A fluorescent immunochromatographic test paper with high sensitivity and wide detection range

Abstract

The present invention belongs to the field of immunoassay technology, and specifically relates to a fluorescent immunochromatographic test paper with high sensitivity and wide detection range. The fluorescent immunochromatographic test paper mainly includes a sample pad, a conjugate pad 1, a conjugate pad 2, a detection line 1, a detection line 2, a quality control line, and a nitrocellulose membrane; by using two pairs of antibody pairs with different reactivities of markers, europium time-resolved fluorescent microspheres and quantum dot microspheres (em: 615nm) are used as detection markers respectively. The detection ability of antigens of different concentrations is improved by utilizing the characteristics of different markers and the reactivity of different antibody pair combinations. The low-concentration sensitivity is greatly improved, and the high-concentration detection range is greatly expanded.

Description

Technical Field

[0001] The invention belongs to the technical field of immunoassay analysis, and in particular relates to a fluorescent immunochromatographic test paper with high sensitivity and a wide detection range. Background Art

[0002] The sensitivity and detection range of the test are affected by factors such as the reactivity of the antibody to the combination and the type of marker. Antibodies with good reactivity tend to have high sensitivity, but often have poor detection performance for high-concentration samples due to the existence of the Hooke effect; antibodies with slow reactivity have poor detection performance for low-concentration samples, but good detection performance for high-concentration samples; for the selection of fluorescent markers, the higher the fluorescence efficiency of the marker, the higher the sensitivity of the test.

[0003] Regarding fluorescent markers, fluorescent microspheres integrate multiple fluorescent molecules into one microsphere, which greatly improves the fluorescence efficiency and has incomparable advantages for high-sensitivity detection. However, the increase in the volume of the marker will advance the node of the Huoke effect, resulting in the inability to accurately measure high-concentration samples and shortening the detection range of the method.

[0004] Compared with fluorescent microspheres, small-volume fluorescent molecules or microspheres have lower fluorescence efficiency and do not have an advantage in detecting low-concentration markers. However, they can delay the node of the Hooke's effect of the immune response and have obvious advantages in measuring high-concentration markers. Summary of the invention

[0005] In view of the problems existing in the current fluorescent immunochromatographic test paper, the present invention uses two pairs of antibody pairs with different reactivity as markers, and uses europium time-resolved fluorescent microspheres and quantum dot microspheres (em: 615nm) as detection markers. Combining the detection performance of various materials, a fluorescent immunochromatographic test paper with high sensitivity and wide detection range is provided.

[0006] A fluorescent immunochromatographic test paper with high sensitivity and wide detection range, comprising a sample pad, a conjugate pad 1, a conjugate pad 2, a detection line 1, a detection line 2, a quality control line, and a nitrocellulose membrane;

[0007] The conjugate pad 1 is coated with europium time-resolved fluorescent microspheres modified with antibody 1; the conjugate pad 2 is coated with quantum dot microspheres modified with antibody 2; the detection line 1 is coated with capture antibody 1 and matched with the conjugate pad 1; the detection line 2 is coated with capture antibody 2 and matched with the conjugate pad 2; no cross reaction occurs between the combination of antibody 1 and antibody 2.

[0008] The sample pad is in the front, followed by: conjugate pad 1, conjugate pad 2, detection line 1, detection line 2, and quality control line.

[0009] The fluorescent immunochromatographic test paper also includes a bottom plate and absorbent paper.

[0010] In the above-mentioned fluorescent immunochromatographic test paper, the sample pad is overlapped on the upper surface of one end of the conjugate pad 1, the conjugate pad 1 is overlapped on the upper surface of one end of the conjugate pad 2, the conjugate pad 2 is overlapped on the upper surface of one end of the nitrocellulose membrane, and the absorbent paper is overlapped on the upper surface of the other end of the nitrocellulose membrane. The nitrocellulose membrane exposed between the end of the conjugate pad 2 and the end of the absorbent paper is coated with a detection line 1, a detection line 2 and a quality control line parallel to the end surface of the bottom plate.

[0011] The sample pad is made of glass fiber, polyester fiber, blood filter membrane and other materials; the binding pads 1 and 2 are made of glass fiber, polyester fiber and other materials.

[0012] The application of the above fluorescent immunochromatographic test paper in detecting procalcitonin PCT.

[0013] Europium time-resolved fluorescent microspheres and quantum dot microspheres (em: 615nm) both have the characteristics of wide excitation spectra, and when used as markers, they can be used for fluorescence quantitative detection using the same detection conditions. The detection line signals of fluorescent immunochromatographic test strips made using the two markers can be measured at the same time using the same fluorescence reader.

[0014] The present invention uses europium time-resolved fluorescent microspheres (200nm, 500nm, etc.) and quantum dot microspheres (Ex: 615, 10nm) as detection markers. By setting two detection lines, the detection range and sensitivity are guaranteed at the same time. A method for preparing a fluorescent immunochromatographic test strip that can improve sensitivity and broaden the quantitative detection range is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the structure of the fluorescent immunochromatographic test paper of the present invention;

[0016] Figure 2 This is a fluorescence intensity analysis diagram of different procalcitonin PCT concentrations in Example 1;

[0017] Figure 3 This is a fluorescence intensity analysis diagram of interleukin-6 IL-6 concentration in Example 2;

[0018] Figure 4 This is a graph showing the results of interleukin-6 concentration detection using different markers in Example 3.

[0019] Beneficial Effects of the Invention

[0020] 1. Wide detection range

[0021] The fluorescent immunochromatographic test paper of the present invention is used to detect procalcitonin PCT, and its detection range can reach 0.02-500ng / mL. The fluorescent immunochromatographic test paper of the present invention is used to detect interleukin 6 IL-6, and its detection range is 5-10000ng / mL.

[0022] 2. High detection sensitivity

[0023] By utilizing the characteristics of different markers and the reactivity of different antibody pairs, the detection capability of antigens at different concentrations is improved. The sensitivity of low concentrations is greatly improved, and the detection range of high concentrations is greatly expanded.

[0024] 3. Fast detection speed and wide applicability

[0025] The fluorescent immunochromatographic test paper of the present invention can be used in different fields by formulating test papers of different specifications according to the selection of sample pad components and detection limit components. DETAILED DESCRIPTION

[0026] Example 1 Detection of procalcitonin PCT

[0027] A fluorescent immunochromatographic test paper with high sensitivity and wide detection range, comprising a sample pad, a conjugation pad 1, a conjugation pad 2, a detection line 1, a detection line 2, and a quality control line; it also comprises a bottom plate and absorbent paper. The sample pad is overlapped on the upper surface of one end of the conjugation pad 1, the conjugation pad 1 is overlapped on the upper surface of one end of the conjugation pad 2, the conjugation pad 2 is overlapped on the upper surface of one end of the nitrocellulose membrane, the absorbent paper is overlapped on the upper surface of the other end of the nitrocellulose membrane, and the nitrocellulose membrane exposed between the end of the conjugation pad 2 and the end of the absorbent paper is coated with the detection line 1, the detection line 2, and the quality control line parallel to the end surface of the bottom plate. See Figure 1 .

[0028] The sample pad is made of glass fiber; the conjugate pads 1 and 2 are made of glass fiber.

[0029] Conjugate pad 1 stores 200 nm europium time-resolved fluorescent microspheres modified with PCT mouse monoclonal antibody P1;

[0030] Conjugate pad 2 stores quantum dots modified with PCT mouse monoclonal antibody P2;

[0031] Detection line T1 is marked with PCT mouse monoclonal antibody P3;

[0032] Detection line T2 is marked with PCT mouse monoclonal antibody P4;

[0033] P1, P2, P3, and P4 are 4 different PCT mouse monoclonal antibodies;

[0034] P1-P3 is a PCT antibody pair combination with high detection sensitivity and fast reaction; P2-P4 is a PCT antibody pair combination with slow reaction;

[0035] Quality control line C is sheep anti-mouse polyclonal antibody.

[0036] Take 50μL of whole blood or 30μL of serum / plasma and drop it onto the sample pad.

[0037] By utilizing the characteristics of different markers and the reactivity of different antibodies to the combination, the detection capability of different concentrations of PCT is improved. The sensitivity of low concentrations is greatly improved, and the detection range of high concentrations is greatly expanded.

[0038] T1 detection line detection range 0.02-50ng / mL, linear correlation coefficient R1 2 =0.9988; T2 test line detection range 10-500ng / mL, linear correlation coefficient R2 2 =0.9991. This test strip expands the PCT detection range to 0.02-500ng / mL. It ensures the sensitivity of the test strip and greatly broadens the detection range.

[0039] Comparative Example 1 Detection of procalcitonin PCT

[0040] Two highly specific and sensitive PCT monoclonal antibodies and one PCT polyclonal antibody were used, among which PCT monoclonal antibody I was a fluorescently labeled antibody, coated on a fluorescent pad, PCT polyclonal antibody and PCT monoclonal antibody II were capture antibodies, coated on the detection area on the chromatographic membrane; the quality control area on the chromatographic membrane was coated with rabbit anti-mouse IgG, which can directly bind to the fluorescently labeled monoclonal antibody I. Antigen-antibody reaction and fluorescent immunochromatography technology were used to detect the content of PCT.

[0041] Insert the test card after the reaction into the Getein1100 fluorescent immunoassay quantitative analyzer, and the instrument's measurement system automatically scans the binding area between the marker and the substance to be tested to obtain an optical signal. The optical signal is then measured and analyzed to quantitatively determine the concentration of the substance to be tested.

[0042] The detection range of the above method is 0.05-100 ng / mL.

[0043] Example 2 Detection of interleukin 6 IL-6

[0044] A fluorescent immunochromatographic test paper with high sensitivity and wide detection range, comprising a sample pad, a conjugation pad 1, a conjugation pad 2, a detection line 1, a detection line 2, and a quality control line; it also comprises a bottom plate and absorbent paper. The sample pad is overlapped on the upper surface of one end of the conjugation pad 1, the conjugation pad 1 is overlapped on the upper surface of one end of the conjugation pad 2, the conjugation pad 2 is overlapped on the upper surface of one end of the nitrocellulose membrane, the absorbent paper is overlapped on the upper surface of the other end of the nitrocellulose membrane, and the nitrocellulose membrane exposed between the end of the conjugation pad 2 and the end of the absorbent paper is coated with the detection line 1, the detection line 2, and the quality control line parallel to the end surface of the bottom plate. See Figure 1 .

[0045] The sample pad is made of polyester fiber; the conjugate pads 1 and 2 are made of polyester fiber.

[0046] Conjugate pad 1 stores 500 nm europium time-resolved fluorescent microspheres modified with IL-6 mouse monoclonal antibody P1;

[0047] Conjugate pad 2 stores IL-6 mouse monoclonal antibody P2 modified quantum dots (em: 615 nm);

[0048] Detection line T1 is marked with IL-6 mouse monoclonal antibody P3;

[0049] Detection line T2 is marked with IL-6 mouse monoclonal antibody P4;

[0050] P1, P2, P3, and P4 are 4 different IL-6 mouse monoclonal antibodies;

[0051] P1-P3 is a combination of IL-6 antibody pairs, with high detection sensitivity and fast reaction; P2-P4 is a combination of IL-6 antibody pairs, with relatively slow reaction;

[0052] Quality control line C is sheep anti-mouse polyclonal antibody.

[0053] By utilizing different marker properties, as well as the reactivity of different antibody pairs in combination, the ability to detect different concentrations of IL-6 was improved.

[0054] The low-concentration detection sensitivity is guaranteed, and the high-concentration detection range is greatly expanded.

[0055] Detection line T1 detection range 5-200pg / mL, linear correlation coefficient R1 2 =0.9981; detection line T2 detection range 100-10000ng / mL, linear correlation coefficient R2 2 =0.9993. This test strip greatly broadens the sensitivity and detection range of the test strip. The detection range is far better than similar products on the market, 5-4000 ng / mL.

[0056] Sensitivity experiment

[0057] Example 3 Detection of interleukin-6 using different markers

[0058] The first option:

[0059] Sample pad 1 stores 500 nm europium time-resolved fluorescent microspheres modified with IL-6 mouse monoclonal antibody P1;

[0060] Sample pad 2 was not treated;

[0061] Detection line T1 is marked with IL-6 mouse monoclonal antibody P3;

[0062] The detection line T2 is not processed;

[0063] P1 and P3 are two different IL-6 mouse monoclonal antibodies;

[0064] Quality control line C is sheep anti-mouse polyclonal antibody.

[0065] Second option:

[0066] Sample pad 1 stores quantum dots modified with IL-6 mouse monoclonal antibody P2;

[0067] Sample pad 2 was not treated;

[0068] Detection line T1 is marked with IL-6 mouse monoclonal antibody P3;

[0069] The detection line T2 is not processed;

[0070] P1 and P3 are two different IL-6 mouse monoclonal antibodies;

[0071] Quality control line C is sheep anti-mouse polyclonal antibody.

[0072] IL-6 was measured in two different ways.

[0073] Scheme 1 detection range 5-200pg / mL, linear correlation coefficient R1 2 =0.9966; detection line T2 detection range 12-200ng / mL, linear correlation coefficient R2 2 =0.9987. This indicates that when fluorescent microspheres are used as markers, the sensitivity of the method will be improved accordingly.

Claims

1. A fluorescent immunochromatographic test paper, characterized in that: It includes a base plate and a sample pad, a conjugation pad 1, a conjugation pad 2, a detection line 1, a detection line 2, a quality control line, a nitrocellulose membrane and absorbent paper, all of which are arranged on the base plate; The conjugate pad 1 is coated with europium time-resolved fluorescent microspheres modified with antibody 1; the conjugate pad 2 is coated with quantum dot microspheres modified with antibody 2; The detection line 1 is coated with a capture antibody 1 and cooperates with a conjugate pad 1; The detection line 2 is coated with a capture antibody 2 and cooperates with a conjugate pad 2; The antibody 1 and the capture antibody 1 are an antibody combination, the antibody 2 and the capture antibody 2 are an antibody combination, there is no cross reaction between the two antibody combinations, and the antibody 1, the antibody 2, the capture antibody 1 and the capture antibody 2 recognize the same antigen; One end of the sample pad is overlapped on the upper surface of one end of the conjugate pad 1, the other end of the conjugate pad 1 is overlapped on the upper surface of one end of the conjugate pad 2, the other end of the conjugate pad 2 is overlapped on the upper surface of one end of the nitrocellulose membrane, and the absorbent paper is overlapped on the upper surface of the other end of the nitrocellulose membrane; the nitrocellulose membrane exposed between the end of the conjugate pad 2 and the end of the absorbent paper is coated with a detection line 1, a detection line 2 and a quality control line parallel to the end surface of the bottom plate.

2. The fluorescent immunochromatographic test paper according to claim 1, characterized in that: The sample pad is made of glass fiber, polyester fiber or blood filter membrane; the conjugate pads 1 and 2 are made of glass fiber or polyester fiber.

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