CN111505271A - Preparation method of olaquindox rapid detection test strip - Google Patents

Abstract

The invention relates to a preparation method of a olaquindox rapid detection test strip, which adopts a competition method for detection, utilizes the competition method to prepare the olaquindox test strip, adopts a marked monoclonal antibody-artificial antigen interception detection mode, sprays a T line on the olaquindox complete antigen, and then utilizes the competition method for detection, the detection method is simpler, the detection time is short, the detection speed is faster, the prepared olaquindox rapid detection test strip has small volume and light weight, is convenient to carry and store, can be operated by ordinary personnel, is suitable for the field detection of mass samples, and is easy to popularize and apply; the sample pad is treated by the developing agent, so that the sensitivity of the test strip can be improved, the matrix effect can be improved, the false positive rate can be reduced, the non-specificity can be reduced, the better fluidity of the liquid to be tested can be ensured, and the like; the preparation of the colloidal gold uses a glass instrument to be cleaned by aqua regia, so that the generation of colloidal gold particles can be effectively prevented from being interfered by a small amount of pollution on the surface of glass, the yield is high, and the production cost is low.

Description

A kind of preparation method of test strip for rapid detection of olaquindox

technical field

The invention relates to the technical field of preparation of test strips for rapid detection, in particular to a preparation method of test strips for rapid detection of olaquindox.

Background technique

Immunochromatographic test paper mainly adopts three detection methods: direct method, indirect method and competitive method. The direct method is mostly used for the detection of macromolecular substances. Its mechanism of action is to use the antibody of the macromolecular substance as the T line, and goat anti-mouse (IgG) as the C line. The antibody on the binding pad is combined to form an antigen-antibody complex, and then combined with the T line to form an antibody-antigen-antibody complex to aggregate and develop color. This method can also be called double-antibody sandwich method; the indirect method is also used for macromolecular substances. The mechanism of action is to use the specific antigen as the T line, and use the colloidal gold-labeled antibody or SPA to prepare the gold-labeled probe as the chromogenic reagent. It binds to the probe, and then binds to the specific antibody immobilized on the T line to develop color, and the final judgment result is consistent with that of the direct method. The competition method is mainly used for the detection of organic small molecule compounds that do not contain protein components. These small molecules can react with antibodies, that is, they are immunoreactive, but they are not immunogenic. Generally, small molecules are synthesized into macromolecules. To detect it, the mechanism of action of the competition method is to use the synthesized macromolecules as the T line and goat anti-mouse (IgG) as the C line. When the sample solution contains small molecules (haptens), the antibody on the binding pad takes precedence over the small molecule. Molecular binding without binding to macromolecules on the T-line aggregates and develops color. The detection mode of the competition method has two basic modes: the labeled monoclonal antibody-artificial antigen interception mode and the labeled artificial antigen-monomeric antibody interception mode. The labeled monoclonal antibody-artificial antigen interception mode is to use colloidal gold to label the monoclonal antibody that specifically recognizes the target antigen to prepare a binding pad. The binding pad is prepared with colloidal gold-labeled artificial antigen, the T line is fixed with a monoclonal antibody that specifically recognizes the target antigen, and the C line is sprayed with goat anti-mouse.

With the progress of the times, with people's continuous exploration, continuous scientific research, and continuous development, there are many detection methods developed by people, mainly including high performance liquid chromatography, liquid chromatography-mass spectrometry (HLPC-MS), Fluorescent microsphere immunochromatography, potential analysis, spectrophotometry, external spectrophotometry, near-infrared spectroscopy, and ELISA detection and other immunological analysis methods. However, the detection steps and operation methods of these methods are relatively complicated, requiring the use of instruments, and some even require relevant professional operators to perform operations, which are not suitable for on-site detection of large quantities of samples.

SUMMARY OF THE INVENTION

The object of the present invention is: in view of the above-mentioned problems, the present invention provides a simple process, the test strip has the advantages of simple and fast detection, simple operation method, everyone can operate, easy to carry, and the detection time is short. Preparation method of test strips.

The present invention is achieved through the following technical solutions:

The invention provides a preparation method of a test strip for rapid detection of olaquindox, comprising the following steps:

(1) Preparation of colloidal gold: Using trisodium citrate and chloroauric acid as raw materials, colloidal gold was prepared by trisodium citrate reduction method;

(2) Optimization of the binding concentration of colloidal gold and olaquindix antibody: adjust the pH of colloidal gold to 7.8-8.2, and divide into several parts; add different concentrations of olaquindox antibody to each part of colloidal gold, shake well, and let stand; Add sodium chloride solution to each part of colloidal gold, let stand, observe the color change, and it is concluded that the optimal concentration of colloidal gold combined with olaquindox antibody is 10µL to 15µL of olaquindine antibody added per milliliter of colloidal gold;

(3) Preparation of gold-labeled probes: adjust the pH of colloidal gold to 7.8 to 8.2, add olaquindox antibody according to the optimal concentration of colloidal gold combined with olaquindox antibody in step (2), and shake well; then add 10% Block with BSA and store at 2°C to 8°C to obtain the gold-labeled probe solution;

(4) Purification of gold-labeled probes: Take the gold-labeled probe solution, centrifuge at a speed of 8300 r/min to 8700 r/min, and take the precipitate; reconstitute the precipitate with 10% TBS buffer solution, and store at 2°C Store at ~8°C to obtain purified gold-labeled probes;

(5) Materials and structures required for the test strip: the test strip includes a PVC bottom plate, an NC film, a sample pad, a binding pad and a water-absorbing pad, and the NC film, the sample pad, the binding pad and the water-absorbing pad are all arranged on the PVC Above the bottom plate, from the lower edge to the upper edge of the PVC bottom plate are sequentially arranged: NC film, sample pad, bonding pad and water-absorbing pad;

The preparation of the test strip includes the following steps:

1) Take out the PVC bottom plate;

2) The NC membrane was streaked with quinolidine antigen and goat anti-mouse IgG antibody as the detection line and the quality control line, and dried;

3) An NC membrane is arranged on the upper surface of the PVC bottom plate, and the NC membrane is a nitrocellulose membrane;

4) Treat the sample pad with S17 surfactant with a glucose content of 2%-5% and a concentration of 0.1%-0.5% as a developing agent, and dry it;

5) A sample pad is arranged on the upper surface of the PVC bottom plate, and the sample pad is made of glass fiber film;

6) Evenly spray the purified gold-labeled probe on the binding pad and dry it;

7) A bonding pad is arranged on the upper surface of the PVC bottom plate, and the bonding pad is made of glass fiber film;

8) A water-absorbing pad is arranged on the upper surface of the PVC bottom plate to obtain the test strip for rapid detection of olaquindox.

Further, the glass instrument used in the step (1) is soaked in aqua regia before use, and the glass instrument is taken out, rinsed with clean water and ultrapure water, and dried before use.

Further, the preparation method of colloidal gold in the step (1) is as follows: 1) adding trisodium citrate and secondary water to the container, heating and refluxing under the condition of a water bath above 95°C and stirring constantly; 2) placing the container in the container Add chloroauric acid to the solution, continue to heat and stir until the solution turns wine red, remove it for natural cooling, bottle it, and store it at 2°C to 8°C to obtain the colloidal gold.

Further, in the step (2), 1 mol/L HCl solution and 0.2 mol/L K2CO3 solution are used to adjust the pH of the colloidal gold to 7.8-8.2, and divide them into several parts; add different parts to each part of the colloidal gold. 10% sodium chloride solution was added to each part of colloidal gold, let stand for 2 hours, and the color change was observed, and it was concluded that the optimal concentration of colloidal gold combined with olaquindox antibody was each The amount of olaquindix antibody added in milliliter of colloidal gold is 10µL to 15µL.

Further, in the step (3), the volume ratio of 10% BSA to quinolidine antibody is 4.5-5.5:1.

Further, in the step (4), the centrifugation time is 8 min to 12 min.

Further, in the step (5), the XYZ three-dimensional film scribing instrument HM3035 is used to scribe lines on the NC film.

Further, the processing method of the sample pad in the step (5) is as follows: soak the sample pad with S17 surfactant, and then put the sample pad into an oven at 37° C. to dry for 2 hours.

Further, in the step (5), the purified gold-labeled probe is uniformly sprayed on the binding pad by the XYZ three-dimensional film-scribing gold sprayer HM3035.

The present invention is a method for preparing a test strip for rapid detection of olaquindox. Because olaquindox is a small molecule, it cannot be detected by a direct method or an indirect method. Using the labelled monoclonal antibody-artificial antigen interception detection mode, the complete antigen of olaquindox is sprayed on the T line, and then the competition method is used for detection. Compared with the traditional olaquindox detection, it has the following advantages:

(1) The detection method is simpler, which makes the sample processing simpler, and the detection steps of manual participation are less, and the detection personnel no longer need centralized training, saving time and effort;

(2) The detection time is short and the detection speed is faster;

(3) The prepared olaquindox rapid detection test strip is small in size, light in weight, convenient to carry and store;

(4) There is no need for relevant professional operators to operate, and ordinary personnel can operate it. It is suitable for on-site detection of large quantities of samples, and is easy to popularize and apply.

The present invention provides a method for preparing a test strip for rapid detection of quinolone. In order to ensure that the sample liquid can flow better, each part must overlap to a certain extent during assembly, and the overlap can be about 1.5mm; in order to improve the sensitivity of the test strip To improve the matrix effect, reduce the false positive rate, reduce non-specificity and ensure better fluidity of the liquid to be tested, etc., the sample pad was treated with S17 surfactant with a concentration of 0.5% and sugar 0.4g, that is, a developing agent.

The invention provides a method for preparing a test strip for rapid detection of oquindox. Whether the concentrations of colloidal gold and labeled antibody match each other is a key factor affecting the experimental labeling. If the protein is excessive, it is very likely that the test strip will be dragged and the material will be wasted. ; If there is too little protein labeling, part of the colloidal gold and the antibody will be incompletely combined. Therefore, it is necessary to find the best labeling amount for the combination of protein and colloidal gold, so that the combination of colloidal gold and antibody is the most stable; the preparation method of the test strip for rapid detection of olaquindox of the present invention prepares 10% NaCl as an ionic electrolyte solution , the main purpose is to make some of the colloidal gold markers with unstable binding produce visible coagulation or coagulation due to inactivation. If the colloidal gold markers are inactivated, the solution color of the colloidal gold markers will no longer keep the original wine. There will be obvious changes, such as turning blue, sedimentation, and therefore, by using the NaCl solution, the appropriate optimal protein concentration value can be easily found by visual inspection.

The invention provides a method for preparing a test strip for rapid detection of oquindox. The glass instrument used in the preparation of colloidal gold is first cleaned with aqua regia, which can effectively avoid a small amount of contamination on the glass surface and interfere with the generation of colloidal gold particles, and the yield is high. , the production cost is low.

Description of drawings

Fig. 1 is the picture of repeatability test of Olaquindox rapid detection test strip;

Figure 2 is a picture of the test results of oquindox.

Detailed ways

Any feature disclosed in this specification (including any accompanying claims, abstract), unless expressly stated otherwise, may be replaced by other equivalent or alternative features serving a similar purpose. That is, unless expressly stated otherwise, each feature is but one example of a series of equivalent or similar features.

The "%" referred to in this application refers to the mass percentage.

The "absorbent pad" referred to in this application refers to filter paper.

Example 1:

A preparation method of Olaquindox rapid detection test strip, comprising the following steps:

(1) Preparation of colloidal gold: Using trisodium citrate and chloroauric acid as raw materials, colloidal gold was prepared by trisodium citrate reduction method; the glass instruments used were soaked in aqua regia before use, and the glass instruments were taken out and then separated. Rinse with clean water and ultrapure water, and dry before use; the preparation method of colloidal gold is as follows: 1) Add trisodium citrate and secondary water to the container, heat and reflux in a water bath above 95°C and keep stirring. ; 2) Add chloroauric acid to the container, continue heating and stirring until the solution turns wine red, remove it for natural cooling, bottling, and store at 2°C to 8°C to obtain the colloidal gold;

(2) Optimization of the binding concentration of colloidal gold and quindanol antibody: adjust the pH of colloidal gold to 8 with 1 mol/L HCl solution and 0.2 mol/L K2CO3 solution, and divide into several parts; add to each part of colloidal gold Different concentrations of olaquindox antibody, shake well, and let stand; then add 10% sodium chloride solution to each colloidal gold, let stand for 2 hours, and observe the color change. The addition amount of Olaquindox antibody per milliliter of colloidal gold is 10µL to 15µL;

Specifically, 1000 µL of colloidal gold was added to 6 EP tubes, and then the pH of the colloidal gold was adjusted to 8 with 1 mol/L HCl and 0.2 mol/L K2CO3, shaken well, shaken for 5 minutes, left to stand, and then separately Add 0µL, 2µL, 4µL, 6µL, 10µL, 15µL of Olaquindine antibody, shake well, let stand at 25°C for 4 minutes, add 40 µL of 10% sodium chloride solution, and let stand for 2 hours to observe the color change;

The results showed that the color of the liquid in tubes 1, 2, 3, and 4 to which 0µL, 2µL, 4µL, and 6µL of olaquindix antibodies were added had changes that could be observed with the naked eye. The liquid color has not changed, and the original wine red is maintained. Therefore, it is concluded that the optimal concentration of colloidal gold binding to olaquindix antibody is 10µL to 15µL of olaquindix antibody per milliliter of colloidal gold.

(3) Preparation of gold-labeled probes: adjust the pH of colloidal gold to 8, add olaquindox antibody according to the optimal concentration of colloidal gold binding to olaquindox antibody in step (2), shake well; then add 10% BSA to block , and stored at 2°C to 8°C to obtain a gold-labeled probe solution; the volume ratio of 10% BSA to quinolidine antibody is 5:1;

(4) Purification of gold-labeled probes: take the gold-labeled probe solution, centrifuge at 8500 r/min for 10 min, and take the precipitate; redissolve the precipitate with 10% TBS buffer solution, and heat it at 2℃～8℃ Stored under conditions to obtain purified gold-labeled probes;

(5) Materials and structures required for the test strip: the test strip includes a PVC bottom plate, an NC film, a sample pad, a binding pad and a water-absorbing pad, and the NC film, the sample pad, the binding pad and the water-absorbing pad are all arranged on the PVC Above the bottom plate, from the lower edge to the upper edge of the PVC bottom plate are sequentially arranged: NC film, sample pad, bonding pad and water-absorbing pad;

The preparation of the test strip includes the following steps:

1) Take out the PVC bottom plate;

2) Scribe on the NC membrane with Olaquindox antigen and goat anti-mouse IgG antibody respectively, as the detection line and the quality control line, and dry;

3) An NC membrane is arranged on the upper surface of the PVC bottom plate, and the NC membrane is a nitrocellulose membrane;

4) Treat the sample pad with S17 surfactant with a glucose content of 2% and a concentration of 0.1% as the developing agent, and dry; Glucose makes the glucose content in the developing agent 2%; the processing method of the sample pad is as follows: soak the sample pad with S17 surfactant, and then put the sample pad into an oven at 37°C to dry for 2 h;

5) A sample pad is arranged on the upper surface of the PVC bottom plate, and the sample pad is made of glass fiber film;

6) Spray the purified gold-labeled probe on the binding pad uniformly by XYZ three-dimensional gold-spraying instrument HM3035, and dry it;

7) A bonding pad is arranged on the upper surface of the PVC bottom plate, and the bonding pad is made of glass fiber film;

8) A water-absorbing pad is arranged on the upper surface of the PVC bottom plate to obtain the test strip for rapid detection of olaquindox.

Repeated experiments were carried out with the rapid detection test strips prepared by the present embodiment:

As shown in Figure 1, the results of the samples tested at different times are respectively, and the coincidence rate of the results is 100%. Put the test strip in a package bag with desiccant and store it at 25 degrees Celsius. Within a week, the test results obtained are consistent. When testing, the olaquindox test strip can be placed flat, and the liquid to be tested can be dropped into the small hole where the sample is dropped, or the test strip can be directly inserted into the olaquindox solution. Note that the liquid level cannot exceed the gold-labeled antibody. The position of the complex is then reacted at room temperature for about 2-5 minutes, which can be observed with the naked eye: if the positive is the color of the quality control line, if it is negative, both lines are colored.

The test strips for rapid detection of olaquindox prepared in this example are displayed for detection

In order to verify the practicability of the rapid detection test strips developed by the present invention, different samples are detected. The results showed that the quality control line developed color, as shown in Figure 2. In order to detect the minimum detection amount of the test strip, the sensitivity of the test strip was detected with a series of standard solutions of olaquindox concentration. The result shows that the minimum detection amount obtained is 6.67×10 ^-8 mg/mL, which shows that the olaquindox test developed by the present invention The paper strip can indeed accurately and quickly carry out qualitative detection of olaquindox.

Example 2:

A preparation method of Olaquindox rapid detection test strip, comprising the following steps:

(1) Preparation of colloidal gold: Using trisodium citrate and chloroauric acid as raw materials, colloidal gold was prepared by trisodium citrate reduction method; the glass instruments used were soaked in aqua regia before use, and the glass instruments were taken out and then separated. Rinse with clean water and ultrapure water, and dry before use; the preparation method of colloidal gold is as follows: 1) Add trisodium citrate and secondary water to the container, heat and reflux in a water bath above 95°C and keep stirring. ; 2) Add chloroauric acid to the container, continue heating and stirring until the solution turns wine red, remove it for natural cooling, bottling, and store at 2°C to 8°C to obtain the colloidal gold;

(2) Optimization of the binding concentration of colloidal gold and quindanol antibody: add 1000 µL of colloidal gold to 6 EP tubes, and adjust the pH of colloidal gold to 7.8 with 1 mol/L HCl solution and 0.2 mol/L K2CO3 solution , shake well, shake for 5 minutes, let stand, then add 0µL, 2µL, 4µL, 6µL, 10µL, 15µL of Olaquindox antibody, shake well, let stand at 25°C for 4 minutes, add 40 µL of 10% sodium chloride The solution was allowed to stand for 2 hours to observe the color change. The liquid color of tubes 5 and 6 added with 10 µL and 15 µL of olaquindix antibody did not change, and the original wine red was maintained. The addition amount of Olaquindox antibody per milliliter of colloidal gold is 10µL to 15µL;

(3) Preparation of gold-labeled probes: adjust the pH of colloidal gold to 7.8, add olaquindox antibody according to the optimal concentration of colloidal gold combined with olaquindox antibody in step (2), shake well; then add 10% BSA to block , and stored at 2°C to 8°C to obtain a gold-labeled probe solution; the volume ratio of 10% BSA to quinolidine antibody is 4.5:1;

(4) Purification of gold-labeled probes: Take the gold-labeled probe solution, centrifuge for 8 minutes at a rotational speed of 8300 r/min, and take the precipitate; redissolve the precipitate with 10% TBS buffer solution, and heat it at 2°C to 8°C Stored under conditions to obtain purified gold-labeled probes;

(5) Materials and structures required for the test strip: the test strip includes a PVC bottom plate, an NC film, a sample pad, a binding pad and a water-absorbing pad, and the NC film, the sample pad, the binding pad and the water-absorbing pad are all arranged on the PVC Above the bottom plate, from the lower edge to the upper edge of the PVC bottom plate are sequentially arranged: NC film, sample pad, bonding pad and water-absorbing pad;

The preparation of the test strip includes the following steps:

1) Take out the PVC bottom plate;

2) Scribe on the NC membrane with Olaquindox antigen and goat anti-mouse IgG antibody respectively, as the detection line and the quality control line, and dry;

3) An NC membrane is arranged on the upper surface of the PVC bottom plate, and the NC membrane is a nitrocellulose membrane;

4) Treat the sample pad with S17 surfactant with a glucose content of 5% and a concentration of 0.5% as the developing agent, and dry it; The pads were placed in an oven at 37°C for 2 h;

5) A sample pad is arranged on the upper surface of the PVC bottom plate, and the sample pad is made of glass fiber film;

6) Spray the purified gold-labeled probe on the binding pad uniformly by XYZ three-dimensional gold-spraying instrument HM3035, and dry it;

7) A bonding pad is arranged on the upper surface of the PVC bottom plate, and the bonding pad is made of glass fiber film;

8) A water-absorbing pad is arranged on the upper surface of the PVC bottom plate to obtain the test strip for rapid detection of olaquindox.

Repeated experiments were carried out with the rapid detection test strips prepared by the present embodiment:

Put the test strip in a package bag with desiccant and store it at 25 degrees Celsius. Within a week, the test results obtained are consistent. When testing, the olaquindox test strip can be placed flat, and the liquid to be tested can be dropped into the small hole where the sample is dropped, or the test strip can be directly inserted into the olaquindox solution. Note that the liquid level cannot exceed the gold-labeled antibody. The position of the complex is then reacted at room temperature for about 2-5 minutes, which can be observed with the naked eye: if the positive is the color of the quality control line, if it is negative, both lines are colored.

The test strips for rapid detection of olaquindox prepared in this example are displayed for detection

In order to verify the practicability of the rapid detection test strips developed by the present invention, different samples are detected. The results showed that the quality control line developed color. In order to detect the minimum detection amount of the test strip, the sensitivity of the test strip was detected with a series of standard solutions of olaquindox concentration. The result shows that the minimum detection amount obtained is 6.67×10 ^-8 mg/mL, which shows that the olaquindox test developed by the present invention The paper strip can indeed accurately and quickly carry out qualitative detection of olaquindox.

Example 3:

A preparation method of Olaquindox rapid detection test strip, comprising the following steps:

(1) Preparation of colloidal gold: Using trisodium citrate and chloroauric acid as raw materials, colloidal gold was prepared by trisodium citrate reduction method; the glass instruments used were soaked in aqua regia before use, and the glass instruments were taken out and then separated. Rinse with clean water and ultrapure water, and dry before use; the preparation method of colloidal gold is as follows: 1) Add trisodium citrate and secondary water to the container, heat and reflux in a water bath above 95°C and keep stirring. ; 2) Add chloroauric acid to the container, continue heating and stirring until the solution turns wine red, remove it for natural cooling, bottling, and store at 2°C to 8°C to obtain the colloidal gold;

(2) Optimization of the binding concentration of colloidal gold and quindanol antibody: add 1000 µL of colloidal gold to 6 EP tubes, and adjust the pH of colloidal gold to 8.2 with 1 mol/L HCl solution and 0.2 mol/L K2CO3 solution , shake well, shake for 5 minutes, let stand, then add 0µL, 2µL, 4µL, 6µL, 10µL, 15µL of Olaquindox antibody, shake well, let stand at 25°C for 4 minutes, add 40 µL of 10% sodium chloride The solution was allowed to stand for 2 hours to observe the color change. The liquid color of tubes 5 and 6 added with 10 µL and 15 µL of olaquindix antibody did not change, and the original wine red was maintained. The addition amount of Olaquindox antibody per milliliter of colloidal gold is 10µL to 15µL;

(3) Preparation of gold-labeled probes: adjust the pH of colloidal gold to 8.2, add olaquindox antibody according to the optimal concentration of colloidal gold binding to olaquindox antibody in step (2), and shake well; then add 10% BSA to block , and stored at 2°C to 8°C to obtain a gold-labeled probe solution; the volume ratio of 10% BSA to quinolidine antibody is 5.5:1;

(4) Purification of gold-labeled probes: take the gold-labeled probe solution, centrifuge at 8700 r/min for 12 min, and take the precipitate; redissolve the precipitate with 10% TBS buffer solution, and heat it at 2℃～8℃ Stored under conditions to obtain purified gold-labeled probes;

(5) Materials and structures required for the test strip: the test strip includes a PVC bottom plate, an NC film, a sample pad, a binding pad and a water-absorbing pad, and the NC film, the sample pad, the binding pad and the water-absorbing pad are all arranged on the PVC Above the bottom plate, from the lower edge to the upper edge of the PVC bottom plate are sequentially arranged: NC film, sample pad, bonding pad and water-absorbing pad;

The preparation of the test strip includes the following steps:

1) Take out the PVC bottom plate;

2) Scribe on the NC membrane with Olaquindox antigen and goat anti-mouse IgG antibody respectively, as the detection line and the quality control line, and dry;

3) An NC membrane is arranged on the upper surface of the PVC bottom plate, and the NC membrane is a nitrocellulose membrane;

4) Treat the sample pad with S17 surfactant with a glucose content of 3% and a concentration of 0.3% as a developing agent, and dry it; The pads were placed in an oven at 37°C for 2 h;

5) A sample pad is arranged on the upper surface of the PVC bottom plate, and the sample pad is made of glass fiber film;

6) Spray the purified gold-labeled probe on the binding pad uniformly by XYZ three-dimensional gold-spraying instrument HM3035, and dry it;

7) A bonding pad is arranged on the upper surface of the PVC bottom plate, and the bonding pad is made of glass fiber film;

8) A water-absorbing pad is arranged on the upper surface of the PVC bottom plate to obtain the test strip for rapid detection of olaquindox.

Repeated experiments were carried out with the rapid detection test strips prepared by the present embodiment:

Put the test strip in a package bag with desiccant and store it at 25 degrees Celsius. Within a week, the test results obtained are consistent. When testing, the olaquindox test strip can be placed flat, and the liquid to be tested can be dropped into the small hole where the sample is dropped, or the test strip can be directly inserted into the olaquindox solution. Note that the liquid level cannot exceed the gold-labeled antibody. The position of the complex is then reacted at room temperature for about 2-5 minutes, which can be observed with the naked eye: if the positive is the color of the quality control line, if it is negative, both lines are colored.

The test strips for rapid detection of olaquindox prepared in this example are displayed for detection

In order to verify the practicability of the rapid detection test strips developed by the present invention, different samples are detected. The results showed that the quality control line developed color. In order to detect the minimum detection amount of the test strip, the sensitivity of the test strip was detected with a series of standard solutions of olaquindox concentration. The result shows that the minimum detection amount obtained is 6.67×10 ^-8 mg/mL, which shows that the olaquindox test developed by the present invention The paper strip can indeed accurately and quickly carry out qualitative detection of olaquindox.

Although the present invention has been described in detail above with general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (9)

1. a preparation method of Olaquindox rapid detection test strip, is characterized in that, comprises the following steps: (1) Preparation of colloidal gold: Using trisodium citrate and chloroauric acid as raw materials, colloidal gold was prepared by trisodium citrate reduction method; (2) Optimization of the binding concentration of colloidal gold and olaquindix antibody: adjust the pH of colloidal gold to 7.8-8.2, and divide into several parts; add different concentrations of olaquindox antibody to each part of colloidal gold, shake well, and let stand; Add sodium chloride solution to each part of colloidal gold, let stand, observe the color change, and it is concluded that the optimal concentration of colloidal gold combined with olaquindox antibody is 10µL to 15µL of olaquindine antibody added per milliliter of colloidal gold; (3) Preparation of gold-labeled probes: adjust the pH of colloidal gold to 7.8 to 8.2, add olaquindox antibody according to the optimal concentration of colloidal gold combined with olaquindox antibody in step (2), and shake well; then add 10% Block with BSA and store at 2°C to 8°C to obtain the gold-labeled probe solution; (4) Purification of gold-labeled probes: Take the gold-labeled probe solution, centrifuge at a speed of 8300 r/min to 8700 r/min, and take the precipitate; reconstitute the precipitate with 10% TBS buffer solution, and store at 2°C Store at ~8°C to obtain purified gold-labeled probes; (5) Materials and structures required for the test strip: the test strip includes a PVC bottom plate, an NC film, a sample pad, a binding pad and a water-absorbing pad, and the NC film, the sample pad, the binding pad and the water-absorbing pad are all arranged on the PVC Above the bottom plate, from the lower edge to the upper edge of the PVC bottom plate are sequentially arranged: NC film, sample pad, bonding pad and water-absorbing pad; The preparation of the test strip includes the following steps: 1) Take out the PVC bottom plate; 2) The NC membrane was streaked with quinolidine antigen and goat anti-mouse IgG antibody as the detection line and the quality control line, and dried; 3) An NC membrane is arranged on the upper surface of the PVC bottom plate, and the NC membrane is a nitrocellulose membrane; 4) Treat the sample pad with S17 surfactant with a glucose content of 2%-5% and a concentration of 0.1%-0.5% as a developing agent, and dry it; 5) A sample pad is arranged on the upper surface of the PVC bottom plate, and the sample pad is made of glass fiber film; 6) Evenly spray the purified gold-labeled probe on the binding pad and dry it; 7) A bonding pad is arranged on the upper surface of the PVC bottom plate, and the bonding pad is made of glass fiber film; 8) A water-absorbing pad is arranged on the upper surface of the PVC bottom plate to obtain the test strip for rapid detection of olaquindox. 2. the preparation method of a kind of olaquindox rapid detection test strip according to claim 1, it is characterised in that the glass instrument used in the step (1) is put into aqua regia and soaked before use, and the glass instrument is taken out Rinse with clean water and ultrapure water respectively, and dry before use. 3. The preparation method of a test strip for rapid detection of olaquindox according to claim 1, wherein the preparation method of colloidal gold in the step (1) is: 1) adding trisodium citrate in the container 2) Add chloroauric acid to the container, continue to heat and stir until the solution turns wine red, remove it for natural cooling, bottle it, and place it at 2 The colloidal gold is obtained by storing under the condition of ℃～8℃. 4. the preparation method of a kind of Olaquindox rapid detection test strip according to claim 1, is characterized in that, in described step (2), use the HCl solution of 1 mol/L and the K of 0.2 mol/L CO Solution will be The pH of the colloidal gold was adjusted to 7.8-8.2, and divided into several parts; different concentrations of olaquindox antibodies were added to each part of the colloidal gold, shaken well, and allowed to stand; then 10% sodium chloride solution was added to each part of the colloidal gold, After standing for 2 hours, the color change was observed, and it was concluded that the optimal concentration of colloidal gold combined with olaquindix antibody was 10 μL to 15 μL of olaquindine antibody added per milliliter of colloidal gold. 5 . The method for preparing a test strip for rapid detection of olaquindox according to claim 1 , wherein in the step (3), the volume ratio of 10% BSA to olaquindox antibody is 4.5 to 5.5:1. 6 . 6 . The method for preparing a test strip for rapid detection of olaquindox according to claim 1 , wherein the centrifugation time in the step (4) is 8 min to 12 min. 7 . 7 . The preparation method of a test strip for rapid detection of olaquindox according to claim 1 , wherein in the step (5), a XYZ three-dimensional gold-spraying instrument HM3035 is used to scribe lines on the NC film. 8 . 8 . The preparation method of a test strip for rapid detection of oquindox according to claim 1 , wherein the processing method of the sample pad in the step (5) is: soaking the sample pad with S17 surfactant. 9 . Wet, and then put the sample pad into an oven at 37 °C to dry for 2 h. 9 . The method for preparing a test strip for rapid detection of oquindox according to claim 1 , wherein in the step (5), the purified gold-labeled probe is evenly distributed by an XYZ three-dimensional gold-spraying instrument HM3035 sprayed on the bonding pad.

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