Antibody-directed labeled colloidal gold immunoprobe and preparation method thereof, and lead ion test paper based on antibody-directed labeling

Abstract

The present invention discloses an antibody-oriented colloidal gold immunoprobe and a preparation method thereof, and a lead ion test paper based on antibody-oriented labeling. The antibody-oriented colloidal gold immunoprobe comprises the following components: colloidal gold particles, a connecting protein, and an anti-lead ion monoclonal antibody; wherein the connecting protein is used to connect the colloidal gold particles and the anti-lead ion monoclonal antibody, and the connecting protein comprises a protein that specifically recognizes the Fc fragment of the monoclonal antibody. The present invention aims to solve the problem that the existing lead ion test paper cannot control the spatial orientation of the antibody and the antibody effective labeling is low.

Description

Technical Field

[0001] The invention relates to the technical field of lead ion testing, and in particular to an antibody-directed labeled colloidal gold immunoprobe and a preparation method thereof, as well as a lead ion test paper based on antibody-directed labeling. Background Art

[0002] Lead is one of the heavy metal elements that has a great toxic effect on human health. It is widely used in industries such as paints, batteries and building materials. It can accumulate in the environment and enter the human body through the food chain, seriously endangering people's health. Therefore, the research on the detection technology of heavy metal lead is of great significance to protecting people's health.

[0003] At present, the main detection technologies for heavy metal lead include atomic absorption spectroscopy, atomic emission spectroscopy, inductively coupled plasma mass spectrometry, atomic fluorescence spectroscopy, stripping voltammetry and dithizone colorimetry. The above detection methods are sensitive and accurate, but they require complex instruments and equipment or specialized instrument operators, and are not suitable for rapid on-site detection of lead ions. Therefore, it is urgent to develop a simpler, more efficient and sensitive lead ion detection technology. Colloidal gold immunochromatographic test strip technology is convenient, fast and intuitive, and is widely used in the rapid detection of toxic and harmful substances such as heavy metals.

[0004] However, colloidal gold immunoprobe preparation mainly adopts physical adsorption and chemical binding methods to achieve antibody labeling on the colloidal gold surface. The above labeling method is simple to operate and time-consuming, but it cannot control the spatial orientation of the antibody on the colloidal gold surface, which affects the exposure of the antibody antigen binding site (Fab) and reduces the effective labeling of the antibody on the colloidal gold surface. Therefore, providing a method for detecting lead ions quickly and efficiently that can control the spatial orientation of the antibody is a technical problem that needs to be solved urgently. Summary of the invention

[0005] The main purpose of the present invention is to provide an antibody-directed labeled colloidal gold immunoprobe and a preparation method thereof, as well as a lead ion test paper based on antibody-directed labeling, aiming to solve the problem that the existing lead ion test paper cannot control the spatial orientation of the antibody and has low effective antibody labeling.

[0006] To achieve the above object, the present invention provides an antibody-directed labeled colloidal gold immunoprobe, characterized in that it comprises the following components: colloidal gold particles, connexins and anti-lead ion monoclonal antibodies;

[0007] Wherein, the connecting protein is used to connect the colloidal gold particles and the anti-lead ion monoclonal antibody, and the connecting protein includes a protein that specifically recognizes the Fc fragment of the monoclonal antibody.

[0008] Optionally, the protein that specifically recognizes the Fc fragment of a monoclonal antibody includes recombinant protein A or recombinant protein G.

[0009] The present invention also proposes a method for preparing an antibody-directed labeled colloidal gold immunoprobe, comprising the following steps:

[0010] The pH value of the colloidal gold particles is adjusted to 7-10, the connecting protein is added and stirred evenly, and then the bovine serum albumin solution is added, and the first precipitate is obtained after centrifugation;

[0011] dissolving the first precipitate in a phosphate buffer to obtain a colloidal gold-connectin complex;

[0012] adding anti-lead ion monoclonal antibody to the colloidal gold-connectin complex, and obtaining a second precipitate after centrifugation;

[0013] The second precipitate is dissolved in a phosphate buffer to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0014] Optionally, in the step of adjusting the pH value of the colloidal gold particles to 7-10, adding the connexin, and obtaining the first precipitate after centrifugation,

[0015] The mass ratio of the colloidal gold particles to the connexin is 100:(2-20).

[0016] Optionally, the step of adjusting the pH value of the colloidal gold particles to 7-10, adding connexin, and obtaining the first precipitate after centrifugation includes: adding potassium carbonate solution to the colloidal gold particles, adjusting the pH value to 7-10, adding connexin, reacting at 25-37° C. for 20-60 min, adding a bovine serum albumin solution with a concentration of 0.45-0.55%, reacting at 25-37° C. for 20-60 min, and then centrifuging at a speed of 8000-12000 r / min for 5-15 min, and obtaining the first precipitate after centrifugation 3-8 times.

[0017] Optionally, in the step of adding anti-lead ion monoclonal antibody to the colloidal gold-connectin complex and obtaining a second precipitate after centrifugation,

[0018] The mass ratio of the colloidal gold particles to the anti-lead ion monoclonal antibody is 100:(10-20).

[0019] Optionally, the step of adding anti-lead ion monoclonal antibodies to the colloidal gold-connectin protein complex and obtaining a second precipitate after centrifugation includes: adding anti-lead ion monoclonal antibodies to the colloidal gold-connectin protein complex, reacting at 25-37° C. for 60-150 min, then centrifuging at a speed of 6000-12000 r / min for 6-12 min, and centrifuging 2-4 times to obtain a second precipitate.

[0020] In addition, the present invention also proposes a lead ion test paper based on antibody directional labeling, comprising:

[0021] An antibody-directed labeled colloidal gold immunoprobe, the antibody-directed labeled colloidal gold immunoprobe comprising the following components: colloidal gold particles, a connecting protein and an anti-lead ion monoclonal antibody; wherein the connecting protein is used to connect the colloidal gold particles and the anti-lead ion monoclonal antibody, and the connecting protein comprises a protein that specifically recognizes the Fc fragment of the monoclonal antibody; and,

[0022] A test strip, the test strip comprising a water absorbent pad, a nitrocellulose membrane, a sample pad and a backing plate, wherein the water absorbent pad, the nitrocellulose membrane and the sample pad are sequentially arranged on the backing plate along a first direction, and one end of each of the water absorbent pad and the sample pad connected to the nitrocellulose membrane is arranged at the upper end of the nitrocellulose membrane and at least wraps a portion of the nitrocellulose membrane.

[0023] Optionally, a quality control line and a detection line are sequentially arranged along a first direction on the upper side of the nitrocellulose membrane, and the quality control line and the detection line are extended along a second direction. The material of the quality control line includes rabbit antibodies against mouse monoclonal antibodies or sheep antibodies against mouse monoclonal antibodies, and the material of the detection line includes lead ion antigens.

[0024] Optionally, the concentration of the rabbit antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; and / or,

[0025] The concentration of the goat antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; and / or,

[0026] The concentration of the lead ion antigen is 0.2-0.5 mg / mL.

[0027] In the technical solution of the present invention, the antibody-oriented colloidal gold immunoprobe is used to achieve rapid, effective, sensitive and efficient detection of lead ions, with low detection cost, and can effectively control the spatial orientation of the anti-lead ion monoclonal antibody on the surface of the colloidal gold particles, thereby improving effective labeling; wherein, the connexin serves as a connecting medium between the colloidal gold particles and the anti-lead ion monoclonal antibody, and the connexin can specifically recognize the crystallizable fragment (i.e., Fc fragment) on the anti-lead ion monoclonal antibody, thereby ensuring that the antigen binding site of the anti-lead ion monoclonal antibody can be fully exposed, thereby improving the effective labeling of the anti-lead ion monoclonal antibody on the surface of the colloidal gold particles, reducing the usage of the anti-lead ion monoclonal antibody to a certain extent, and reducing the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0029] Figure 1 A schematic diagram of the principle of antibody-directed labeling of the antibody-directed labeling colloidal gold immunoprobe provided by the present invention;

[0030] Figure 2 A schematic diagram of the process for preparing the antibody-directed labeled colloidal gold immunoprobe provided by the present invention;

[0031] Figure 3 A schematic structural diagram of an embodiment of a test strip provided by the present invention;

[0032] Figure 4 for Figure 3 A schematic diagram of the structure of the test strip from another perspective;

[0033] Figure 5 Characterization diagram of the colloidal gold-connectin complex and the antibody-directed labeled colloidal gold immunoprobe in Example 1 provided by the present invention;

[0034] Figure 6 Characterization diagram of the colloidal gold-connectin complex and the antibody-directed labeled colloidal gold immunoprobe in Example 2 provided by the present invention;

[0035] Figure 7 Characterization diagram of the colloidal gold-connexin complex and the antibody-directed labeled colloidal gold immunoprobe in Example 3 provided by the present invention;

[0036] Figure 8Characterization diagram of the colloidal gold-connexin complex and the antibody-directed labeled colloidal gold immunoprobe in Example 4 provided by the present invention;

[0037] Fig. 9 Characterization diagram of the colloidal gold-connexin complex and the antibody-directed labeled colloidal gold immunoprobe in Example 5 provided by the present invention;

[0038] Fig.10 A lead ion concentration detection diagram of Example 1 provided by the present invention;

[0039] Fig.11 A lead ion concentration detection diagram of Example 2 provided by the present invention;

[0040] Fig.12 A lead ion concentration detection diagram of Example 3 provided by the present invention;

[0041] Fig.13 A lead ion concentration detection diagram of Example 4 provided by the present invention;

[0042] Fig.14 This is a lead ion concentration detection diagram of Example 5 provided by the present invention.

[0043]

[0044] Description of Figure Numbers:

[0045] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0046] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0047] The preparation of colloidal gold immunoprobe mainly adopts physical adsorption and chemical binding methods to achieve the labeling of antibodies on the colloidal gold surface. The above labeling method is simple to operate and time-saving, but it cannot control the spatial orientation of antibodies on the colloidal gold surface, which affects the exposure of the antibody's antigen binding site and reduces the effective labeling of antibodies on the colloidal gold surface.

[0048] In view of this, the present invention provides an antibody-directed labeled colloidal gold immunoprobe. The antibody-directed labeled colloidal gold immunoprobe provided by the present invention is used to detect the lead ion concentration, which can ensure the full exposure of the antibody antigen binding site (Fab), improve the effective labeling of the antibody on the colloidal gold surface, and reduce the amount of antibody used; Figure 1 This is a schematic diagram of an embodiment of the antibody-directed labeled colloidal gold immunoprobe provided by the present invention. Figure 2 This is a schematic diagram of the process of an embodiment of the antibody-directed labeling colloidal gold immunoprobe provided by the present invention. Figure 3 A schematic diagram of an embodiment of the lead ion test paper based on antibody directional labeling provided by the present invention; Figures 4 to 5 Characterization diagram of the colloidal gold-connectin complex and antibody-directed labeled colloidal gold immunoprobe provided by the present invention.

[0049] See also Figure 1 The antibody-directed labeled colloidal gold immunoprobe 100 includes the following components: colloidal gold particles 1, connecting proteins 2 and anti-lead ion monoclonal antibodies 3; wherein the connecting proteins 2 are used to connect the colloidal gold particles 1 and the anti-lead ion monoclonal antibodies 3, and the connecting proteins 2 include proteins that specifically recognize monoclonal antibody Fab fragments.

[0050] In the technical solution of the present invention, the antibody-oriented colloidal gold immunoprobe 100 is used to achieve rapid, effective, sensitive and efficient detection of lead ions, with low detection cost, and can effectively control the spatial orientation of the anti-lead ion monoclonal antibody 3 on the surface of the colloidal gold particle 1, thereby improving effective labeling; wherein, the connexin 2 serves as a connection medium between the colloidal gold particle 1 and the anti-lead ion monoclonal antibody 3, and the connexin 2 can specifically recognize the crystallizable fragment (i.e., Fc fragment) on the anti-lead ion monoclonal antibody 3, thereby ensuring that the antigen binding site of the anti-lead ion monoclonal antibody 3 can be fully exposed, thereby improving the effective labeling of the anti-lead ion monoclonal antibody 3 on the surface of the colloidal gold particle 1, reducing the usage of the anti-lead ion monoclonal antibody 3 to a certain extent, and reducing the detection cost.

[0051] Furthermore, in this embodiment, when selecting the protein that specifically recognizes the Fc fragment of the monoclonal antibody, it is necessary to ensure that the antigen binding site (Fab) of the anti-lead ion monoclonal antibody 3 is fully exposed. Therefore, in one embodiment, the protein that specifically recognizes the Fc fragment of the monoclonal antibody is selected as recombinant protein A, and in another embodiment, the protein that specifically recognizes the Fc fragment of the monoclonal antibody is selected as recombinant protein G; both the recombinant protein A and the recombinant protein G can ensure that the antigen binding site (Fab) of the anti-lead ion monoclonal antibody 3 is fully exposed, thereby improving the effective labeling of the anti-lead ion monoclonal antibody 3 on the surface of the colloidal gold particles 1.

[0052] Also, see Figure 2 The present invention provides a method for preparing an antibody-directed labeled colloidal gold immunoprobe. The antibody-directed labeled colloidal gold immunoprobe 100 prepared by the method for preparing the antibody-directed labeled colloidal gold immunoprobe provided by the present invention is used to detect lead ion concentration, which can ensure full exposure of the antibody antigen binding site (Fab), improve the effective labeling of the antibody on the colloidal gold surface, reduce the amount of antibody used, and reduce the detection cost. The method for preparing the antibody-directed labeled colloidal gold immunoprobe comprises the following steps:

[0053] Step S10, adjusting the pH value of the colloidal gold particles 1 to 7-10, adding the connexin 2 and stirring evenly, then adding the bovine serum albumin 4 solution, and obtaining the first precipitate after centrifugation.

[0054] In this embodiment, before preparing the first precipitate, it is necessary to adjust the pH value of the colloidal gold particles 1 to between 7 and 10. The specific operation is to add potassium carbonate solution to the colloidal gold particles when adjusting the colloidal gold particles 1, and then stir evenly. The purpose of adjusting the pH value to between 7 and 10 is to provide a friendly environment for the connexin 2 so that the connexin 2 can be stably adsorbed on the surface of the colloidal gold particles 1.

[0055] In this embodiment, the purpose of adding the connexin 2 is to serve as a connecting medium between the colloidal gold particles 1 and the anti-lead ion monoclonal antibodies 3. The connexin 2 can specifically recognize the crystallizable fragment (i.e., Fc fragment) on the anti-lead ion monoclonal antibodies, thereby ensuring that the antigen binding site (Fab) of the anti-lead ion monoclonal antibodies 3 can be fully exposed, thereby improving the effective labeling of the anti-lead ion monoclonal antibodies 3 on the surface of the colloidal gold particles 1.

[0056] In addition, during the reaction, the reaction temperature and reaction time have an impact on the quality of the antibody-oriented colloidal gold immunoprobe 100 finally prepared. Specifically, when performing step S10, the following steps can be performed: adding potassium carbonate solution to the colloidal gold particles 1, adjusting the pH value to 7-10, adding connexin 2, reacting at 25-37°C for 20-60 minutes, then adding a bovine serum albumin 4 solution with a concentration of 0.45-0.55%, reacting at 25-37°C for 20-60 minutes, and then centrifuging at a speed of 8000-12000 r / min for 5-15 minutes, and obtaining the first precipitate after centrifugation 3-8 times. The purpose of multiple centrifugation is to ensure that the free connexin 2 and the bovine serum albumin 4 can be fully separated from the first precipitate, saving the waste of the anti-lead ion monoclonal antibody 3 in the subsequent steps and saving manufacturing costs.

[0057] In this embodiment, the mass ratio of the colloidal gold particles 1 to the connexin 2 is 100:(2-20).

[0058] Step S20, dissolving the first precipitate in a phosphate buffer to obtain a colloidal gold-connexin complex.

[0059] In this embodiment, the concentration of the phosphate buffer is 0.01 mol / L.

[0060] Step S30, adding the anti-lead ion monoclonal antibody 3 to the colloidal gold-connectin complex, and obtaining a second precipitate after centrifugation.

[0061] Specifically, when performing step S30, the following steps can be performed: adding the anti-lead ion monoclonal antibody 3 to the colloidal gold-connected protein complex, reacting at 25 to 37° C. for 60 to 150 min, then centrifuging at a speed of 6000 to 12000 r / min for 6 to 12 min, centrifuging 2 to 4 times, and obtaining a second precipitate.

[0062] Wherein, the mass ratio of the colloidal gold particles 1 to the anti-lead ion monoclonal antibody 3 is 100:(10-20).

[0063] Step S40 , dissolving the second precipitate in a phosphate buffer to obtain the antibody-directed labeled colloidal gold immunoprobe 100 .

[0064] In this embodiment, the concentration of the phosphate buffer is 0.01 mol / L.

[0065] In addition, the present invention also provides a lead ion test paper based on antibody directional labeling. The lead ion test paper based on antibody directional labeling provided by the present invention is used to test the lead ion concentration, which can achieve rapid, sensitive and efficient detection of lead ions. The lead ion test paper based on antibody directional labeling includes an antibody directional labeling colloidal gold immunoprobe 100 and a test strip 200; the specific structure and preparation method of the antibody directional labeling colloidal gold immunoprobe 100 refer to the above embodiment. Since the antibody directional labeling colloidal gold immunoprobe 100 adopts all the technical solutions of all the above embodiments, it at least has all the beneficial effects brought by the technical solutions of the above embodiments, which will not be repeated here. The test strip 200 includes a water absorbent pad 10, a nitrocellulose membrane 20, a sample pad 30 and a backing plate 40, wherein the water absorbent pad 10, the nitrocellulose membrane 20 and the sample pad 30 are sequentially arranged on the backing plate 40 along a first direction, and the ends of the water absorbent pad 10 and the sample pad 30 connected to the nitrocellulose membrane 20 are arranged at the upper end of the nitrocellulose membrane 20, and at least part of the nitrocellulose membrane 20 is wrapped.

[0066] In this embodiment, one end of each of the absorbent pad 10 and the sample pad 30 connected to the nitrocellulose membrane 20 is arranged at the upper end of the nitrocellulose membrane 20, and at least wraps a portion of the nitrocellulose membrane 20. The purpose of such a setting is to facilitate the diffusion of the detection reagent during detection, so that the detection liquid can quickly diffuse onto the nitrocellulose membrane 20, thereby improving the detection efficiency. In addition, in order to ensure the reliability of the detection result, the absorbent pad 10 at least wraps the nitrocellulose membrane 20 with a length of 1 to 3 mm, and the sample pad 30 at least wraps the nitrocellulose membrane 20 with a length of 1 to 3 mm. It should be noted that within this range, the detection liquid can quickly diffuse onto the nitrocellulose membrane 20, and will not affect the detection of the nitrocellulose membrane 20. If the length of the sample pad 30 or the absorbent pad 10 wrapped around the nitrocellulose membrane 20 is less than 1 mm, the diffusion time of the detection liquid will be prolonged and the detection efficiency will be reduced; if the length of the absorbent pad 10 or the sample pad 30 wrapped around the nitrocellulose membrane 20 is greater than 3 mm, it will affect the feedback of the test results of the nitrocellulose membrane 20, resulting in unreliable test data.

[0067] See also Figure 3, a quality control line 201 and a detection line 202 are sequentially arranged on the upper side of the nitrocellulose membrane 20 along the first direction, the quality control line 201 and the detection line 202 are extended along the second direction, the material of the quality control line 201 includes rabbit antibodies against mouse monoclonal antibodies or sheep antibodies against mouse monoclonal antibodies, and the material of the detection line 202 includes lead ion antigens. Specifically, in practical applications, the quality control line 201 is arranged on the side of the nitrocellulose membrane 20 close to the water absorbent pad 10, and the detection line 202 is arranged on the side of the nitrocellulose membrane 20 close to the sample pad 30. Such an arrangement is more in line with the use habits of the detection personnel, is convenient for detection, and improves detection efficiency.

[0068] Furthermore, the concentration of the rabbit antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; the concentration of the goat antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; and the concentration of the lead ion antigen is 0.2-0.5 mg / mL.

[0069] Furthermore, the scratching volume of the quality control line 201 is 0.5-0.6 μL / cm, and the scratching volume of the detection line 202 is 0.5-0.6 μL / cm.

[0070] Specifically, in this embodiment, the test strip 200 can be prepared by the following method: the absorbent pad 10, the nitrocellulose membrane 20 and the sample pad 30 are respectively adhered along a first direction on the sticky side surface of the backing plate 40, wherein it should be noted that during the pasting process, the absorbent pad 10 overlaps with the nitrocellulose membrane 20 by 1 to 3 mm, and the sample pad 30 overlaps with the nitrocellulose membrane 20 by 1 to 3 mm.

[0071] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0072] Example 1

[0073] (1) Preparation of antibody-directed labeled colloidal gold immunoprobe

[0074] Potassium carbonate solution was added dropwise to the colloidal gold particles, the pH value was adjusted to 7.3, recombinant protein G was added at a mass ratio of 100:18 of colloidal gold particles and recombinant protein G, the reaction was carried out at 37°C for 30 minutes, a 0.5% bovine serum albumin solution was added, the reaction was carried out at 37°C for 30 minutes, and the mixture was centrifuged at a speed of 9500 r / min for 13 minutes. After seven centrifugal treatments, the first precipitate was obtained.

[0075] The first precipitate was dissolved in a phosphate buffer having a concentration of 0.01 mol / L to obtain a colloidal gold-connectin complex.

[0076] Anti-lead ion monoclonal antibodies were added to the colloidal gold-connectin protein complex at a mass ratio of 100:17.5, and the mixture was reacted at 37° C. for 120 min. The mixture was then centrifuged at 8000 r / min for 10 min, and centrifuged three times to obtain a second precipitate and a supernatant.

[0077] The second precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L (the amount of the phosphate buffer was 10% of the total volume of the second precipitate and the supernatant) to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0078] (2) Preparation of test strips

[0079] A water-absorbing pad, a nitrocellulose membrane and a sample pad are respectively adhered to the sticky side of the liner along the first direction, wherein the overlapping distances between the water-absorbing pad and the sample pad and the nitrocellulose membrane are both 2 mm, the quality control line is coated with 0.5 mg / mL of anti-mouse monoclonal antibody sheep antibody, and the detection line is coated with 0.3 mg / mL of lead ion antigen, and the film-stripping volume of the quality control line and the detection line is 0.6 μL / cm.

[0080] (3) Detection of lead ion concentration

[0081] Obtain a lead ion standard solution, add disodium ethylenediaminetetraacetic acid chelating agent to the lead ion standard solution, react at 37°C for 10 minutes, then add a potassium hydroxide solution with a concentration of 1 mol / L, adjust the pH to 7.4, and dilute to a lead ion standard solution with a certain concentration gradient (wherein the lead ion standard solution with a concentration gradient is 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL and 10 ng / mL). Add the lead ion standard solution with the above concentration gradient and the antibody-directed labeled colloidal gold immunoprobe solution to the microwell and mix evenly, then insert the sample pad end of the lead ion test strip into the microwell, react at room temperature for 15 minutes, and observe with the naked eye whether the quality control line is colored. At the same time, compare the color of the test line on the test strip with the standard color card, observe the color depth of the test line, and the lead ion concentration can be obtained.

[0082] It should be noted that, in the present invention, the standard color card refers to the color presented by the test line on the test strip when the lead ion standard solution is 0 ng / mL.

[0083] Please refer to the test results Figure 5 and Fig.10 ,Depend on Figure 5The characterization diagram shows that the recombinant protein G successfully linked the colloidal gold particles and the anti-lead ion monoclonal antibody. Fig.10 It can be seen that the test strips can clearly detect different concentrations of lead ions (it should be noted that Fig.10 The 0, 2, 4, 6, 8, and 10 in the figure represent the lead ion standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, and 10 ng / mL, respectively).

[0084] Example 2

[0085] (1) Preparation of antibody-directed labeled colloidal gold immunoprobe

[0086] Potassium carbonate solution was added dropwise to the colloidal gold particles, the pH value was adjusted to 9.8, recombinant protein A was added in a mass ratio of 100:3 of colloidal gold particles and recombinant protein A, the reaction was carried out at 28°C for 50 minutes, a bovine serum albumin solution with a concentration of 0.55% was added, the reaction was carried out at 28°C for 50 minutes, and then the mixture was centrifuged at a speed of 11000 r / min for 12 minutes. After six centrifugal treatments, the first precipitate was obtained.

[0087] The first precipitate was dissolved in a phosphate buffer having a concentration of 0.01 mol / L to obtain a colloidal gold-connectin complex.

[0088] Anti-lead ion monoclonal antibodies were added to the colloidal gold-connectin protein complex at a mass ratio of 100:12.3, and the mixture was reacted at 28°C for 150 minutes. The mixture was then centrifuged at 10,000 r / min for 8 minutes, and centrifuged three times to obtain a second precipitate and a supernatant.

[0089] The second precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L (the amount of the phosphate buffer was 10% of the total volume of the second precipitate and the supernatant) to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0090] (2) Preparation of test strips

[0091] A water absorbent pad, a nitrocellulose membrane and a sample pad are respectively adhered along a first direction on one sticky side of the liner, wherein the overlapping distances between the water absorbent pad and the sample pad and the nitrocellulose membrane are both 2 mm, the quality control line is coated with 0.5 mg / mL of anti-mouse monoclonal antibody sheep antibody, the detection line is coated with 0.3 mg / mL of lead ion antigen, and the synovial volume of the quality control line and the detection line is 0.6 μL / cm.

[0092] (3) Detection of lead ion concentration

[0093] Obtain a lead ion standard solution, add disodium ethylenediaminetetraacetic acid chelating agent to the lead ion standard solution, react at 28°C for 10 minutes, then add a potassium hydroxide solution with a concentration of 1 mol / L, adjust the pH to 7.4, and dilute to a lead ion standard solution with a certain concentration gradient (wherein the lead ion standard solution with a concentration gradient is 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL and 10 ng / mL). Add the lead ion standard solution with the above concentration gradient and the antibody-directed labeled colloidal gold immunoprobe solution to the microwell and mix evenly, then insert the sample pad end of the lead ion test strip into the microwell, react at room temperature for 15 minutes, and observe with the naked eye whether the quality control line is colored. At the same time, compare the color of the test line on the test strip with the standard color card, observe the color depth of the test line, and the lead ion concentration can be obtained.

[0094] Please refer to the test results Figure 6 and Fig.11 ,Depend on Figure 6 The characterization diagram shows that the recombinant protein A successfully linked the colloidal gold particles and the anti-lead ion monoclonal antibody. Fig.11 It can be seen that the test strips can clearly detect different concentrations of lead ions (it should be noted that Fig.11 The 0, 2, 4, 6, 8, and 10 in the figure represent the lead ion standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, and 10 ng / mL, respectively).

[0095] Example 3

[0096] (1) Preparation of antibody-directed labeled colloidal gold immunoprobe

[0097] Potassium carbonate solution was added dropwise to the colloidal gold particles, the pH value was adjusted to 7.5, recombinant protein G was added in a mass ratio of 100:20 of colloidal gold particles and recombinant protein G, the reaction was carried out at 25°C for 20 minutes, a bovine serum albumin solution with a concentration of 0.45% was added, the reaction was carried out at 25°C for 20 minutes, and then the mixture was centrifuged at a speed of 8000 r / min for 5 minutes. After three centrifugal treatments, the first precipitate was obtained.

[0098] The first precipitate was dissolved in a phosphate buffer having a concentration of 0.01 mol / L to obtain a colloidal gold-connectin complex.

[0099] Anti-lead ion monoclonal antibodies were added to the colloidal gold-connectin protein complex at a mass ratio of 100:20, and the mixture was reacted at 25° C. for 60 min. The mixture was then centrifuged at 6000 r / min for 6 min, and centrifuged twice to obtain a second precipitate and a supernatant.

[0100] The second precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L (the amount of the phosphate buffer was 10% of the total volume of the second precipitate and the supernatant) to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0101] (2) Preparation of test strips

[0102] A water absorbent pad, a nitrocellulose membrane and a sample pad are respectively adhered along a first direction on one sticky side of the liner, wherein the overlapping distances between the water absorbent pad and the sample pad and the nitrocellulose membrane are both 1 mm, the quality control line is coated with 0.45 mg / mL rabbit antibody of anti-mouse monoclonal antibody, the detection line is coated with 0.3 mg / mL lead ion antigen, and the synovial volume of the quality control line and the detection line is 0.6 μL / cm.

[0103] (3) Detection of lead ion concentration

[0104] Obtain a lead ion standard solution, add disodium ethylenediaminetetraacetic acid chelating agent to the lead ion standard solution, react at 25°C for 10 minutes, then add a potassium hydroxide solution with a concentration of 1 mol / L, adjust the pH to 7.4, and dilute to a lead ion standard solution with a certain concentration gradient (wherein the lead ion standard solution with a concentration gradient is 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL and 10 ng / mL). Add the lead ion standard solution with the above concentration gradient and the antibody-directed labeled colloidal gold immunoprobe solution to the microwell and mix evenly, then insert the sample pad end of the lead ion test strip into the microwell, react at room temperature for 15 minutes, and observe with the naked eye whether the quality control line is colored. At the same time, compare the color of the test line on the test strip with the standard color card, observe the color depth of the test line, and the lead ion concentration can be obtained.

[0105] Please refer to the test results Figure 7 and Fig.12 ,Depend on Figure 7 The characterization diagram shows that the recombinant protein G successfully linked the colloidal gold particles and the anti-lead ion monoclonal antibody. Fig.12 It can be seen that the test strips can clearly detect different concentrations of lead ions (it should be noted that Fig.12 The 0, 2, 4, 6, 8, and 10 in the figure represent the lead ion standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, and 10 ng / mL, respectively).

[0106] Example 4

[0107] (1) Preparation of antibody-directed labeled colloidal gold immunoprobe

[0108] Potassium carbonate solution was added dropwise to the colloidal gold particles, the pH value was adjusted to 10, recombinant protein A was added in a mass ratio of 100:2 of colloidal gold particles and recombinant protein A, the mixture was reacted at 30°C for 60 minutes, a bovine serum albumin solution with a concentration of 0.55% was added, the mixture was reacted at 30°C for 60 minutes, the mixture was centrifuged at a speed of 12000 r / min for 15 minutes, and the first precipitate was obtained after three centrifugal treatments.

[0109] The first precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L to obtain a colloidal gold-connectin complex.

[0110] Anti-lead ion monoclonal antibodies were added to the colloidal gold-connectin protein complex at a mass ratio of 100:10, and the mixture was reacted at 30°C for 150 minutes. The mixture was then centrifuged at 12000 r / min for 12 minutes, and centrifuged four times to obtain a second precipitate and a supernatant.

[0111] The second precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L (the amount of the phosphate buffer was 10% of the total volume of the second precipitate and the supernatant) to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0112] (2) Preparation of test strips

[0113] A water absorbent pad, a nitrocellulose membrane and a sample pad are respectively adhered along a first direction on one sticky side of the liner, wherein the overlapping distances between the water absorbent pad and the sample pad and the nitrocellulose membrane are both 2 mm, the quality control line is coated with 0.55 mg / mL of anti-mouse monoclonal antibody sheep antibody, the detection line is coated with 0.3 mg / mL of lead ion antigen, and the synovial volume of the quality control line and the detection line are both 0.6 μL / cm.

[0114] (3) Detection of lead ion concentration

[0115] Obtain a lead ion standard solution, add disodium ethylenediaminetetraacetic acid chelating agent to the lead ion standard solution, react at 30°C for 10 minutes, then add a potassium hydroxide solution with a concentration of 1 mol / L, adjust the pH to 7.4, and dilute to a lead ion standard solution with a certain concentration gradient (wherein the lead ion standard solution with a concentration gradient is 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL and 10 ng / mL). Add the lead ion standard solution with the above concentration gradient and the antibody-directed labeled colloidal gold immunoprobe solution to the microwell and mix evenly, then insert the sample pad end of the lead ion test strip into the microwell, react at room temperature for 15 minutes, and observe with the naked eye whether the quality control line is colored. At the same time, compare the color of the test line on the test strip with the standard color card, observe the color depth of the test line, and the lead ion concentration can be obtained.

[0116] Please refer to the test results Figure 8 and Fig.13 ,Depend on Figure 8 The characterization diagram shows that the recombinant protein A successfully linked the colloidal gold particles and the anti-lead ion monoclonal antibody. Fig.13 It can be seen that the test strips can clearly detect different concentrations of lead ions (it should be noted that Fig.13 The 0, 2, 4, 6, 8, and 10 in the figure represent the lead ion standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, and 10 ng / mL, respectively).

[0117] Example 5

[0118] (1) Preparation of antibody-directed labeled colloidal gold immunoprobe

[0119] Potassium carbonate solution was added dropwise to the colloidal gold particles, the pH value was adjusted to 9, recombinant protein A was added in a mass ratio of 100:9 of colloidal gold particles and recombinant protein A, the reaction was carried out at 35° C. for 40 minutes, a 0.5% bovine serum albumin solution was added, the reaction was carried out at 35° C. for 40 minutes, and then the mixture was centrifuged at a speed of 10,000 r / min for 10 minutes. After five centrifugal treatments, a first precipitate was obtained.

[0120] The first precipitate was dissolved in a phosphate buffer having a concentration of 0.01 mol / L to obtain a colloidal gold-connectin complex.

[0121] Anti-lead ion monoclonal antibodies were added to the colloidal gold-connectin protein complex at a mass ratio of 100:20, and the mixture was reacted at 35° C. for 10 min. The mixture was then centrifuged at 10,000 r / min for 10 min, and centrifuged three times to obtain a second precipitate and a supernatant.

[0122] The second precipitate was dissolved in a phosphate buffer with a concentration of 0.01 mol / L (the amount of the phosphate buffer was 10% of the total volume of the second precipitate and the supernatant) to obtain an antibody-directed labeled colloidal gold immunoprobe.

[0123] (2) Preparation of test strips

[0124] A water absorbent pad, a nitrocellulose membrane and a sample pad are respectively adhered along a first direction on one sticky side of the liner, wherein the overlapping distances between the water absorbent pad and the sample pad and the nitrocellulose membrane are both 2 mm, the quality control line is coated with 0.52 mg / mL rabbit antibody of anti-mouse monoclonal antibody, the detection line is coated with 0.4 mg / mL lead ion antigen, and the synovial volume of the quality control line and the detection line is 0.6 μL / cm.

[0125] (3) Detection of lead ion concentration

[0126] Obtain a lead ion standard solution, add disodium ethylenediaminetetraacetic acid chelating agent to the lead ion standard solution, react at 35°C for 10 minutes, then add a potassium hydroxide solution with a concentration of 1 mol / L, adjust the pH to 8, and dilute to a lead ion standard solution with a certain concentration gradient (wherein the lead ion standard solution with a concentration gradient is 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL and 10 ng / mL). Add the lead ion standard solution with the above concentration gradient and the antibody-directed labeled colloidal gold immunoprobe solution to the microwell and mix evenly, then insert the sample pad end of the lead ion test strip into the microwell, react at room temperature for 15 minutes, and observe with the naked eye whether the quality control line is colored. At the same time, compare the color of the test line on the test strip with the standard color card, observe the color depth of the test line, and the lead ion concentration can be obtained.

[0127] Please refer to the test results Fig. 9 and Fig.14 ,Depend on Fig. 9 The characterization diagram shows that the recombinant protein A successfully linked the colloidal gold particles and the anti-lead ion monoclonal antibody. Fig.14 It can be seen that the test strips can clearly detect different concentrations of lead ions (it should be noted that Fig.14 The 0, 2, 4, 6, 8, and 10 in the figure represent the lead ion standard solutions of 0 ng / mL, 2 ng / mL, 4 ng / mL, 6 ng / mL, 8 ng / mL, and 10 ng / mL, respectively).

[0128] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. An antibody-directed labeled colloidal gold immunoprobe, characterized in that: The invention comprises the following components: colloidal gold particles, connexin and anti-lead ion monoclonal antibody; Wherein, the connecting protein is used to connect the colloidal gold particles and the anti-lead ion monoclonal antibody, and the connecting protein includes a protein that specifically recognizes the Fc fragment of the monoclonal antibody; The method for preparing the antibody-directed labeled colloidal gold immunoprobe comprises the following steps: The pH value of the colloidal gold particles is adjusted to 7-10, the connecting protein is added and stirred evenly, and then the bovine serum albumin solution is added, and the first precipitate is obtained after centrifugation; dissolving the first precipitate in a phosphate buffer to obtain a colloidal gold-connectin complex; adding anti-lead ion monoclonal antibody to the colloidal gold-connectin complex, and obtaining a second precipitate after centrifugation; The second precipitate is dissolved in a phosphate buffer to obtain an antibody-directed labeled colloidal gold immunoprobe.

2. The antibody-directed labeled colloidal gold immunoprobe according to claim 1, characterized in that: The protein that specifically recognizes the Fc fragment of the monoclonal antibody includes recombinant protein A or recombinant protein G.

3. A method for preparing an antibody-directed labeled colloidal gold immunoprobe, characterized in that: The following steps are involved: The pH value of the colloidal gold particles is adjusted to 7-10, the connecting protein is added and stirred evenly, and then the bovine serum albumin solution is added, and the first precipitate is obtained after centrifugation; dissolving the first precipitate in a phosphate buffer to obtain a colloidal gold-connectin complex; adding anti-lead ion monoclonal antibody to the colloidal gold-connectin complex, and obtaining a second precipitate after centrifugation; The second precipitate is dissolved in a phosphate buffer to obtain an antibody-directed labeled colloidal gold immunoprobe.

4. The method for preparing the antibody-directed labeled colloidal gold immunoprobe according to claim 3, characterized in that: In the step of adjusting the pH value of the colloidal gold particles to 7-10, adding the connexin, and obtaining the first precipitate after centrifugation, The mass ratio of the colloidal gold particles to the connexin is 100:(2-20).

5. The method for preparing the antibody-directed labeled colloidal gold immunoprobe according to claim 3, characterized in that: The steps of adjusting the pH value of the colloidal gold particles to 7-10, adding connexin, and obtaining the first precipitate after centrifugation include: dropping a potassium carbonate solution into the colloidal gold particles, adjusting the pH value to 7-10, adding connexin, reacting at 25-37° C. for 20-60 minutes, adding a bovine serum albumin solution with a concentration of 0.45-0.55%, reacting at 25-37° C. for 20-60 minutes, and then centrifuging at a speed of 8000-12000 r / min for 5-15 minutes, and obtaining the first precipitate after 3-8 centrifugation.

6. The method for preparing the antibody-directed labeled colloidal gold immunoprobe according to claim 3, characterized in that: In the step of adding anti-lead ion monoclonal antibody to the colloidal gold-connectin complex and obtaining a second precipitate after centrifugation, The mass ratio of the colloidal gold particles to the anti-lead ion monoclonal antibody is 100:(10-20).

7. The method for preparing the antibody-directed labeled colloidal gold immunoprobe according to claim 3, characterized in that: The step of adding anti-lead ion monoclonal antibody to the colloidal gold-connectin protein complex and obtaining a second precipitate after centrifugation comprises: adding anti-lead ion monoclonal antibody to the colloidal gold-connectin protein complex, reacting at 25-37° C. for 60-150 minutes, then centrifuging at a speed of 6000-12000 r / min for 6-12 minutes, and centrifuging 2-4 times to obtain a second precipitate.

8. A lead ion test paper based on antibody directional labeling, characterized in that: include: An antibody-directed labeled colloidal gold immunoprobe, wherein the antibody-directed labeled colloidal gold immunoprobe is the antibody-directed labeled colloidal gold immunoprobe as claimed in any one of claims 1 to 2 or the antibody-directed labeled colloidal gold immunoprobe prepared by the method for preparing an antibody-directed labeled colloidal gold immunoprobe as claimed in any one of claims 3 to 7; as well as, A test strip, the test strip comprising a water absorbent pad, a nitrocellulose membrane, a sample pad and a backing plate, wherein the water absorbent pad, the nitrocellulose membrane and the sample pad are sequentially arranged on the backing plate along a first direction, and one end of each of the water absorbent pad and the sample pad connected to the nitrocellulose membrane is arranged at the upper end of the nitrocellulose membrane and at least wraps a portion of the nitrocellulose membrane.

9. The lead ion test paper based on antibody directional labeling as claimed in claim 8, characterized in that: A quality control line and a detection line are sequentially arranged on the upper side of the nitrocellulose membrane along a first direction, and the quality control line and the detection line are extended along a second direction. The material of the quality control line includes rabbit antibodies against mouse monoclonal antibodies or sheep antibodies against mouse monoclonal antibodies, and the material of the detection line includes lead ion antigens.

10. The lead ion test paper based on antibody directional labeling according to claim 9, characterized in that: The concentration of the rabbit antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; and / or, The concentration of the goat antibody against the mouse monoclonal antibody is 0.45-0.55 mg / mL; and / or, The concentration of the lead ion antigen is 0.2-0.5 mg / mL.

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