same as that in Example 1. The obtained calibration card has an accuracy of 2.5%, a repeatability of 2%, and a service life of more than half a year.

[0045] The present invention can prepare an immune colloidal gold calibration card with good stability and long time effectiveness, and can more conveniently and quickly perform optical path calibration on a colloidal gold POCT instrument.

[0046] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0047] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these 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 preparing an immunocolloidal gold calibration card, characterized in that: The following steps are involved: (1) Synthesis of colloidal gold: Using the trisodium citrate reduction method, add 100 mL of 0.01% chloroauric acid solution into a cleaned and dried 250 mL beaker, heat to boiling under stirring with a magnetic stirrer, keep boiling for 2 min, then add 1.5 mL of 1% trisodium citrate solution, continue heating and boiling for 10 min, cool to room temperature, restore to the original volume with distilled water, and store at 4°C for later use; (2) Preparation of colloidal gold labeled antibody: adjust the pH of the prepared colloidal gold solution to 6.5 with 0.2 mol / L K2CO3, dilute the antibody to a concentration of 1 mg / mL with double distilled water or PBS, and then add it dropwise to the colloidal gold solution under stirring at room temperature to make the amount of antibody in the colloidal gold solution 50 uL / mL, keep stirring for 20 min, add 10% BSA stabilizer to make the concentration of BSA stabilizer in the colloidal gold solution 50 μL / mL, continue stirring at room temperature for 25 min, finally, centrifuge at 4°C, 4000 r / min for 30 min, resuspend the precipitate with 0.2 M phosphate buffer, repeat centrifugation twice, and concentrate for later use; (3) Calibration card streaking: The gold-labeled antibody solution was diluted 1, 2, 5, and 10 times with 0.01 mol / L PB, and streaked on the nitrocellulose membrane using a fully automatic streaking instrument. The quality control line was the colloidal gold-labeled antibody solution diluted 2 times, and the detection lines were the colloidal gold-labeled antibody solution diluted 1, 5, and 10 times, respectively. The streaked reagent strips were placed in an oven at 37°C for drying, cut, and packaged for later use.