High Sensitivity Immunochromatographic Detection Kit

Abstract

The present invention relates to a high-sensitivity immunochromatographic detection kit, comprising an antibody detection liquid holding device and a test strip, wherein the antibody detection liquid holding device contains a corresponding antibody detection liquid; the reaction pad, sample pad, and water-absorbing pad are assembled in sequence and staggered on a bottom plate, one end of the reaction pad is connected to the sample pad, and the other end is connected to the water-absorbing pad, and the reaction pad is pre-coated with a detection line and a quality control line separated from each other, characterized in that: a biological bridge is combined on the test strip and the marker. The present invention further relates to a method for performing immunochromatographic detection using the kit. By performing immunochromatographic testing with the kit of the present invention, the detection sensitivity is improved and the amount of antibody used is reduced.

Description

Technical Field

[0001] The invention belongs to the field of immunological detection, and in particular, relates to a high-sensitivity immunochromatographic detection kit. Background Art

[0002] Immunochromatography, also known as lateral flow chromatography, is widely used in food safety, clinical medical testing and other fields due to its advantages of rapid detection, simple operation and low cost. According to the difference in molecular weight of the substance to be tested, the detection mode of immunochromatography is divided into the competitive method suitable for small molecules and the double antibody sandwich method suitable for substances with large molecular weight.

[0003] The existing immunochromatography technology has the problems of low detection sensitivity and low antibody utilization rate. The main reasons are as follows: 1. It is related to the connection between the marker and the antibody. The connection between the marker and the antibody is disordered, that is, the marker can be connected to the constant region of the antibody to fully expose the variable region (the local area that binds to the antigen); it can also be connected to the variable region of the antibody to make the antibody ineffective or reduce its effect; 2. The marker is directly connected to the antibody. Since the distance between the two is too close, steric hindrance is formed, which reduces the binding efficiency of the antibody and antigen; 3. During the chromatography process, due to the fast flow rate, the antibody and antigen are not fully bound at the detection line.

[0004] In order to solve the above problems, the present invention provides a high-sensitivity test kit, in which a "biological bridge" is provided between the marker and the detection line. The immunochromatographic test using the test kit improves the detection sensitivity and reduces the amount of antibodies used. Summary of the invention

[0005] The object of the present invention is to provide a high-sensitivity immunochromatographic detection kit, comprising a test strip and an antibody detection liquid containing device, wherein the device contains the corresponding antibody detection liquid; the test strip is composed of a reaction pad, a sample pad, and a water-absorbing pad assembled in sequence on a bottom plate, and the reaction pad is pre-coated with a detection line and a quality control line separated from each other, and is characterized in that: a biological bridge is combined in both the test strip and the antibody detection liquid.

[0006] There may be multiple biological bridges, for example, two or three, one of which is located on the reaction pad of the test strip and combined with the detection line, and the other biological bridges are located in the antibody detection solution and combined with the marker.

[0007] One end of the reaction pad of the test strip is connected to the sample pad, and the other end is connected to the absorbent pad; the reaction pad, the sample pad and the absorbent pad may be staggered by 1.5-2.5 mm, for example, 2 mm.

[0008] The "biobridge" is a biological product that can specifically bind to the constant region of an antibody and has high affinity. The biobridge is an anti-antibody, protein A, protein G and other molecules that can specifically bind to an antibody. The biobridge ensures that at least one functional region is a constant region that specifically binds to the detection antibody.

[0009] The antibody detection solution comprises an antibody 1, an antibody 2 and a biological bridge 1 coupled with a marker.

[0010] The antibody 1 and antibody 2 may be monoclonal antibodies, polyclonal antibodies, or single-chain antibodies, preferably monoclonal antibodies; the antibody 1 and antibody 2 are antibodies of different species.

[0011] The marker can be latex particles, or colloidal gold, colloidal carbon, colloidal selenium, superparamagnetic particles, fluorescent particles, up-conversion luminescent particles and the like.

[0012] The biological bridge 1 is a molecule such as an anti-antibody, protein A, protein G, etc. that can specifically bind to the constant region of the antibody 1.

[0013] There is a biological bridge 1 between the marker and the antibody 1, and the biological bridge 1 can specifically bind to the constant region of the antibody 1 with high affinity, which can improve the utilization rate of the antibody 1. The variable region of the antibody 1 can be fully exposed, and the antibody 1 can be kept away from the marker, which can reduce steric hindrance to a certain extent, thereby improving the detection sensitivity.

[0014] The detection line of the test strip contains a biological bridge 2. The biological bridge 2 is an anti-antibody, protein A, protein G and other molecules that can specifically bind to the constant region of the antibody 2; the quality control line contains an antibody 3 that can specifically bind to the biological bridge 1.

[0015] The antibody 3 may be a monoclonal antibody or a polyclonal antibody, preferably a polyclonal antibody.

[0016] In one embodiment, the kit of the present invention is a double antibody sandwich model. In this model, there are two biological bridges, which specifically bind to the two antibodies in the double antibody sandwich, that is, one biological bridge is connected to the marker, and the other biological bridge is connected to the test strip detection line.

[0017] Before the test object is added to the test strip for chromatography, it is first mixed with antibody 1, antibody 2 and biological bridge 1 coupled with a marker to react fully; and during the chromatography process, there is a biological bridge at the detection line position that can specifically bind to antibody 2 and has a high binding force with antibody 2.

[0018] Compared with the traditional method of directly coating the antibody 2 on the reaction pad, the technical solution of the present invention has a higher utilization rate of the antibody 2 and a higher detection sensitivity.

[0019] The present invention also aims to provide a highly sensitive immunochromatographic detection method, which is characterized by using the kit of the present invention for detection.

[0020] In the present invention, after the marker is connected to the "biological bridge", the constant region of the antibody is specifically bound by the biological bridge, so that the variable region of the antibody can be fully exposed, and the antibody is kept away from the marker, reducing the steric hindrance to a certain extent. The position of the detection line is fixed with a "biological bridge". Since the binding affinity of the "biological bridge" to the antibody is higher, the binding efficiency of the "biological bridge" to the antibody is higher during chromatography, thereby improving the detection sensitivity. During the chromatography process, the position of the detection line has a "biological bridge" that can specifically bind to the antibody, and the binding force with the antibody is high, which can improve the utilization rate of the antibody and the detection sensitivity. The reason why the present invention has the advantages is that, of the two ends of the antibody, one end is a binding region, that is, a region that plays a role, and the other end is a constant region that does not play a role. When the traditional chemical coupling method couples the antibody and the marker, the problem is that the binding region of the antibody may be coupled to the marker, resulting in waste of the antibody; it is also possible that the middle section of the antibody is coupled to the marker, thereby weakening the binding force of the antibody. The biological bridge added in the present invention can directionally bind to the constant region of the antibody, allowing the binding region to be fully exposed to the reaction system. Therefore, the addition of the biological bridge can reduce the amount of antibody used and improve the test sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A test strip of the kit according to the present invention.

[0022] Figure 2 It is a schematic diagram of a kit for double antibody sandwich mode testing of the present invention, which includes I: an antibody detection liquid containing device (containing the corresponding antibody detection liquid) and II: a test strip.

[0023] Description of reference numerals:

[0024] 1—antibody 1; 2—antibody 2; 3—biobridge 1; 4—marker; 5—sample pad; 6—test line; 7—biobridge 2; 8—quality control line; 9—reaction pad; 10—absorbent pad; 11—bottom plate; I—antibody test liquid storage device; II—test strip. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific examples, but the scope of the present invention is not limited thereto. The reagents used in the examples of the present invention are all commercially available, and unless otherwise specified, all operating methods are conventional operating methods.

[0026] Embodiment 1:

[0027] Human enkephalin detection

[0028] Preparation of Enkephalin Antibody Detection Solution:

[0029] (1) 2.5 mg of superparamagnetic composite particles with a particle size of 200 nm (Quantum Design, USA, product number 100001), 0.96 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC, Thermo, USA, product number 22980), 1.15 mg of N-hydroxysuccinimide (NHS, Thermo, USA, product number 24500) and 1 mL of 0.1 M pH 4.7 2-(N-morpholino)ethanesulfonic acid (MES, Sigma-Aldrich, USA, product number M3671) buffer (1.066 g of MES, 0.45 g of NaCl dissolved in 50 mL of pure water, adjusted to pH 4.7) were mixed and reacted at 37° C. for 30 min to obtain activated superparamagnetic composite particles;

[0030] (2) Adsorbing the magnetic particles with a magnet, discarding the supernatant, and washing twice with the above-mentioned MES buffer; mixing the activated superparamagnetic composite particles obtained in step (1), 0.08 mg of "biobridge 1" (sheep anti-mouse IgG Fc antibody in this embodiment) and a 50 mM boric acid buffer with a pH of 8.5, and reacting at 37° C. for 3 h to obtain a coupled label reaction solution;

[0031] (3) Add 50mM boric acid buffer containing BSA (Sigma-Aldrich, USA, product number A4612) at pH=8.5 to the reaction solution in step (2) to make the final concentration of BSA 5%, react at 37°C for 30min to obtain a reaction solution containing blocked magnetic particles; adsorb the magnetic particles with a magnet, discard the supernatant, and wash twice with 0.8mL boric acid buffer at pH8.5; finally, add 0.5mL phosphate buffer at pH8.5 containing 1% BSA, 0.5% Tween-20, and 1% sucrose to obtain a superparamagnetic composite particle labeling solution. Add 0.01mg anti-enkephalin monoclonal antibody and 0.05mg anti-enkephalin rabbit polyclonal antibody to the solution to obtain an enkephalin antibody detection solution.

[0032] Preparation of enkephalin test strips:

[0033] (1) Treatment of the reaction pad: Use coating buffer (0.01 M PBS solution containing 2% BSA, pH 7.2, filtered through a 0.22 μm filter membrane) to adjust the concentration of "Bio-Bridge 2" (sheep anti-rabbit IgG antibody) to 1 mg / mL (as the test line), and the concentration of mouse IgG antibody to 1 mg / mL (as the quality control line). Spray the two on a nitrocellulose membrane at a distance of 5 mm at a rate of 1 μL / cm, dry at 37°C, add a desiccant and seal for later use.

[0034] (2) Assembly of the test paper: Paste the reaction pad, absorbent pad, and sample pad on the bottom plate in sequence, staggered by 2 mm, to form a test paper.

[0035] (3) Cutting of test strips: Use a strip cutter to cut the above test paper into 3 mm wide test strips.

[0036] Enkephalin detection:

[0037] Take 10 μL of serum and 90 μL of enkephalin antibody detection solution and mix them in a 0.5 mL centrifuge tube, insert the enkephalin test strip into the centrifuge tube (with the sample pad at the bottom), and chromatograph for 10 minutes at room temperature.

[0038] After a series of debugging, the detection limit of this method is 5μg / mL. When the test line and the quality control line appear brown at the same time, it proves that the enkephalin contained in the sample is higher than the detection limit; when the test line has no color and the quality control line appears brown, it proves that the enkephalin contained in the sample is lower than the detection limit; when both the test line and the quality control line have no color, the test strip is invalid.

Claims

1. A high-sensitivity immunochromatographic detection kit, comprising an antibody detection liquid containing device (I) and a test strip (II), wherein the antibody detection liquid containing device contains the corresponding antibody detection liquid; the test strip is assembled in sequence and staggered by a reaction pad (9), a sample pad (5), and a water-absorbing pad (10) on a bottom plate (11), one end of the reaction pad (9) is connected to the sample pad (5), and the other end is connected to the water-absorbing pad (10), and the reaction pad (9) is pre-coated with a detection line (6) and a quality control line (8) separated from each other, characterized in that: A biological bridge is combined in both the test strip and the antibody detection liquid; the antibody detection liquid comprises an antibody 1, an antibody 2 and a biological bridge 1 coupled with a marker; the detection line of the test strip contains the biological bridge 2, and the quality control line contains an antibody 3 that can specifically bind to the biological bridge 1; the antibody 1 and the antibody 2 are antibodies of different species, and the antibody 1 and the antibody 2 are monoclonal antibodies, or polyclonal antibodies, or single-chain antibodies; the antibody 3 is a monoclonal antibody or a polyclonal antibody; the biological bridge 1 is an anti-antibody, protein A, or protein G molecule that can specifically bind to the constant region of the antibody 1; the biological bridge 2 is an anti-antibody, protein A, or protein G molecule that can specifically bind to the constant region of the antibody 2.

2. The kit according to claim 1, characterized in that: The biological bridge is an anti-antibody, protein A, and protein G molecule that can specifically bind to the antibody.

3. The kit according to claim 1, characterized in that: The kit is a double antibody sandwich model.

4. The kit according to claim 1, characterized in that: The label is latex particles, colloidal gold, colloidal carbon, colloidal selenium, superparamagnetic particles, fluorescent particles or up-conversion luminescent particles.

5. A highly sensitive non-diagnostic immunochromatographic detection method, characterized in that: The detection is carried out using the kit described in any one of claims 1 to 4.

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