CN111505281A - A sensitizing immunochromatographic kit for sensitive detection of novel coronavirus antibodies - Google Patents

Abstract

The invention relates to a sensitivity enhanced immunochromatographic kit for sensitively detecting a novel coronavirus antibody, which introduces a controllable copper growth technology to amplify the optical signal intensity of a colloidal gold probe in a detection line on the basis of a colloidal gold immunochromatographic test strip, thereby realizing high-sensitivity detection of an object to be detected. The kit comprises a colloidal gold immunochromatographic test strip and a copper growth solution, wherein the colloidal gold immunochromatographic test strip is sequentially fixed on a PVC plastic bottom plate through a sample pad, a combination pad, a nitrocellulose membrane and absorbent paper; the copper growth solution comprises a copper growth solution A and a copper growth solution B. The kit provided by the invention has the advantages of simple operation, high sensitivity, good stability, high detection speed (20-30min), easy result reading, no need of other auxiliary instruments and equipment, and can be used for on-site rapid and ultrasensitive detection of clinical and emergency events and large-scale epidemiological investigation.

Description

A sensitizing immunochromatographic kit for sensitive detection of novel coronavirus antibodies

technical field

The invention relates to the technical field of immunochromatographic analysis, in particular to a sensitized immunochromatographic kit for sensitive detection of novel coronavirus antibodies, the technical principle of which is a double-antigen sandwich method.

Background technique

Colloidal gold immunochromatographic test strips have been widely used in clinical diagnosis, food safety testing and environmental monitoring due to their simplicity, rapidity, convenience and naked eye detection. Traditional colloidal gold immunochromatographic test strips mainly use colloidal gold with a particle size of 20-40 nm as a signal probe. However, due to its small particle size, its colorimetric signal intensity is relatively weak, resulting in a low detection sensitivity. The relatively low detection sensitivity limits its application in ultrasensitive detection to a certain extent. In recent years, with the rapid development of nanoscience and technology, various new nanomaterials such as fluorescent microspheres, quantum dots, quantum dot microspheres, upconversion fluorescent nanoparticles, carbon nanomaterials, and magnetic materials have been reported. It can be used to replace colloidal gold as a detection probe to improve the detection sensitivity of traditional immunochromatographic test strips, but the synthesis of these nanoprobes is relatively complex, expensive and difficult to be used in the market. Compared with these new nanomaterials, colloidal gold has always occupied a major position in immunochromatographic test strips due to its advantages of simple synthesis, easy modification, good biocompatibility, stable optical properties and easy reading, especially in commercial applications. In chemical production, its market share is over 90%. Therefore, how to improve the detection sensitivity of traditional colloidal gold immunochromatographic test strips is of great significance to further expand its application in ultrasensitive detection.

The signal amplification strategy based on the test strip detection line has received extensive attention of researchers because it does not require the synthesis of additional auxiliary signal amplification materials. This type of strategy mainly includes the following categories: 1) Double gold signal amplification technology: On the basis of traditional colloidal gold immunochromatographic test strips, by introducing signal amplification gold label pads, it can use antigen-antibody reaction and biotin- Streptavidin system specifically binds gold-labeled antibody conjugates on the detection line, resulting in further aggregation of colloidal gold nanoparticles, thereby amplifying the colorimetric signal of colloidal gold, such as patent CN102507929A; 2) Enzyme enhancement technology: in On the basis of traditional colloidal gold immunochromatographic test strips, the introduction of horseradish peroxidase or nanozymes with peroxidase activity catalyzes the oxidation of corresponding substrates such as tetramethylbenzidine (TMB) to generate colored The precipitate is deposited on the detection line, so that the colorimetric signal of colloidal gold is amplified; 3) Gold or silver staining enhancement technology: On the basis of traditional colloidal gold immunochromatographic test strips, in the presence of a reducing agent, gold Or silver ions are easy to take colloidal gold as the core, gather on the surface of colloidal gold and reduce to gold or silver atoms, so that nano-sized colloidal gold grows into larger particles through the aggregation and reduction of gold or silver ions, so that the ratio of colloidal gold is reduced. The color signal is amplified, such as patent CN102135536A. The first two methods often easily affect the immunological properties of the colloidal gold probe due to the need for double labeling of the colloidal gold probe, resulting in poor methodological stability. The growth of metal nanoshells in the gold or silver staining enhancement technology belongs to the layer-by-layer growth mode. The growth rate is not only related to the number of gold nanoparticles on the T and C lines of the test strip, but also closely related to the concentration of the growth solution and the growth time. As a result, the growth process is uncontrollable, and the stability and reproducibility of the detection results are poor, which greatly limits the popularization and application of this method in practice. In addition, the ability of these methods to improve detection sensitivity is limited, only 1-2 orders of magnitude. Therefore, the sensitized colloidal gold immunochromatographic test strip still cannot realize the ultrasensitive detection of the low-concentration analyte. The invention proposes to use the polymer-mediated controllable copper growth technology to significantly enhance the optical signal intensity of the colloidal gold probe on the test strip detection line, and the color of the strip changes from zero to strong, from weak to strong, and can achieve ultra-low Qualitative detection with naked eye at target concentration. After searching, there is no published literature report on the use of controlled copper growth technology to sensitize the detection sensitivity of traditional colloidal gold immunochromatographic test strips.

SUMMARY OF THE INVENTION

The purpose of the present invention is that the present invention aims to solve the current defect of low detection sensitivity due to the weak signal intensity of the 20-40nm colloidal gold probe of the existing colloidal gold immunochromatographic test strip, using polymer-mediated colloidal gold probe The controllable copper growth deposition on the needle surface significantly amplifies the optical signal intensity of the colloidal gold probe on the test strip detection line, providing a new technology to improve the detection sensitivity of the traditional colloidal gold immunochromatography method, and realize the detection of ultra-low concentration target substances. Perform a quick on-site inspection.

For achieving the above object, the technical scheme adopted in the present invention is:

A sensitizing immunochromatographic kit for sensitive detection of novel coronavirus antibodies, the kit comprises a colloidal gold immunochromatographic test strip and a copper growth solution, and the colloidal gold immunochromatographic test strip consists of a sample pad, a binding pad , nitrocellulose membrane, and absorbent paper are sequentially fixed on the PVC plastic bottom plate, and the binding pad is a glass fiber sprayed with colloidal gold-labeled novel coronavirus (SARS-CoV-2) recombinant antigen and colloidal gold-labeled rabbit IgG antibody The nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with SARS-CoV-2 recombinant antigen, and the quality control line is coated with an anti-rabbit secondary antibody solution; the copper growth solution It includes copper growth solution A and copper growth solution B. Copper growth solution A is a pre-formed copper ion coordination growth solution formed by polymer and divalent copper ions. Copper growth solution B is a mixed solution of sodium ascorbate and trisodium citrate.

The present invention also provides a preparation method of a sensitized immunochromatographic kit for sensitive detection of novel coronavirus antibodies, comprising the following steps:

1) Preparation of colloidal gold-labeled recombinant antigen: Take 1 mL of citric acid-coated colloidal gold solution with a particle size of 30-40 nm, adjust the pH of the solution to 7.0-8.0 with potassium carbonate solution with a concentration of 0.2 M; add 5 μL to the solution The concentration of SARS-CoV-2 recombinant antigen solution was 1～2mg/mL, and the reaction was shaken for 30min at room temperature; then 100 μL of 1～2mg/mL bovine serum albumin solution was added to the above mixed solution and the reaction was continued at room temperature for 30min; at 4 Under the condition of ℃, centrifuge at 10000rpm for 20min, discard the supernatant, and redissolve the precipitate in 0.01M pH7.4 phosphate buffer solution (PBS) to obtain the final product colloidal gold-labeled recombinant antigen; colloidal gold-labeled rabbit IgG antibody uses the same After the method was completed, the SARS-CoV-2 recombinant antigen solution was replaced with a rabbit IgG antibody solution.

2) Preparation of colloidal gold immunochromatographic test strips: a) The preparation of the binding pad is to spray the colloidal gold-labeled recombinant antigen and colloidal gold-labeled rabbit IgG antibody on the glass fiber membrane; b) It has a detection line and a quality control The preparation of the nitrocellulose membrane of the line is by spraying the SARS-CoV-2 recombinant antigen with a concentration of 1-2 mg/mL and the anti-rabbit secondary antibody solution with a concentration of 1-2 mg/mL respectively on the nitrocellulose membrane to draw two parallel lines, SARS-CoV-2. - The CoV-2 recombinant antigen solution was streaked as the detection line (T line), and the anti-rabbit secondary antibody solution was streaked as the quality control line (C line); c) Colloidal gold immunochromatography test strip assembly: the sample pad, with The binding pad of the gold-labeled detection antibody, the nitrocellulose membrane with T and C lines, and the absorbent paper were respectively pasted onto the plastic backing plate in turn. stored at temperature;

3) Configuration of copper growth solution: a) Copper growth solution A: Accurately weigh 200-400 mg of copper chloride and 100-200 mg of polymer and dissolve them in 10 mL of ultrapure water, respectively, and then mix the two to prepare copper growth A. b) Copper growth solution B: Accurately weigh 1-2 g of sodium ascorbate and 1 mg of trisodium citrate and dissolve them in 10 mL and 1 mL of ultrapure water, respectively, and then mix the two to prepare copper growth solution B.

Further, the polymer is polyethyleneimine PEI, polyacrylic acid PAA, polyvinylpyrrolidone PVP or polylysine PL.

The present invention also provides a method for using a sensitized immunochromatographic kit for sensitive detection of novel coronavirus antibodies, comprising the following steps:

1) Take 100-120 μL of the sample solution to be tested and add it to the sample hole of the test card of the test strip, and react at room temperature for 5-10 minutes;

2) after the above-reacted test strips were washed with phosphate buffer and ultrapure water in turn, soaked in copper growth A solution for reaction at room temperature for 5 min;

3) Next, transfer the test strip to the copper growth solution B and continue to react for 2 minutes, so that the polymer-mediated copper nanoshell layer is controlled to grow and deposit on the surface of colloidal gold, which significantly enhances the optical signal intensity of colloidal gold, and the experimental results are observed with the naked eye.

The invention is suitable for ultrasensitive detection of disease-related antigens and antibody markers, and the disease-related markers include: tumor markers such as carcinoembryonic antigen, prostate specific antigen, alpha-fetoprotein; inflammatory markers such as C-reactive protein, Procalcitonin, interleukin-6; cardiac markers such as troponin, myoglobin, N-terminal atrial natriuretic peptide and N-terminal brain natriuretic peptide; infectious disease markers such as novel coronavirus antigens and antibodies, HIV antigens and Antibodies, Ebola virus antigens and antibodies, Middle East respiratory syndrome coronavirus antigens and antibodies, hepatitis B virus-related antigens and antibodies; pathogens such as viruses, pathogenic bacteria, etc., especially suitable for on-site rapid ultrasensitive detection of objects to be tested. The sample processing can be carried out according to the conventional processing method.

Compared with the prior art, the beneficial effects of the present invention are:

1. On the basis of traditional colloidal gold immunochromatographic test strips, the present invention adopts polymer-mediated controllable copper growth signal amplification technology to improve the detection sensitivity of colloidal gold immunochromatographic test strips, and also retains its advantages. specific characteristics.

2. The polymer-mediated controllable copper growth signal amplification technology adopted in the present invention involves the controllable reduction deposition of divalent copper ions induced by sodium ascorbate under the mediation of polymers on the surface of colloidal gold probes. The principle is to immerse the colloidal gold test strip after the conventional detection in the copper growth liquid A and B in turn. At this time, the pre-adsorbed copper ions are reduced to copper and deposited on the surface of the colloidal gold probe to achieve signal enhancement. In this scheme, the number of copper nanoparticles reduced and deposited in the detection area is related to the number of pre-adsorbed copper ions, while the number of pre-adsorbed copper ions is only related to the number of colloidal gold probes in the detection area of the test strip. Controlled growth of particles. Compared with the traditional gold-silver growth-sensitizing test strips, the method has higher sensitivity and better accuracy and reliability of detection results. In addition, the copper chloride, polymer, sodium ascorbate and trisodium citrate involved in the signal amplification system have all achieved commercial large-scale production, so this method has low reagent cost, high stability and good reproducibility, and has good commercialization prospects.

3. The polymer-mediated controllable copper growth signal amplification technology used in the present invention can be applied to all colloidal gold immunochromatographic test strips.

4. The sensitized immunochromatographic test strip constructed by the present invention is especially suitable for ultra-sensitive detection of trace target analytes, and has broad application prospects in clinical and on-site detection of emergencies, such as the early stage of infectious diseases. screening, etc.

Description of drawings

Fig. 1 is a schematic diagram of a test strip structure, for (A) is a structure diagram of a traditional colloidal gold immunochromatographic test strip, and (B) is a schematic diagram of the principle of the method of the present invention;

Figure 2 is a schematic diagram of the detection of antibodies in the serum of patients with novel coronavirus infection: traditional colloidal gold immunochromatographic test strips (top) and sensitization test strips constructed by the present invention (bottom).

Detailed ways

Specific embodiments of the present invention will be described in detail below. In order to avoid unnecessary details, well-known structures or functions will not be described in detail in the following embodiments.

Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The test reagent consumables used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified.

In the following experiments, phosphate buffered saline (PBS, 0.05M, pH 7.4) was prepared as follows: NaCl 40g, _Na2HPO4 13.5g, _{KH2PO4 1.0g} , _KCl 1.0g dissolved in 1L ultrapure water . The pH was adjusted to 7.4 with 0.1M NaOH.

The novel coronavirus (SARS-CoV-2) recombinant antigen involved in the test was purchased from Jiangxi Yeli Medical Equipment Co., Ltd. Rabbit IgG antibody and anti-rabbit secondary antibody were provided by Beijing Rejing Biotechnology Co., Ltd. The copper chloride, polyethyleneimine PEI, sodium ascorbate and trisodium citrate can be purchased from the market.

Example 1

A detection kit for sensitizing colloidal gold immunochromatographic test strips, which includes colloidal gold immunochromatographic test strips and copper growth solution; as shown in A in Figure 1, the colloidal gold immunochromatographic test strips The strip includes a sample pad, binding pad, NC membrane and absorbent paper, the binding pad is provided with a novel coronavirus recombinant antigen labeled with colloidal gold, and the nitrocellulose membrane is provided with a detection line (T line) and a quality control line (C line); The copper growth solution includes copper growth A solution and B solution.

The kit was prepared as follows:

1. Preparation of colloidal gold immunochromatographic test strips

1. Preparation of colloidal gold-labeled recombinant antigen:

Take 1 mL of citric acid-coated colloidal gold solution with a particle size of 35 nm, and adjust the pH of the solution to 7.4 with potassium carbonate solution with a concentration of 0.2 M; ) reconstituted antigen solution, shake at room temperature for 30 minutes; then add 100 μL of 1.5 mg/mL bovine serum albumin solution to the above mixed solution and continue shaking at room temperature for 30 minutes; at 4°C, centrifuge at 10,000 rpm for 20 minutes, discard The supernatant and the precipitate were redissolved in 0.01M pH7.4 phosphate buffered solution (PBS) to obtain the final product colloidal gold-labeled recombinant antigen. Colloidal gold-labeled rabbit IgG antibodies were accomplished in a similar manner.

2. Preparation of colloidal gold immunochromatographic test strips:

a) The preparation of the binding pad is to spray the colloidal gold-labeled recombinant antigen and the colloidal gold-labeled rabbit IgG antibody on the glass fiber membrane;

b) The nitrocellulose (NC) membrane with detection line and quality control line was prepared by spraying 1.5 mg/mL SARS-CoV-2 recombinant antigen and 1.5 mg/mL anti-rabbit secondary antibody solution on the NC membrane respectively Draw two parallel lines, the SARS-CoV-2 recombinant antigen solution is drawn as the detection line (T line), and the anti-rabbit secondary antibody solution is drawn as the quality control line (C line);

c) Assembly of colloidal gold immunochromatographic test strips: Paste the sample pad, the binding pad with gold-labeled detection antibody, the NC film with T and C lines, and the absorbent paper on the plastic backing plate in turn, and the adjacent overlapping parts The length is 2mm, cut into 4mm wide test strips, sealed and dried, and stored at temperature.

2. Preparation of colorimetric signal amplification reagents

3. Configuration of copper growth solution:

a) Copper growth liquid A: Accurately weigh 300 mg of copper chloride and 150 mg of polyethyleneimine PEI and dissolve them in 10 mL of ultrapure water, respectively, and then mix the two to prepare liquid A;

b) Copper growth liquid B: Accurately weigh 1.5 g of sodium ascorbate and 1 mg of trisodium citrate and dissolve them in 10 and 1 mL of ultrapure water, respectively, and then mix the two to prepare liquid B.

Example 2

The method of the present invention was performed by using the kit described in Example 1 to verify the detection effect of the method on antibodies against novel coronavirus (SARS-CoV-2).

Referring to the process shown in B in Figure 1, use a pipette to accurately take 120 μL of serum sample solutions containing different concentrations of antibodies, add them to the sample wells of the test strip detection card, and react at room temperature for 10 minutes; After washing with phosphate buffer and ultrapure water, soak them in copper growth solution A for 5 min at room temperature; then transfer the test strips to copper growth solution B and continue to react for 2 min, and interpret the experimental results with the naked eye.

As shown in Figure 2, when using commercial colloidal gold test strips purchased by Jiangxi Yeli Medical Co., Ltd. to test 10 clinical serum samples of patients collected by the First Affiliated Hospital of Nanchang University, we found that all test strips were only qualitative The control line (C line) has an obvious red band, but the detection line (T line) has no red band. However, after performing the method of the present invention, 7 of the test samples (numbers: 27, 30, 31, 32, 40, 41, 43) showed a red band visible to the naked eye on the T line, indicating that the serum to be tested contains novel coronavirus antibodies.

Further comparison and analysis with the clinical nucleic acid detection results showed that the nucleic acid detection of the patients corresponding to the above 7 serum samples showed positive results, indicating that the method established by the present invention has higher detection sensitivity and can detect the traditional colloidal gold immunochromatographic test. The "false negative" samples that were not successfully detected by the note effectively avoided the missed detection. In addition, 3 serum samples with negative clinical nucleic acid confirmation (No. 17, 60, 61) showed no visible bands on their T lines after amplification, indicating that the amplification method has good specificity.

Conclusion: The detection test described in Example 2 shows that the new method mentioned in the present invention has higher detection sensitivity than traditional colloidal gold immunochromatographic test strips for antibody detection in serum samples of clinical patients with novel coronavirus infection , more suitable for ultra-sensitive detection of trace antibodies in clinical serum samples, further proves that the new method of sensitizing traditional colloidal gold immunochromatographic test strips based on polymer-mediated controllable copper growth technology provided by the present invention is feasible of.

Although the present invention uses novel coronavirus (SARS-CoV-2) antibody as a model analyte to explore the feasibility of polymer-mediated controllable copper growth technology to enhance the detection sensitivity of immunochromatographic test strips on a double-antigen sandwich immunochromatography platform , but the content defined by the present invention is not limited to the new coronavirus (SARS-CoV-2) antibody, and all other target analytes such as biological macromolecules, nucleic acids and pathogens detected by the method of the present invention are included in the patent. within the scope of protection. In addition, in addition to using traditional antigen-antibody reactions, the present invention can also use new biological recognition reactions that have appeared in recent years, such as nucleic acid hybridization, nucleic acid aptamers, ligand receptors, etc. to construct corresponding ultra-sensitive analysis methods for the detection of target analytes . Therefore, the development and extension of the present invention based on these recognition molecules also fall within the protection scope of the present invention.

Claims (4)

1. A sensitivity enhanced immunochromatographic kit for sensitively detecting a novel coronavirus antibody is characterized by comprising a colloidal gold immunochromatographic test strip and a copper growth solution, wherein the colloidal gold immunochromatographic test strip is sequentially fixed on a PVC plastic base plate through a sample pad, a binding pad, a nitrocellulose membrane and absorbent paper, the binding pad is a glass fiber membrane sprayed with a novel coronavirus (SARS-CoV-2) recombinant antigen marked by colloidal gold and a rabbit IgG antibody marked by colloidal gold, the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with the SARS-CoV-2 recombinant antigen, and the quality control line is coated with an anti-rabbit secondary antibody solution; the copper growth solution comprises a copper growth solution A and a copper growth solution B, wherein the copper growth solution A is a copper ion coordination growth solution formed by a polymer and divalent copper ions in advance, and the copper growth solution B is a mixed solution of sodium ascorbate and trisodium citrate.

2. A preparation method of a sensitization type immunochromatography kit for sensitively detecting novel coronavirus antibodies is characterized by comprising the following steps:

1) the preparation of the colloidal gold labeled recombinant antigen comprises the steps of taking citric acid coated colloidal gold solution with the particle size of 1M L being 30-40 nm, adjusting the pH value of the solution to 7.0-8.0 by potassium carbonate solution with the concentration of 0.2M, adding L SARS-CoV-2 recombinant antigen solution with the concentration of 5 mu L being 1-2 mg/M to the solution, oscillating for 30min at room temperature, then adding 100 mu L1-2 mg/M L bovine serum albumin solution to the mixed solution, continuing oscillating for 30min at room temperature, centrifuging for 20min at 10000rpm under the condition of 4 ℃, abandoning supernatant, redissolving precipitates in 0.01M Phosphate Buffer Solution (PBS) with the pH of 7.4 to obtain the final product colloidal gold labeled recombinant antigen, completing the preparation of the colloidal gold labeled rabbit IgG antibody by adopting the same method, and replacing the SARS-CoV-2 recombinant antigen solution with the rabbit IgG antibody solution.

2) A) preparing a binding pad by spraying a recombinant antigen marked by colloidal gold and a rabbit IgG antibody marked by colloidal gold on a glass fiber membrane, b) preparing a nitrocellulose membrane with a detection line and a quality control line by respectively spraying L SARS-CoV-2 recombinant antigen with the concentration of 1-2 mg/m and L anti-rabbit secondary antibody solution with the concentration of 1-2 mg/m on the nitrocellulose membrane to draw two parallel lines, marking the SARS-CoV-2 recombinant antigen solution as the detection line (T line), and marking the anti-rabbit secondary antibody solution as the quality control line (C line), C) assembling the colloidal gold immunochromatographic test strip, namely respectively and sequentially pasting the sample pad, the binding pad with the gold-marked detection antibody, the nitrocellulose membrane with the T and C lines and absorbent paper on a plastic lining board, cutting the adjacent overlapped part into test strips with the length of 2mm, sealing and drying, and storing at the temperature;

3) the preparation method of the copper growth solution comprises the steps of a) accurately weighing 200-400 mg of copper chloride and 100-200 mg of polymer to be respectively dissolved in 10m L ultrapure water, and then mixing the two solutions to prepare the copper growth solution A, B) accurately weighing 1-2 g of sodium ascorbate and 1mg of trisodium citrate to be respectively dissolved in 10m L and 1m L ultrapure water, and then mixing the two solutions to prepare the copper growth solution B.

3. The sensitization immunochromatographic kit for sensitively detecting a novel coronavirus antibody in accordance with claim 2, wherein the polymer is polyethyleneimine PEI, polyacrylic acid PAA, polyvinylpyrrolidone PVP or polylysine P L.

4. The use method of the sensitization type immunochromatography kit for sensitively detecting the novel coronavirus antibody is characterized by comprising the following steps of:

1) adding 100-120 mu L sample solution to be detected into a sample adding hole of a detection card of the test strip, and reacting for 5-10 min at room temperature;

2) washing the reacted test strip with phosphate buffer solution and ultrapure water in sequence, and soaking the test strip in copper growth A solution for reaction for 5min at room temperature;

3) and then transferring the test strip to a copper growth solution B for continuous reaction for 2min, so that the polymer mediated copper nano shell layer can be controllably grown and deposited on the surface of the colloidal gold, the optical signal intensity of the colloidal gold is obviously enhanced, and the experimental result is observed by naked eyes.

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