A colloidal gold labeling processing method and its application

Abstract

The present invention provides a colloidal gold-labeled processing method and its application, and relates to the field of biological detection technology. The colloidal gold-labeled processing method is to pre-treat an unqualified first colloidal gold-labeled product to obtain a pre-treatment liquid; the pre-treatment liquid is mixed with a second colloidal gold-labeled product to obtain a third colloidal gold-labeled product; the OD520 of the first colloidal gold-labeled product is less than 0.2; the OD520 of the second colloidal gold-labeled product is greater than 0.2; the OD520 of the third colloidal gold-labeled product is 0.2-0.6; and the pre-treatment is carried out by adding sugar and / or alcohol. The colloidal gold-labeled processing method of the present invention can reuse the unqualified labeled colloidal gold-labeled product, and by detecting the sensitivity of the final test strip, it is found that the test result is higher than the standard requirement, and the processing method is simple, can save costs, and improve production capacity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and specifically relates to a colloidal gold labeling processing method and application thereof. Background Art

[0002] Immunogold technology is a solid-phase labeling immunoassay technology that uses colloidal gold as a marker and utilizes specific antigen-antibody reactions. Following the fluorescein, radioisotope and enzyme detection technologies, immunogold technology has been widely used in research fields such as biomedical immunohistochemistry and cell biology. The principle of colloidal gold labeling is essentially the coating process in which biological macromolecules such as antibody proteins are adsorbed onto the surface of colloidal gold particles. The adsorption mechanism may be that the negative charge on the surface of colloidal gold and the positive charge on the protein molecule attract each other by electrostatic force, forming a strong bond within the van der Waals force. In addition, the roughness of colloidal gold particles is also an important condition that is conducive to adsorption. Since this labeling process mainly relies on physical adsorption, it has no obvious effect on the biological activity of protein molecules.

[0003] The principle of colloidal gold labeling of proteins is generally believed to be that when the pH value is equal to or slightly alkaline to the isoelectric point of proteins, proteins are electrically neutral. At this time, the electrostatic interaction between protein molecules and colloidal gold particles is small, but the surface tension of protein molecules is the largest. They are in a weak hydration state and are easier to adsorb on the surface of gold particles. Since protein molecules are firmly bound to the surface of gold particles, a protein layer is formed, which prevents the mutual contact of colloidal gold particles and keeps colloidal gold in a stable state. If the pH value is lower than the isoelectric point of proteins, proteins are positively charged and colloidal gold is negatively charged. The two are very easy to electrostatically combine to form large polymers. If the pH value is higher than the isoelectric point of proteins, proteins are negatively charged, and the negative charge of gold particles repel each other and cannot combine with each other. In order to prevent the aggregation and precipitation of proteins and colloidal gold, bovine serum albumin, ovalbumin, polyethylene glycol or gelatin are often used as stabilizers. The separation and purification of colloidal gold protein markers by propylene dextran S-400 chromatography is only suitable for BSA as a stabilizer.

[0004] Colloidal gold labeling technology is affected by the batch of labeled protein, the batch of colloidal gold, and the preparation and operation of the operator's solution, and it cannot guarantee that every labeled batch of products is qualified. When the OD value range of the colloidal gold particle labeling is less than 0.2, it cannot meet the requirements, and such labeled batch products often need to be discarded.

[0005] Therefore, there is an urgent need for a colloidal gold labeling treatment method that can save colloidal gold labeled products and adjust unqualified colloidal gold labeled products to meet performance requirements. Summary of the invention

[0006] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Numerical ranges recited by endpoints include all numbers and fractions within the corresponding ranges, as well as the recited endpoints.

[0007] It should be noted that:

[0008] The OD values ​​in the present invention are all measured at 520 nm;

[0009] The second colloidal gold labeled product (OD520 greater than 0.2) described in the present invention also refers to a high-efficiency labeled product.

[0010] The present invention aims at the problems existing in the prior art and provides a colloidal gold labeling treatment method and its application. The treatment method adds sugar alcohol substances to the unqualified colloidal gold labeling product for pretreatment, and then uses the high-efficiency colloidal gold labeling product to adjust the pretreated colloidal gold labeling product, so that the unqualified colloidal gold labeling product can be reused, saving costs and improving production capacity.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] The present invention provides a colloidal gold labeling processing method, the processing method comprising the following steps:

[0013] S1, pre-treating the unqualified first colloidal gold labeled product to obtain a pre-treated solution;

[0014] S2, mixing the pretreatment solution and the second colloidal gold labeled product to obtain a third colloidal gold labeled product;

[0015] The OD520 of the first colloidal gold labeled product is less than 0.2;

[0016] The OD520 of the second colloidal gold labeled product is greater than 0.2;

[0017] The OD520 of the third colloidal gold labeled product is 0.2-0.6.

[0018] Preferably, the pretreatment method is pretreatment by adding sugar and / or alcohol.

[0019] Preferably, the sugar includes at least one of trehalose, sucrose, lactose, fructose, glucose and dextran; the alcohol includes at least one of mannitol, sorbitol, inositol, xylitol, lactitol, maltitol and erythritol.

[0020] More preferably, the sugar includes at least one of trehalose, sucrose and lactose; and the alcohol includes at least one of mannitol, sorbitol, inositol, xylitol and lactitol.

[0021] Preferably, the mass concentration of the added sugar and / or alcohol is 0.1%-10%.

[0022] It is worth noting that the mass concentration of sugar or alcohol is: the final concentration of sugar or alcohol in the pretreatment solution during the pretreatment step.

[0023] More preferably, the mass concentration of the sugar and / or alcohol is 4%-8%.

[0024] Most preferably, the mass concentration of the sugar and / or alcohol is 5%.

[0025] Preferably, the volume ratio of the pretreatment solution to the second colloidal gold labeled product is 1:9-9:1.

[0026] Further preferably, the volume ratio of the pretreatment solution to the second colloidal gold labeled product is 1:9-6:4.

[0027] Most preferably, the volume ratio of the pretreatment solution to the second colloidal gold labeled product is 5:5.

[0028] Preferably, the processing method further comprises the steps of preparing a colloidal gold solution, preparing a labeling solution, preparing a gold label binding pad, and preparing a test strip.

[0029] Specifically, the preparation method of the colloidal gold solution includes: using a reduction method, taking 100 ml of a 0.01% chloroauric acid aqueous solution and heating it to boiling, accurately adding 0.7 ml of a 1% trisodium citrate aqueous solution while stirring, the golden yellow chloroauric acid aqueous solution turns purple-red within 2 minutes, continuing to boil for 15 minutes, and after cooling, restoring the original volume with distilled water. When the absorption peak value is 525 nm, the colloidal gold particle size is about 40 nm.

[0030] Preferably, the labeling solution comprises a colloidal gold preservation solution and a colloidal gold diluent.

[0031] Preferably, the components of the colloidal gold preservation solution include PBS, sucrose, Tween 80, and sodium azide; the components of the colloidal gold diluent include Tis-HCl, sucrose, Tween 80, trehalose, casein, and sodium azide.

[0032] Further preferably, the components in the colloidal gold preservation solution include 0.1M PBS, 5% sucrose, 2% Tween 80, and 0.5mg / ml sodium azide;

[0033] The components in the colloidal gold diluent include 20mmol / L Tis-HCl, 5% sucrose, 1% Tween 80, 3% trehalose, 0.3% casein, and 0.5mg / ml sodium azide.

[0034] Preferably, the pH of the Tis-HCl is 9.

[0035] Preferably, the labeling solution further comprises sodium carbonate, 20% BSA, and PBS solution.

[0036] Preferably, the pH of the PBS is 7.4, and the concentration of the sodium carbonate is 0.1M.

[0037] Preferably, the gold-labeled binding pad is prepared by spraying the adjusted qualified labeled product onto the gold-labeled binding pad, and then drying at a constant temperature of 37°C.

[0038] Preferably, the test strips are prepared by spraying films, treating sample pads, assembling large plates and cutting into strips.

[0039] Among them, spraying requires the preparation of spraying liquid T line solution and spraying liquid C line solution.

[0040] Specifically, the spray liquid T line solution and the spray liquid C line solution need to be prepared with 0.1M PBS.

[0041] The present invention also provides an application of the above-mentioned treatment method in preparing colloidal gold immunochromatography products.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The colloidal gold labeling treatment method of the present invention is to perform sugar and / or alcohol pretreatment, so that unqualified labeled colloidal gold labeling products can be reused. By detecting the sensitivity of the final test strip, it is found that the detection result is higher than the standard requirement. The treatment method is simple, can save costs, and improve production capacity. DETAILED DESCRIPTION

[0044] The present invention is described below through specific embodiments to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto, and the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0045] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0046] Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0047] PVC board: commonly available in the market, item number is SM31-25;

[0048] NC membrane: commonly available in the market, product number is NC-140;

[0049] Sample pad: commonly available in the market, item number SB-08;

[0050] Gold label pad: commonly available in the market, item number SB-06;

[0051] Absorbent pad: commonly available in the market, item number SX30;

[0052] Troponin I (cTnI) antibody: commonly available in the market, with product numbers cTnI101 and cTnI102;

[0053] Sheep anti-mouse IgG polyclonal antibody: commonly available in the market, product number M-IgG202;

[0054] Troponin I (cTnI) antigen: commonly available on the market, product number is cTnI301.

[0055] Example 1 Adjusting Colloidal Gold Particles

[0056] 1. Preparation of labeling solution:

[0057] ①Prepare 0.1M sodium carbonate and set aside.

[0058] ②Prepare 20% BSA and set aside.

[0059] ③Prepare 50mmol / L PBS with pH 7.4 and set aside.

[0060] ④Prepare colloidal gold preservation solution: 0.1M PBS, 5% sucrose, 2% Tween 80, 0.5mg / ml sodium azide.

[0061] ⑤Prepare colloidal gold diluent: pH 9.0 20mmol / L Tis-HCl, 5% sucrose, 3% trehalose, 1% Tween 80, 0.3% casein, 0.5mg / ml sodium azide.

[0062] 2. Colloidal gold particle labeling:

[0063] ① Take 20mL of colloidal gold solution, add 100μL of 0.1mol / L sodium carbonate solution, and leave at room temperature for 30 minutes;

[0064] ②Add 200 μL of 1 mg / mL troponin I (cTnI) antibody and mix well, and leave at room temperature for 30 minutes;

[0065] ③Add 200 μL of 20% BSA solution and mix well, then place at room temperature for 30 minutes;

[0066] ④ Then centrifuge at 12000rpm for 20 minutes and discard the supernatant after centrifugation;

[0067] ⑤ Add 10 mL of 50 mmol / L PBS buffer at pH 7.4 to resuspend, and centrifuge at 12000 rpm for 20 minutes; ⑥ After centrifugation, discard the supernatant, resuspend the precipitate in 2.0 mL of colloidal gold preservation solution to obtain a colloidal gold resolution;

[0068] ⑦ Dilute the colloidal gold complex solution obtained in step ⑥ with the colloidal gold diluent at a ratio of 1:10, and measure the OD value at 520 nm to find that it is 0.1;

[0069] ⑧ The colloidal gold complex solution in ⑦ was pretreated by adding mannitol, wherein the final concentration of mannitol was 5%, and the second colloidal gold particles (OD520 was 0.5) were mixed with the product of the colloidal gold complex solution added with mannitol in a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 respectively;

[0070] ⑨ The products after mixing in different proportions were then tested, and the OD values ​​of the mixed particles were measured to be 0.2, 0.2, 0.2, 0.3, 0.3, 0.4, 0.4, 0.4, and 0.5, respectively. Alternatively, the products were prepared into gold-labeled binding pads and then assembled into test strips to test their sensitivity.

[0071] 3. Colloidal gold solution spraying:

[0072] ① Set the spraying volume to 3μL / mm and the moving speed to 60mm / s, and spray the qualified mixed particles onto the gold label pad;

[0073] ②After spraying, transfer the gold label pad to a 37℃ constant temperature box for drying overnight;

[0074] ③ Collect the dried gold-labeled binding pad and store it in a drying oven for later use.

[0075] Example 2 Preparation of test strips

[0076] 1. Spray film:

[0077] ①Prepare 0.1M PBS;

[0078] ②Prepare the T-line solution for spraying: Use 0.1M PBS to prepare the T-line, prepare the mixed troponin I (cTnI) antibody to a concentration of 1.00 mg / mL, and the final concentration of PBS is 0.01M;

[0079] ③Prepare the C-line solution for the spraying liquid: Use 0.1M PBS to prepare the C-line, prepare the goat anti-mouse IgG polyclonal antibody to a concentration of 1.00mg / mL, and the final concentration of PBS is 0.01M;

[0080] ④Spraying: Set the spraying volume to 0.1μL / mm and the moving speed to 60mm / s for spraying;

[0081] ⑤ Transfer the sprayed NC membrane plate to a 37°C constant temperature box for drying overnight. After completion, collect it in a drying box and store it for later use.

[0082] 2. Sample pad processing:

[0083] Prepare 0.01M PBST solution, immerse the sample pad in the solution, put the sample pad into the gauze rack after it is soaked, and place it in a 37°C oven to dry overnight. After completion, collect it in a drying oven and store it for later use.

[0084] 3. Assembly of large panels:

[0085] Paste the large plate sprayed with NC film, sample pad, gold label pad and absorbent pad in the order of test strips.

[0086] 4. Cut into strips:

[0087] The large plate was cut into test strips with a width of 3.0-4.0 mm using a strip cutter.

[0088] Test Example 1

[0089] 1. Test object:

[0090] Troponin I (cTnI) recombinant antigen was used.

[0091] 2. Test steps:

[0092] ① The concentration preparation method of the enterprise reference product is as shown in the following table. A control group and an experimental group pretreated with added sugar, alcohol, and amino acids are set up, and the control group is high-efficiency labeled colloidal gold.

[0093] Table 1 Concentration of reference products of enterprises

[0094]

[0095] ② Aspirate 75μL of each sample, lay each test strip flat, and add each sample to the sample pad area of ​​the test strip.

[0096] ③ Reaction reading: After 10-20 minutes of reaction, read the color development result on the test strip with the naked eye.

[0097] 3. Rules for interpretation of results:

[0098] Observe the color development of the reaction strips, and the color depth is represented by "+"; "1+" represents color development but weak color, "2+" represents obvious color development but not deep color, "3+" represents deep color development, "4+" represents very deep color development, "5+" represents dark color development, and "-" represents no color development. Three parallel test strips with different concentrations of troponin I (cTnI) recombinant antigen are used as the judgment results. The specific results are shown in Table 2. When the color development result of the reagent strip is 3+ or above, it indicates that the performance of the reagent strip is qualified.

[0099] Table 2 Test results of adding different substances to the unqualified marking solution

[0100]

[0101]

[0102] 4. Result analysis:

[0103] Judging from the test results, adding sugar alcohols can improve sensitivity, adding sugars is consistent with the detection results of high-efficiency markers, while amino acids such as arginine can reduce sensitivity.

[0104] Test Example 2

[0105] 1. Test object:

[0106] Troponin I (cTnI) recombinant antigen was used.

[0107] 2. Test steps:

[0108] ①The preparation method of the enterprise reference product is shown in the following table:

[0109] Table 3 Concentration of reference products of enterprises

[0110]

[0111] ② Aspirate 75μL of each sample, place each test strip flat, and add each sample to the sample pad area of ​​the test strip. ③ Read the reaction results. After 10-20 minutes of reaction, read the color results on the test strip with the naked eye.

[0112] 3. Rules for interpretation of results:

[0113] Observe the color development degree of the reaction bands. The color depth is represented by "+", where "1+" means there is color development but the color is weak, "2+" means the color development is obvious but not deep, "3+" means the color development is relatively deep, "4+" means the color development is very deep, "5+" means the color development is so deep that it turns dark, and "-" means no color development. For 3 parallel test strips, different concentrations of recombinant antigen of cardiac troponin I (cTnI) are used to determine the results. When the color development result of the test strip is 3+ or above, it indicates that the performance of the test strip is qualified.

[0114] Table 4 Test results of different volume ratios of the second colloidal gold complex solution and the colloidal gold complex solution added with mannitol

[0115]

[0116] Table 5 Test results of different volume ratios of the second colloidal gold complex solution and the colloidal gold complex solution without added mannitol

[0117]

[0118] 4. Result analysis:

[0119] From the test results, the detection results of the colloidal gold complex solution added with mannitol and the second colloidal gold complex solution are stronger than those of the colloidal gold complex solution without added mannitol and the second colloidal gold complex solution. Moreover, for the detection results of the former after being processed according to the ratio, the OD values are all within the range of 0.2 - 0.6, meeting the requirements for the colloidal gold labeled product in the preparation of the test strip. When detecting the prepared test strips, the results show that according to the method of the present invention, the effects after the dilution ratio of 4:6 are all better, and the dilution ratio of 5:5 is the optimal combination.

[0120] Test Example 3

[0121] 1. Test method:

[0122] Use the test strips prepared in the examples of the present invention, the test strips prepared with normal qualified labeling, and the test strips prepared with unqualified labeling for detection respectively.

[0123] 2. Test samples:

[0124] Use 10 volunteer serum samples for testing. The cTnI concentrations of the 10 volunteer serum samples are determined using the New Industries kit (YZB / Guangdong 0524 - 2007 "Cardiac Troponin I (Troponin I) Assay Kit (Chemiluminescence Method)"), and the determination results are as follows in the table.

[0125] Table 6 Determination results of test samples

[0126] Serial number Fixed value result (ng / ml) 1 53 2 1.5 3 1.3 4 35 5 21 6 1.8 7 32 8 2.1 9 38 10 1.6

[0127] 3. Test strip testing steps:

[0128] The test strips prepared according to the method of the present invention, the test strips prepared with normal marking and qualified, and the test strips prepared with unqualified marking are all tested using the following steps.

[0129] ① Aspirate 75μL of each sample, lay each test strip flat, and add each sample to the sample pad area of ​​the test strip.

[0130] ② Reaction reading: After 10-20 minutes of reaction, read the color development result on the test strip with the naked eye.

[0131] 4. Rules for interpretation of results:

[0132] Observe the color development of the reaction strips. The color development depth is indicated by "+"; "1+" means that the color is weak, "2+" means that the color is obvious but not deep, "3+" means that the color is deep, "4+" means that the color is very deep, "5+" means that the color is so deep that it is dark, and "-" means that there is no color. Three parallel test strips are used, and different concentrations of human serum protein are used as the judgment results. When the color development result of the reagent strip is 3+ or above, it indicates that the performance of the reagent strip is qualified.

[0133] 5. Sample test results:

[0134] Table 7 Serum test results

[0135]

[0136]

[0137] 6. Result analysis:

[0138] From the test results, the test strips prepared by adding mannitol and using the second colloidal gold particles have the same test results as the test strips prepared by normal qualified marking; the test strips prepared by using the second colloidal gold particles without adding mannitol have a weaker color than the test strips prepared by normal qualified marking, but are stronger than the test strips prepared by unqualified marking. This shows that this method can meet the product performance requirements, while the test results of the test strips prepared by unqualified marking cannot meet the performance requirements, and the test strips of this method have better discrimination.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A colloidal gold labeling method, characterized in that: The processing method comprises the following steps: S1, pre-treating the unqualified first colloidal gold labeled product to obtain a pre-treated solution; S2, mixing the pretreatment solution and the second colloidal gold labeled product to obtain a third colloidal gold labeled product; The OD520 of the first colloidal gold labeled product is less than 0.2; The OD520 of the second colloidal gold labeled product is greater than 0.2; The OD520 of the third colloidal gold labeled product is 0.2-0.6; The pretreatment method is to add sugar and / or alcohol for pretreatment; The sugar includes at least one of trehalose, sucrose, lactose, fructose, glucose and dextran; the alcohol includes at least one of mannitol, sorbitol, inositol, xylitol, lactitol, maltitol and erythritol; The mass concentration of the added sugar and / or alcohol is 0.1%-10%.

2. The processing method according to claim 1, characterized in that: The sugar includes at least one of trehalose, sucrose and lactose; the alcohol includes at least one of mannitol, sorbitol, inositol, xylitol and lactitol.

3. The processing method according to claim 1, characterized in that: The mass concentration of the added sugar and / or alcohol is 4%-8%.

4. The processing method according to claim 1, characterized in that: The volume ratio of the pretreatment liquid to the second colloidal gold labeled product is 1:9-9:

1.

5. The processing method according to claim 4, characterized in that: The volume ratio of the pretreatment liquid to the second colloidal gold labeled product is 1:9-6:

4.

6. The processing method according to claim 1, characterized in that: The method also includes preparing a labeling solution; the labeling solution includes a colloidal gold preservation solution and a colloidal gold diluent; the components in the colloidal gold preservation solution include PBS, sucrose, Tween 80, and sodium azide; the components in the colloidal gold diluent include Tis-HCl, sucrose, Tween 80, trehalose, casein, and sodium azide.

7. Use of the treatment method according to any one of claims 1 to 6 in the preparation of colloidal gold immunochromatography products.

8. The processing method according to claim 6, characterized in that: The components of the colloidal gold preservation solution include 0.1 M PBS, 5% sucrose, 2% Tween 80, and 0.5 mg / ml sodium azide.

9. The processing method according to claim 6, characterized in that: The components in the colloidal gold diluent include 20mmol / L Is-HCL, 5% sucrose, 1% Tween 80, 0.3% casein, and 0.5mg / ml sodium azide.

10. The processing method according to claim 6, characterized in that: The pH of the Tis-HCl is 9, and the pH of the PBS is 7.4.

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