CN101470114B - Sensitization detection method of colloidal gold immunity chromatography and use thereof - Google Patents

Abstract

A colloidal gold immunochromatography method for detection of sensitivity enhancement, when conventional colloidal gold immunochromatography detects a sample to be tested, after adding the sample to be tested, a signal amplification reagent 10 is added between the gold-labeled antibody layer and the detection line ~80 μL, the detection line obtained by conventional colloid immunochromatography is processed to enhance the signal, and after reacting at room temperature for 1 to 10 minutes, if the sample to be tested contains an antibody, a detection line and a double line of control line are formed on the detection carrier. black or gray black, this is positive, if it does not contain anti-principle, the control line is black or gray black, and the detection line has no color, this is negative; using the method of the present invention, it can significantly improve the colloidal gold immunochromatography kit to detect biological The sensitivity of macromolecules, rapid color development, and less background interference than conventional silver staining methods have very wide application prospects and development value.

Description

Sensitive detection method and application of colloidal gold immunochromatography

technical field

The invention relates to a macromolecular substance detection method, in particular to a sensitivity enhancement method for gold standard immunochromatographic detection of biomacromolecular substances and a method for making a kit by using the same.

Background technique

At present, the simple, fast, highly selective and highly sensitive detection of biological macromolecules has always been the direction of people's efforts. This is in the rapid diagnosis of diseases, health quarantine, animal and plant quarantine, food safety testing, environmental monitoring, etc. areas are important. Among them, the immunoassay (Immunoassay) based on the specific reaction of antigen and antibody has high selectivity and convenience, and its principle is simple, it can be produced and used without large-scale experimental equipment or large investment, and the average sample testing cost It is far lower than the instrumental analysis method and other characteristics, making it the first choice for the detection of many specific substances.

Under the action of reducing agents such as trisodium citrate, white phosphorus, ascorbic acid, tannic acid, etc., chloroauric acid (HAuCl ₄ ) polymerizes into gold particles of a specific size, and forms a stable colloidal state due to electrostatic interaction, so it is called colloidal gold. Since the surface of colloidal gold is negatively charged, it can be firmly combined with the positive charges of macromolecules such as proteins due to electrostatic adsorption without affecting the biological characteristics of proteins. In 1971, Faulk and Taytor introduced colloidal gold into the immunoassay method, forming the immunogold labeling technique (Immunogoldlabeling techique), which is to use colloidal gold as a tracer for antigen antibody labeling, and mainly use nitrocellulose membrane as a carrier to realize immunodetection. Immunogold labeling technology is mainly divided into two modes: (1) Immunofiltration analysis (Flowthrough-immunofiltration assay, IFA): the immune reaction is carried out by vertically penetrating the nitrocellulose membrane immobilized with the ligand (flow through type ); (2) Lateral flow-immunochromatographic assay (ICA): the analysis principle is the same as that of IFA, except that the flow of the reaction liquid is not a straight penetrating flow, but a chromatographic effect through capillary phenomenon Lateral flow type was applied in 1990.

Although the above two methods are immunogold standard techniques based on nitrocellulose membrane as the carrier, their detection principles are quite different. The main performance is: after ICA is added to the test solution, the test solution chromatographically diffuses under the action of capillary to realize the separation of the sample matrix, and there is almost no sample matrix interference in the detection line; while IFA relies on vertical penetration washing, the detection spots are easy to diffuse, Difficult to read results. Therefore, most of the widely used commercial test strips (or kits) products adopt the principle of immunochromatography.

However, the biggest disadvantage of immunogold labeling detection method is the low detection sensitivity, which limits the scope of practical application. In the past, people mainly used silver staining technology to overcome the problem of low detection sensitivity. That is, the detection line on the sample pad is treated with silver nitrate and a specific reducing agent as a signal amplification reagent. However, the signal amplification effect of this method is not obvious, mainly because the background of the detection signal is blurred and the background color is high, so it is difficult to judge the detection result by naked eyes. Therefore, it is almost difficult to see colloidal gold immunochromatography products based on silver staining technology at present.

Contents of the invention

The technical problem solved by the present invention is to provide a sensitive detection method of colloidal gold immunochromatography. This method is based on the conventional colloidal gold immunochromatography assay method, using nano Gold particles process the detection line obtained by conventional colloidal gold immunochromatography to enhance the signal, which can significantly enhance the detection signal of conventional colloidal gold immunochromatography with clean background and low background value, which can improve the detection sensitivity by 8 to 200 times , thereby overcoming the problem of low sensitivity of the traditional colloidal gold immunochromatographic detection method.

Another object of the present invention is to provide a preparation method of a colloidal gold immunochromatography high-sensitivity detection kit and its product and application. The kit is prepared by using the above-mentioned enhanced detection method of colloidal gold immunochromatography.

A signal amplification reagent for colloidal gold immunochromatography, which is a mixed reagent in which a sensitizing reagent A and a sensitizing reagent B are mixed in a volume ratio of 1:1 to 1:5, and the sensitizing reagent A is by weight The percent concentration is 0.1%-10% chloroauric acid solution, and the sensitizing reagent B is a reducing agent solution. Wherein the reducing agent solution is preferably a molar concentration of 0.1 to 10mM hydroxylamine hydrochloride solution or a weight percentage concentration of 0.1 to 0.5% ascorbic acid solution, and can also be other reducing agents, such as trisodium citrate, white phosphorus, tannic acid, etc. . Wherein the weight percent concentration of the chloroauric acid solution is preferably 1-4%. Wherein the molar concentration of the hydroxylamine hydrochloride solution is preferably 2-6 mM, or the weight percent concentration of the ascorbic acid solution is preferably 0.15-0.3%.

A sensitivity-enhancing detection method of colloidal gold immunochromatography. When the conventional colloidal gold immunochromatography detects the sample to be tested, the signal is added between the gold-labeled antibody layer and the detection line after adding the sample to be tested. Amplify the reagent by 10-80 μL, process the detection line obtained by conventional colloidal gold immunochromatography to enhance the signal, react at room temperature for 1-10 minutes, and observe the results.

When the method of the present invention is specifically applied, wherein the sample to be tested is an extract of animal secretions, serum, tissue organs, etc., the sample to be tested is treated with a pretreatment method such as elution, centrifugal dilution, etc., and 40-80 μL of the treated liquid droplet is taken On the sample well of the conventional colloidal gold immunochromatography kit, each sample was repeated 2 to 3 times. After reacting for 1 to 10 minutes at room temperature, 100 μL of phosphate washing solution PBST was added to the sample well, and the chromatography strip was washed. Then quickly take 10-80 μL of the pre-mixed chloroauric acid solution and reducing agent solution and drop it on the sample well or the well of the sensitizing reagent; wherein the developing reagent contains a weight percent concentration of 1- 2% nonylphenol polyoxyethylene ether NP40 and 0.1-1% surfactant Tween 20 in 0.1mM phosphate buffer PB.

According to the method of the present invention can prepare special-purpose test strip and/test kit, preparation method can be the following steps:

The front end of the upper surface of a plastic backing plate is pasted with a layer of absorbent paper layer at the sample loading end, and after the absorbent paper layer at the sample loading end, the gold-labeled antibody layer and the detection layer are continuously lapped until the rear end of the plastic backing plate. The rear end of the upper surface of the detection layer is pasted with a layer of absorbent paper at the absorbent end that can absorb at least 200 μL of liquid. The gold-labeled antibody layer is a nitrocellulose membrane layer coated with antibodies, and the detection layer is a nitrocellulose membrane layer. , the position between the gold-labeled antibody layer and the absorbent paper layer at the water-absorbing end is coated with a detection line and a control line in sequence. After preparation, it is cut into strips according to the specifications of 4 mm wide and 8 cm long, and then assembled In a plastic box. Multiple strips can be set in a large box, or one strip can be set in a small box, and then matched with the required reagents. The upper surface of the box is provided with three openings: a hole for adding a sample, a hole for adding a sensitizing reagent, and an observation hole. The position between the antibody layer and the detection line, the observation hole is facing the absorbent paper layer at the absorbent end.

Wherein the preparation method of the gold-labeled antibody layer comprises the following steps:

(1). Preparation of colloidal gold solution

a. Reagents and their volume ratios for preparing colloidal gold solution:

Double distilled water 100mL

1mL freshly prepared aqueous solution of chloroauric acid with a concentration of 1 to 5% by weight

1.0-1.5 mL of trisodium citrate aqueous solution with a concentration of 1-5% by weight;

b. Preparation method

Heat 100mL of the above-mentioned double-distilled water to 65°C with microwave, add 1ml of freshly prepared chloroauric acid aqueous solution with a concentration of 1 to 5% by weight, heat to 95°C with microwave, quickly add the required concentration of 1% by weight ～5% trisodium citrate aqueous solution, stirring constantly, under the action of sodium citrate reducing agent, chloroauric acid, the color of its colloidal gold solution is from black → blue → purple → wine red, and after cooling, a wine red 20 ~ 40nm Colloidal gold solution with particle size, the colloidal gold synthesized at this time can be stored at 4°C for 12 months;

(2). Preparation of gold-labeled antibody layer

Get the colloidal gold solution 10mL of above-mentioned preparation, be that the potassium carbonate aqueous solution of 1% is adjusted pH to 8.5～9.2 (preferably 8.7～9.2) with weight percentage concentration, dissolve 0.2g potassium chloride, 1.44g disodium hydrogen phosphate and 0.24g potassium dihydrogen phosphate, adjust the pH of the solution to 7.4 with hydrochloric acid, add water to 1L, shake well to make 0.1mM phosphate buffer PB, dilute the antibody with the phosphate buffer PB to a protein content of 0.1 ～1mg/mL, take 0.1～0.5mL, centrifuge at 13000rpm, 4°C for 30min, take out the corresponding volume from the centrifuged supernatant so that the minimum protein content of the liquid after adding the colloidal gold solution is 10～20μg/mL ( Preferably 10-16 μg/mL), under rapid stirring, slowly add the antibody dropwise to the above pH-adjusted colloidal gold solution, and let it stand at room temperature for 5 minutes; then add 1 mL of filtered BSA solution with a concentration of 10% by weight, and continue Stir for 10-15 minutes, then centrifuge at 10,000 rpm and 4°C for 30 minutes, discard the supernatant to obtain a precipitate; Tris solution (TBS, containing 1% BSA) to dissolve the precipitate, centrifuge at 10000rpm/min and 4°C for 30min, discard the supernatant, and use 10mL of 0.02% by weight respectively Sodium azide, 1% sucrose, 1% BSA, and a tris solution with a pH of 8.2 were used to resuspend the loose precipitate at the bottom to obtain a gold-labeled antibody probe solution; finally, the gold-labeled antibody probe solution was sprayed with gold The instrument sprays gold on the nitrocellulose membrane until the liquid begins to ooze out, and dries at 37°C for 2 hours to form a gold-labeled antibody layer.

The preparation method of the detection layer includes: coating the detection line formed by the specific polyclonal or monoclonal antibody for the sample to be tested on the nitrocellulose membrane and the detection line formed by the specific IgG antibody for the gold-labeled antibody. Control line: The specific preparation method is to take polyclonal antibody or monoclonal antibody, adjust the concentration to 1mg/mL with a developing reagent, and spray the detection line in the middle of the nitrocellulose membrane with a film sprayer; then take IgG antibody 1mg/mL, and use a film sprayer Spray the control line on the middle section of the nitrocellulose membrane, 0.5cm away from the detection line, set the amount of sprayed film at 20μL/10cm, and dry the sprayed film at 37°C for 2 hours.

The method and products such as test strips prepared by the method can be widely used in biomacromolecule detection or medical diagnosis.

The method of the present invention uses a sensitizing reagent to increase sensitivity, utilizes the characteristics that gold atoms generated by the oxidation-reduction reaction of chloroauric acid and hydroxylamine hydrochloride can be adsorbed by colloidal gold, and locates the deposition site of colloidal gold for color enhancement, thereby improving the quality of the organisms to be tested. Detection sensitivity of macromolecules. Using the method of the invention can significantly improve the sensitivity of the colloidal gold immunochromatography kit for detecting biological macromolecules, and has rapid color development and less background interference than conventional silver staining methods, and has very wide application prospects and development value. Using the sensitizing reagent of the present invention for one or more treatments, compared with the original colloidal gold immunochromatography assay, the sensitivity can be significantly improved by 8-200 times, and the operation is simple and fast, no special equipment is required, and it can be widely used It is used in clinical diagnosis, antibody and antigen detection, health quarantine, environmental detection and other fields, and has a very wide application prospect and development value.

Description of drawings

Fig. 1 is the top view of the kit prepared in embodiment 7;

Fig. 2 is A-A sectional view of Fig. 1;

Fig. 3 is the result that commercially available colloidal gold test strip detects avian influenza virus NP expression protein in embodiment 2;

Fig. 4 is the result that utilizes colloidal gold sensitization method of the present invention to detect avian influenza virus NP expression protein in embodiment 2;

Fig. 5 is the result that commercially available colloidal gold test strip detects avian influenza virus in chicken embryo allantoic fluid in embodiment 3;

Fig. 6 utilizes colloidal gold sensitization method of the present invention to detect the result of avian influenza virus in chicken embryo allantoic fluid in embodiment 3;

Fig. 7 is the result that commercially available colloidal gold test strip detects infecting chicken avian influenza virus in embodiment 4;

Fig. 8 is the result utilizing the colloidal gold sensitization method of the present invention to detect infected chicken avian influenza virus in embodiment 4;

Fig. 9 is the result that commercially available colloidal gold test strip detects pregnant woman's urine sample in embodiment 5;

Fig. 10 is the result that utilizes colloidal gold sensitization method of the present invention to detect the urine sample of pregnant woman in embodiment 5;

Fig. 11 is the result that commercially available colloidal gold test strip detects Salmonella enteritidis nutrient solution in embodiment 6;

Fig. 12 is the result of utilizing colloidal gold sensitization method of the present invention to detect Salmonella enteritidis culture solution in embodiment 6;

Fig. 13 is the result that self-made colloidal gold test strip detects Newcastle disease virus live virus in embodiment 10;

Fig. 14 is the result of the test strip in Example 10 when using the kit of the present invention to detect live Newcastle disease virus.

Detailed ways

Embodiment 1: the preparation of various reagents

1. Chlorauric acid aqueous solution: Weigh 1.0 g of chloroauric acid, dissolve it in 100 mL of double-distilled water to form a chloroauric acid aqueous solution with a concentration of 1% by weight, and shake well.

2. Trisodium citrate aqueous solution: Weigh 1.0-1.5 g of trisodium citrate, dissolve it in 100 mL of double-distilled water to form an aqueous solution of trisodium citrate with a concentration of 1-1.5% by weight, and shake well.

3. Phosphate buffered saline (PBS): Dissolve 8g of sodium chloride (NaCl), 0.2g of potassium chloride (KCl), 1.44g of disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 0.24g of potassium dihydrogen phosphate in 800mL of distilled water (KH ₂ PO ₄ ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1L, and shake well.

4. Phosphate buffer (PB): Dissolve 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 0.24g potassium dihydrogen phosphate (KH ₂ PO ₄ ) in 800mL distilled water, and use Hydrochloric acid (HCl) to adjust the pH value of the solution to 7.4, add water to 1L, shake well.

5. Phosphate washing solution (PBST): Dissolve 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 0.24g potassium dihydrogen phosphate in 800mL distilled water (KH ₂ PO ₄ ), adjust the pH value of the solution to 7.4 with hydrochloric acid (HCl), add water to 1 L, then add 200 μL of surfactant Tween20, and shake well.

6. Polyclonal antibody or monoclonal antibody: dilute with PBS so that the weight percent concentration of polyclonal antibody or monoclonal antibody in the solution is 1 mg/mL.

7. Development reagent: Add 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 0.24g potassium dihydrogen phosphate (KH ₂ PO ₄ ) with 800mL distilled water, and dissolve with hydrochloric acid (HCl) Adjust the pH of the solution to 7.4, add water to 1 L, shake well, and prepare 0.1 mM phosphate buffer (PB, Phosphate Buffer). Add nonylphenol ethoxylate (NP40) at a concentration of 1% by weight and Tween 20 surfactant at 0.5%.

8. Sensitizers:

Sensitizing reagent A: prepare chloroauric acid with a weight percent concentration of 0.1% to 10%, and dilute it with a developing reagent; the preferred weight percent concentration of chloroauric acid in the chloroauric acid aqueous solution is 1 to 4%;

Sensitizing reagent B: prepare hydroxylamine hydrochloride with a molar concentration greater than 0.1-10 mM or an aqueous solution of ascorbic acid with a concentration greater than 0.1-0.5% by weight, and dilute with developing reagents. The preferred molar concentration of hydroxylamine hydrochloride is 5 mM, and the preferred weight percent concentration of ascorbic acid in the ascorbic acid aqueous solution is 0.15%.

9. IgG antibody: dilute with PBS to make the weight percent concentration of IgG antibody in the solution 1 mg/mL.

10. BSA (bovine serum albumin) solution: dilute with double distilled water so that the weight percent concentration of BSA in the solution is 10%.

Embodiment 2: Contrast test of avian influenza virus NP expression protein sensitization detection

The artificially expressed avian influenza virus NP protein was diluted 200 times with a developing agent, and then serially diluted until it was diluted to 200× ²⁸ times. The commercially available avian influenza virus general-purpose gold standard detection test strip (Anigen Company) was used to detect Samples of each concentration were measured, each sample was measured twice, and one sample was taken from each batch of determination as a negative control. In the test, the test line and the control line show clear dark red, which means the sample is positive.

Using the sensitization detection method provided by the present invention, the sensitization reagent A (0.5% chloroauric acid) is mixed with the sensitization reagent B (10mM hydroxylamine hydrochloride) according to the volume ratio of 1:5 to make a signal amplification reagent, respectively Take 50 μL of the signal amplification reagent for treatment. The specific treatment method is to add the signal amplification reagent between the gold-labeled antibody layer and the detection line of the test strip. After the negative control treatment showed a negative result, one test strip of each concentration sample was sensitized in parallel. The detection line and the control line show clear gray and black, which means the sample is positive. When using test strips to detect, the minimum 1600-fold dilution sample can see the reaction band (see Figure 3), after the detection test strip is sensitized, the minimum 25600-fold dilution sample can see the reaction band (see Figure 4), the The gold standard sensitization method can increase the bottom line of detection of avian influenza virus NP protein by 16 times.

Example 3: Colloidal gold-enhanced detection test for H9N2 subtype avian influenza virus

Inoculate chicken embryos with H9N2 subtype avian influenza virus, harvest chicken embryo allantoic fluid and carry out serial doubling dilution with developing agent, until diluted to ^2-12 times, use commercially available avian influenza virus universal gold standard detection test strip (Anigen Company ) to measure the allantoic fluid samples of each dilution, each sample was measured twice, and one sample was taken from each batch of determination as a negative control. In the test, the test line and the control line show clear dark red, which means the sample is positive.

Using the sensitization detection method provided by the present invention, the sensitization reagent A (1% chloroauric acid) and the sensitization reagent B (10mM hydroxylamine hydrochloride) are mixed according to the volume ratio of 1: 2 to make a signal amplification reagent, respectively Take 40 μL of the signal amplification reagent for treatment. The specific treatment method is to add the signal amplification reagent between the gold-labeled antibody layer and the detection line of the test strip. After the negative control treatment showed a negative result, one test strip of each concentration sample was sensitized in parallel. The detection line and the control line show clear gray and black, which means the sample is positive. When using test strips for detection, the detection limit before the sensitization is that the hemagglutinin titer (HAU) is ^2-5 (see Figure 5), and the HAU is ^2-12 after the test strips are sensitized. And there is no bottom limit (see Fig. 6), the comparative test results show that the detection of H9N2 subtype avian influenza virus colloidal gold can increase the sensitivity more than ²⁷ times.

Example 4: Colloidal gold-enhanced detection test for H9N2 subtype avian influenza virus in virus-infected samples

Chickens were inoculated with H9N2 subtype avian influenza virus, and throat swabs and cloacal swabs were collected for detection. After the collected samples were eluted from the centrifuge, they were diluted with a developing agent, and the samples of each concentration were measured with commercially available avian influenza virus general-purpose gold standard detection test strips (Anigen Company). Each sample was measured twice, and each batch of determination was reserved 1 as a negative control. In the test, the test line and the control line show clear dark red, which means the sample is positive.

Using the sensitization detection method provided by the present invention, the sensitization reagent A (5% chloroauric acid) and the sensitization reagent B (5mM hydroxylamine hydrochloride) are mixed according to the volume ratio of 1: 1 to make a signal amplification reagent, respectively Take 80 μL of the signal amplification reagent for treatment. The specific treatment method is to add the signal amplification reagent between the gold-labeled antibody layer and the detection line of the test strip. The detection line and the control line show clear gray and black, which means the sample is positive. When using test strips for detection, that is, only No. 10 chicken throat swabs can be seen before the sensitization (see Figure 7), and after the test strips are sensitized, No. Obvious bands can be seen in chicken throat swabs (see Figure 8), which improves the detection sensitivity and can detect virus infection as early as possible in practical applications.

Embodiment 5: Sensitization test to colloidal gold early pregnancy detection test paper

Take the urine of pregnant women who are pregnant for 4 months and carry out doubling dilution with the developing agent, and use commercially available colloidal gold early pregnancy detection test paper (produced by Blue Cross Bio-Pharmaceutical Co., Ltd.) to measure the samples of each concentration, and measure each sample twice, each batch One strip was retained for the determination as a negative control. In the test, the test line and the control line show clear dark red, which means the sample is positive.

Using the sensitization detection method provided by the present invention, the sensitization reagent A (0.1% chloroauric acid) and the sensitization reagent B (0.1% ascorbic acid) are mixed according to the volume ratio of 1:5 to make a signal amplification reagent, respectively Take 10 μL of the signal amplification reagent for treatment. The specific treatment method is to add the signal amplification reagent between the gold-labeled antibody layer and the detection line of the test strip. The detection line and the control line show clear gray and black, which means the sample is positive. When using test strips to detect, the naked eye can see a positive band when it is diluted to ²⁹ when it is not sensitized, and the rest are all negative (see Figure 9). When the detection test strip is sensitized and diluted to ^2.15 , a positive band can be seen with the naked eye, and the sensitization multiple is ^2.6 times (see Figure 10).

Embodiment 6: Salmonella sensitization detection comparative test

The Salmonella Enteritidis broth culture solution was serially diluted until it was diluted to ^2-4 times, and the samples of each dilution were measured with commercially available Salmonella universal gold standard detection test strips (Dalian Prekang Co., Ltd.), and each sample The assay was performed twice, and one strip was retained as a negative control for each batch of assays. In the test, the test line and the control line show clear dark red, which means the sample is positive.

Using the sensitization detection method provided by the present invention, the sensitization reagent A (1% chloroauric acid) is mixed with the sensitization reagent B (0.5% ascorbic acid) according to the volume ratio of 1:3 to make a signal amplification reagent, respectively Take 60 μL of the signal amplification reagent for treatment. The specific treatment method is to add the signal amplification reagent between the gold-labeled antibody layer and the detection line of the test strip. The detection line and the control line show clear gray and black, which means the sample is positive. When using test strips for detection, only the stock solution sample (ID marked 0) can see the reaction band (see Figure 11), after the test strip is sensitized, the minimum 8-fold diluted sample (ID marked -3) Reaction bands can be seen (see Figure 12), and this gold standard sensitization method can increase the detection bottom line of Salmonella enteritidis by 8 times.

Example 7: Preparation and Application of Colloidal Gold Immunochromatography High Sensitivity Detection Kit

As shown in Figures 1 and 2, a colloidal gold immunochromatographic high-sensitivity detection kit includes a box body 1 and a test strip placed therein, and the upper surface of the box body 1 is provided with three openings: add Sample hole 2, add sensitizing reagent hole 3 and observation hole 4, described test paper strip comprises plastic backing plate 11, is pasted with one deck sample end absorbent paper layer 5 at the front end of the upper surface of plastic backing plate 11, described adding After the absorbent paper layer 5 at the sample end, the gold-labeled antibody layer 6 and the detection layer 9 are continuously overlapped until the rear end of the plastic backing plate 11, and the rear end of the upper surface of the detection layer 9 is pasted with a layer of water-absorbing end that can absorb at least 200 μL of liquid Water-absorbing paper layer 10, the gold-labeled antibody layer 6 is a nitrocellulose film layer coated with antibodies, and the detection layer 9 is a nitrocellulose film layer, which absorbs water at the gold-labeled antibody layer 6 and the water-absorbing end. The position between the paper layers 10 is coated with a detection line 7 and a control line 8 in sequence, the sample loading hole 2 is facing the absorbent paper layer 5 at the sample loading end, and the sensitizing reagent adding hole 3 is facing the gold-labeled antibody layer 6 The position between the detection line 7 and the observation hole 4 is facing the absorbent paper layer 10 at the absorbent end.

Wherein the preparation method of the gold-labeled antibody layer 6 comprises the following steps:

(1). Preparation of colloidal gold solution

a. Reagents and their volume ratios for preparing colloidal gold solution:

Double distilled water 100mL

1mL freshly prepared aqueous solution of chloroauric acid with a concentration of 1 to 5% by weight

1.0-1.5 mL of trisodium citrate aqueous solution with a concentration of 1-5% by weight;

b. Preparation method

Heat 100mL of the above-mentioned double-distilled water to 65°C with microwave, add 1ml of freshly prepared chloroauric acid aqueous solution with a concentration of 1 to 5% by weight, heat to 95°C with microwave, quickly add the required concentration of 1% by weight ～5% trisodium citrate aqueous solution, stirring constantly, under the action of sodium citrate reducing agent, chloroauric acid, the color of its colloidal gold solution is from black → blue → purple → wine red, and after cooling, a wine red 20 ~ 40nm Colloidal gold solution with particle size, the colloidal gold synthesized at this time can be stored at 4°C for 12 months;

(2). Preparation of gold-labeled antibody layer 6

Take 10 mL of the above-prepared colloidal gold solution, adjust the pH to 8.5 to 9.2 with an aqueous solution of potassium carbonate with a concentration of 1% by weight, and dissolve 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate and 0.24 g of potassium dihydrogen phosphate with 800 mL of distilled water , adjust the pH value of the solution to 7.4 with hydrochloric acid, add water to 1L, shake well to prepare 0.1mM phosphate buffer PB, dilute the antibody with the phosphate buffer PB to a protein content of 0.1-1mg/mL, take Centrifuge 0.1-0.5mL at 13,000rpm and 4°C for 30min, take out the corresponding volume from the centrifuged supernatant so that the minimum protein content of the liquid after adding to the colloidal gold solution is 10-20μg/mL, slowly stir the antibody Add dropwise to the above pH-adjusted colloidal gold solution, and leave it at room temperature for 5 minutes; then add 1 mL of filtered BSA solution with a concentration of 10% by weight, and continue stirring for 10 to 15 minutes, and then respectively at 10,000 rpm and 4°C Centrifuge for 30min, discard the supernatant to obtain the precipitate; then use 10mL of tris solution (TBS, containing 1% BSA) dissolves the precipitate, centrifuges at 10000rpm/min and 4°C for 30min, discards the supernatant, and uses 10mL of sodium azide, 1% sucrose, 1% BSA and Re-suspend the loose sediment at the bottom with a tris solution with a pH of 8.2 to obtain a gold-labeled antibody probe solution; finally, spray the gold-labeled antibody probe solution on the nitrocellulose membrane with gold spraying equipment until the liquid begins to ooze out , and dried at 37° C. for 2 hours to form a gold-labeled antibody layer 6 .

Wherein the preparation method of the detection layer 9 includes: the detection line 7 formed by the specific polyclonal or monoclonal antibody for the sample to be tested and the specific IgG antibody for the gold-labeled antibody are coated on the nitrocellulose membrane Formed control line 8: The specific preparation method is to take polyclonal antibody or monoclonal antibody, adjust the concentration to 1mg/mL with a developing reagent, spray the detection line 7 in the middle of the nitrocellulose membrane with a film sprayer; then take IgG antibody 1mg/mL, Use a film spraying machine to spray the control line 8 on the middle section of the nitrocellulose membrane, 0.5cm away from the detection line, set the amount of spray film at 20μL/10cm, and dry the spray film at 37°C for 2 hours.

When using this kit, take chicken organ or anal secretion solution, serum, whole blood, liver and spleen and other visceral extracts, dilute it with a developer reagent to 40-80 μL, and drop it on the sample addition of the immunochromatography kit. On well 2, each sample was repeated 2 to 3 times; after reacting at room temperature for 1 to 10 minutes, pre-mix the sensitizing reagent A and B according to the volume ratio of 1:1 to 1:5, and quickly take 10 to 80 μL and drop it on the Add the sensitizing reagent to well 3, and react at room temperature for 1 to 10 minutes. If the above secretion, serum, whole blood or organs contain antibodies, a detection line will be formed on the detection carrier, and the double line of the control line will be clear black. Positive, otherwise the control line is black, and the detection line is negative if there is no color.

Embodiment 8: Determination of the optimal pH value of the colloidal gold labeling of the colloidal gold immunochromatography high-sensitivity detection kit in embodiment 7

Take several 5mL test tubes, _add 1mL colloidal gold solution respectively, adjust the pH value of the solution to 3, 4, 5 _, 6, 7, 8, 9, 10, 11, 12, 13, 14; take a 96-well culture plate, add 100 μL of the above colloidal gold solutions into the wells according to the pH value from low to high, and repeat three times; add 3 μL weight percentage to each well The purified monoclonal antibody with a concentration of 1mg/mL was mixed evenly in the wells, and placed at room temperature for 15 minutes; 20 μL of 10% NaCl was added to each well, mixed evenly in the wells, and placed at room temperature for 10 minutes; observe the color change of colloidal gold , and measure its OD _520nm and OD _580nm respectively, and record the pH(X) when the difference between OD _520nm and OD _580nm is the largest; repeat the previous steps, and then further refine the pH value gradient, and set the pH value gradient as X -0.6; X-0.3; X; X+0.3; X+0.6; X+1, observe the color change of colloidal gold until it is left at room temperature for 2 hours, measure its OD _{520 nm} and OD _580nm values respectively, and record them in OD _520nm and The pH value at which the OD _580nm difference is the largest, based on which the suitable pH value for labeling is judged to be 8.5-9.2, and the preferred pH value is 8.7-9.2.

Embodiment 9: Determination of the minimum protein stable amount of the colloidal gold label of the colloidal gold immunochromatography high-sensitivity detection kit in embodiment 7

Salt precipitation assay: Use colorimetry to determine the optimum pH for gold labeling, adjust the pH value of colloidal gold to 8.7-9.2 with 0.1mol/LK ₂ CO ₃ , remove aggregates by high-speed centrifugation at 10,000rpm/min for antibodies, and use 0.01 for antigen or antibody Dilute the mol/L PB buffer solution (pH 8.0) to 0.2mg/mL, and the diluted antigen, antibody and other reagents should be operated according to Table 1.

Table 1 Determination of minimum protein content of stable colloidal gold by salt precipitation method

Add the reagents according to the above table and let it rest for 1-2 hours. The tubes containing small amounts of protein will turn from red to blue, while the solution in the test tubes with the added protein reaching or exceeding the minimum stable amount will remain red. The protein content that keeps the red color constant is the optimum protective amount of protein, and on this basis, an increase of 20% is the actual amount of protein used to stabilize the colloidal gold. The results show that when the protein content is 10-20 μg/mL, it is considered suitable; when the protein content is 10-16 μg/mL, it is more suitable.

Embodiment 10: The detection example of the colloidal gold immunochromatography high-sensitivity detection kit in embodiment 7: Newcastle disease virus live toxin sensitivity enhancement detection comparison test

Chicken embryos were inoculated with Newcastle disease virus, and the allantoic fluid of harvested chicken embryos was serially diluted with a developing agent until it was diluted to ^2-12 times. The self-prepared Newcastle disease virus gold standard detection test strip (coated antibody was rabbit high Serum-free, the labeled antibody is a specific monoclonal antibody) were measured on allantoic fluid samples of each dilution, each sample was measured twice, and one sample was reserved for each batch of determination as a negative control. In the detection, the test line and the control line show clear dark red, which means the sample is positive (see Figure 13).

Using the colloidal gold immunochromatography high-sensitivity detection kit provided by the present invention, the sensitizing reagent A (4% chloroauric acid) and the sensitizing reagent B (0.15 ascorbic acid) are mixed according to the volume ratio of 1:3 to prepare For the signal amplification reagent, take 20 μL of the signal amplification reagent and drop them into the sensitizing reagent well on the kit, and observe the observation hole 4 after reacting for 10 minutes. The detection line and the control line show clear gray and black, which means the sample is positive. The test results show that using the colloidal gold immunochromatography high-sensitivity detection kit provided by the present invention to detect live Newcastle disease virus can increase the sensitivity by more than ²⁷ times (see Figure 14).

The above-mentioned embodiments are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. All such modifications and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (4)

1. A signal amplification reagent for colloidal gold immunochromatography, characterized in that: it is a mixed reagent that sensitizing reagent A and sensitizing reagent B mix by a volume ratio of 1: 1～1: 5, said The sensitizing reagent A is a chloroauric acid solution with a weight percentage concentration of 1-4%, and the sensitizing reagent B is an ascorbic acid solution with a weight percentage concentration of 0.15%-0.3%. 2. signal amplifying reagent according to claim 1, is characterized in that: it is the mixing reagent that sensitizing reagent A and sensitizing reagent B are mixed by the volume ratio of 1: 3, and described sensitizing reagent A is weight percent concentration It is a 4% chloroauric acid solution, and the sensitizing reagent B is a 0.15% ascorbic acid solution by weight. 3. A sensitivity-enhancing detection method of colloidal gold immunochromatography, characterized in that: when colloidal gold immunochromatography detects the sample to be tested, after adding the sample to be tested, between the gold-labeled antibody layer and the detection line Add 10-80 μL of the signal amplification reagent described in claim 1 or 2, process the detection line obtained by colloidal gold immunochromatography to enhance the signal, and react at room temperature for 1-10 minutes to observe the results; the colloidal gold immunochromatography Chromatography test results are not intended for diagnosis and treatment of disease. 4. the application of the signal amplification reagent described in claim 1 or 2 in colloidal gold immunochromatography, the detection result of colloidal gold immunochromatography is not used for the diagnosis and treatment of disease.

Images