A detection system using dry chemiluminescence method

Abstract

The present invention is a detection system using dry chemiluminescence method, which effectively solves the technical problems of long reaction time, easy cross-infection and inaccurate detection in the prior art. It includes a reagent card, and the reagent card includes a dry chromatography test strip and a detection card shell. The detection card shell is buckled above and below the dry chromatography test strip. The reagent card is used in combination with a starting liquid and a detection instrument. The dry luminescence detection system of the present invention not only retains the advantages of high sensitivity, good accuracy and strong anti-interference ability of chemiluminescence detection, but also uses chromatography technology for separation and avoids complex operation steps such as washing. It is simple and convenient, with a fast detection speed. During the whole operation process, the starting liquid after luminescence will also be absorbed by the blotting paper in the test paper card, without liquid discharge, and the operation is convenient.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro immunodiagnosis in medicine, and specifically to a detection system using dry chemiluminescence method. Background Art

[0002] In recent years, chemiluminescence technology has advanced by leaps and bounds in the field of in vitro diagnosis. However, the coating carriers of traditional chemiluminescence technology are polyethylene 96-well plates or magnetic microspheres. The antigen-antibody reaction occurs in liquid, with a long reaction time. After the reaction is completed, 3 to 5 washings are required to wash away unreacted antibody impurities, and then a substrate or reaction solution is added for luminescence, and the number of photons is measured in an instrument.

[0003] The washing in the operation uses a large amount of washing solution and generates a large amount of potentially infectious liquid. During the washing process, the same washing system is used to wash the reaction solution instruments of different samples, which is prone to cross-contamination and reduces the detection accuracy. Moreover, the whole process takes a long time and the fully automatic instrument is huge.

[0004] Fluorescent immunochromatography technology has the advantages of fast speed and simple operation. However, fluorescence is excitation light, with low luminescence intensity and large detection deviation. When detecting, the fluorescent substances are stacked on the test line and the quality control line, and the light is severely blocked from each other when irradiated by laser, and the two lines also affect each other. In addition, there is no washing step, and more impurities such as miscellaneous proteins on the chromatographic strip can also generate fluorescence, thus affecting the detection result. The detection result is the fluorescence ratio of the test line and the quality control line, and the corresponding random error introduced is large, and the detection is inaccurate.

[0005] On this basis, the present invention provides a detection system using dry chemiluminescence method to solve this problem. Summary of the Invention

[0006] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a detection system using dry chemiluminescence method, which effectively solves the technical problems of long reaction time, easy cross-infection and inaccurate detection in the current technology.

[0007] A detection system using dry chemiluminescence method, characterized in that it includes a reagent card, the reagent card includes a dry chromatography test strip and a detection card shell, the detection card shell is buckled above and below the dry chromatography test strip, and the reagent card is matched with a starting solution and a detection instrument.

[0008] Preferably, the dry chromatography test strip includes a back plate, and a sample treatment pad, a substrate labeling pad, a coating pad, and a blotting paper are sequentially adhered to the back plate from left to right by an adhesive.

[0009] Preferably, the sample treatment pad is made by soaking glass fiber paper with lectin and 0.1M phosphate buffer solution, and then drying it.

[0010] Preferably, the substrate labeling pad is prepared by spraying the antibody or antigen to be detected labeled with acridinium ester on glass fiber paper and then drying it.

[0011] Preferably, the coating pad is prepared by dotting the antibody or coupled antigen solution of the object to be detected on the nitrocellulose membrane, and then drying it to form coating dots.

[0012] Preferably, the detection card housing comprises a bottom shell and an upper cover, the bottom shell is mounted on the lower surface of the back plate, and the upper cover is placed above the dry chromatography test strip.

[0013] Preferably, the upper cover is provided with a sample addition hole at the sample processing pad and a detection hole at the coating pad.

[0014] Preferably, the starting solution is a mixed solution containing hydrogen peroxide, potassium hydroxide and an enhancer.

[0015] Preferably, the detection instrument includes a detection module and a calculation module.

[0016] The present invention provides a method for an in vitro immunodiagnostic reagent of a dry luminescence detection system that can be conveniently, quickly and accurately detected in various occasions. The method only requires a trace amount of sample and does not require steps such as washing to quickly detect the content of antigen or antibody. The present invention has low background luminescence, high signal-to-noise ratio, few interference factors of luminescence reaction, rapid and concentrated luminescence release, high luminescence efficiency, high luminescence intensity, easy to bind to protein, and the photon yield does not decrease after binding, thereby increasing sensitivity.

[0017] The dry luminescence detection system of the present invention not only retains the advantages of high sensitivity, good accuracy and strong anti-interference ability of chemiluminescence detection, but also utilizes chromatography technology for separation and avoids complex operation steps such as washing. It is simple and convenient, with a fast detection speed. During the entire operation process, the starting liquid after luminescence will also be absorbed by the absorbent paper in the test paper card, without liquid discharge, and is easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a three-dimensional schematic diagram of the overall structure of the reagent card of the present invention.

[0019] Figure 2 This is a standard curve diagram of the present invention drawn by taking the natural logarithm of the antigen standard concentration and the average value of the measured photon number. DETAILED DESCRIPTION

[0020] The above and other technical contents, features and effects of the present invention are described in detail below with reference to the attached Figure 1 The detailed description of the embodiments will clearly show that the structural contents mentioned in the following embodiments are all based on the drawings in the specification.

[0021] The exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.

[0022] The present invention is a detection system using dry chemical luminescence method, which includes a reagent card. The reagent card includes a dry chromatography test strip and a detection card shell. The dry chromatography test strip is inserted into the detection card shell, and the detection card shell is buckled above and below the dry chromatography test strip. When in specific use, the reagent card also needs to be used in conjunction with a starting solution and a detection instrument.

[0023] The dry chromatography test strip includes a long strip-shaped back plate 1. On the back plate 1, a sample treatment pad 2, a substrate labeling pad 3, a coating pad 4, and a blotting paper 5 are successively adhered from left to right by non-drying glue, and their widths are all cut into equal widths as required.

[0024] The sample treatment pad 2 is made by soaking glass fiber paper with lectin and 0.1M phosphate buffered saline solution, and then drying it in a constant temperature air blast drying oven.

[0025] The substrate labeling pad 3 is made by spraying an acridinium ester-labeled analyte antibody or antigen on glass fiber paper, and then drying it in a constant temperature air blast drying oven.

[0026] The coating pad 4 is made by dot-spraying an analyte antibody or conjugate antigen solution on a nitrocellulose membrane, and then drying it in a constant temperature air blast drying oven to form a coating line or coating dots.

[0027] The detection card shell includes a bottom shell 6 and an upper cover 7. The bottom shell 6 is installed on the lower surface of the back plate 1, and the upper cover 7 is installed above the dry chromatography test strip. A strip-shaped fixing groove is reserved between the bottom shell 6 and the upper cover 7 to fix the dry chromatography test strip.

[0028] On the upper cover 7, a sample addition hole 8 is opened at the position of the sample treatment pad 2, and a detection hole 9 is opened at the position of the coating pad 4. The detection hole 9 is circular. The sample addition hole 8 is used to add a blood sample, and the detection hole 9 is used to observe the detection result.

[0029] The starting solution is a mixed solution containing hydrogen peroxide, potassium hydroxide, and an enhancer.

[0030] The detection instrument mainly includes a detection module and a calculation module. The detection module moves up and down a light guide probe with a liquid addition steel needle, and then adds the starting solution through a hollow stainless steel needle. The light guide probe transmits the light generated by the test strip card to a photoelectric converter, and after passing through a filter, the number of photons at a wavelength of 430 nm is detected. The calculation module simulates the detected concentration based on the detected number of photons, and calculates the monitored concentration of the sample through a regression equation.

[0031] In order to have a clearer understanding of the technical features, objectives, and effects of the present invention, the specific implementation manners are described below, but not limited to this product. The following are more detailed embodiments:

[0032] In the detection system of the dry chemical luminescence method of the present invention, it is a dry luminescence reagent for C-reactive protein (CRP).

[0033] Preparation of each component of the reagent card:

[0034] (1) Preparation of the sample treatment pad:

[0035] Dilute concanavalin A to a 0.1 mg / ml solution with 0.01 M PB, then soak glass fiber paper, and after completely wetting, dry it at 37 °C to make the sample treatment pad.

[0036] (2) Preparation of the coating pad:

[0037] Dilute the coated monoclonal antibody of CRP to 0.1 mg / ml with the coating diluent (0.1 M CB, 0.138 M NaCl, 0.005 M EDTA-Na-2H2O, pH 7.5), then spray it onto the nitrocellulose membrane to form a thick line, and then dry it at 37 °C to make the coating pad.

[0038] (3) Preparation of the substrate labeling pad:

[0039] Label the monoclonal antibody of CRP with acridinyl sulfonamide, dilute it to 0.08 mg / ml with the release buffer (containing 20% sucrose, 5% trehalose, 0.5% N,O-bis(trimethylsilyl)acetamide (BSA), 0.1% sodium azide), then spray it onto the glass fiber membrane, and then dry it at 37 °C to make the substrate labeling pad.

[0040] (4) Preparation of the reagent card: According to the combination method attached Figure 1 , on the plastic bottom plate with adhesive, use the overlapping method. First, paste the nitrocellulose membrane (which is the coating pad after formation), then paste the substrate labeling pad and the absorbent paper at the left and right ends of the nitrocellulose membrane respectively, and then paste the sample treatment pad at the left end of the substrate labeling pad. Cut the pasted test strip into a 35-mm dry chromatography test strip, and put the dry chromatography test strip into the detection card shell. Align the sample treatment pad with the sample adding hole and align the detection line on the coating pad of the nitrocellulose membrane with the detection hole.

[0041] Preparation of the starting solution:

[0042] Take purified water to prepare a solution containing 0.15 M sodium hydroxide, 0.5% urea peroxide and 1% octyltrimethylammonium chloride.

[0043] Quantitative detection of the reagent card:

[0044] (1) Standard curve drawing:

[0045] Add CRP antigen standard products with different concentrations (take five different concentrations, namely 1, 10, 20, 100, 200 mg / L respectively, and set 5 replicates for each concentration) into the sample addition hole on the prepared CRP dry chromatography test strip, and perform membrane chromatography at 37 °C for 10 minutes. Turn on the detection instrument, inject the starting solution into the starting solution bottle of the detection instrument, adjust the instrument so that the starting solution is injected 50 μL each time, and put the test strip card after chromatography into the instrument to read the photon count as shown in the following table:

[0046] Table 1 Detection results of CRP standard products:

[0047]

[0048]

[0049] Table 1

[0050] Take the natural logarithm of the antigen standard product concentration and the average value of the measured photon count respectively to draw a standard curve. The standard curve graph is attached to the specification Figure 2 。

[0051] Use the sample with unknown concentration to detect the photon count according to the method for detecting the standard product and calculate the result according to the standard curve as shown in Table 3.

[0052]

[0053] Table 3 Comparison of the advantages and disadvantages between the detection system of the present invention and the prior art methods:

[0054]

[0055]

[0056] The present invention provides a method for an in vitro immunodiagnostic reagent of a dry luminescence detection system that can conveniently, quickly and accurately detect on various occasions. A method that only requires a small amount of sample and can quickly detect the antigen or antibody content without steps such as rinsing. The background luminescence of the present invention is low, the signal-to-noise ratio is high, there are few interference factors in the luminescence reaction, the luminescence release is rapid and concentrated, the luminescence efficiency is high, the luminescence intensity is large, it is easy to be coupled with proteins and the photon yield does not decrease after coupling, thereby increasing the sensitivity.

[0057] The dry luminescence detection system of the present invention not only retains the advantages of high sensitivity, good accuracy and strong anti-interference ability of chemiluminescence detection, but also uses chromatography technology for separation and avoids complex operation steps such as washing. It is simple and convenient, has a fast detection speed, and during the whole operation process, the starting solution after luminescence will also be absorbed by the blotting paper in the test strip card without liquid discharge, and the operation is convenient.

[0058] The beneficial effects of the present invention are:

[0059] 1. The sample processing pad uses plant lectin, which can make red blood cells agglutinate on the sample processing pad when using whole blood sample testing, so as to separate blood cells from plasma. It has higher separation efficiency and faster speed than traditional blood filter membranes, and requires less test samples.

[0060] 2. It adopts dry luminescent chromatography separation technology, which does not require the use of cleaning fluid, saves a lot of water, and has no wastewater discharge (the added start-up fluid will be absorbed by the absorbent paper on the test strip after testing). The equipment can be used on mobile testing equipment such as testing vehicles.

[0061] 3. Dry luminescence eliminates the tedious incubation and cleaning operations of wet luminescence, significantly improves detection efficiency, the equipment is simple and has a low failure rate, and results can be obtained in about 15 minutes, so it can be used in emergency and other fields.

[0062] 4. Adopt acridinium ester self-luminescence, which emits light quickly (completed within 0.9S), has high luminous intensity and low interference. The detection head is pressed and stuck for detection, and the starting liquid filling and detection are carried out simultaneously on the detection head to avoid light leakage.

Claims

1. A detection system using dry chemiluminescence method, characterized in that, It includes a reagent card, which comprises a dry chromatography test strip and a detection card shell. The detection card shell is buckled above and below the dry chromatography test strip. The reagent card is used in combination with a starting liquid and a detection instrument; The dry chromatography test strip includes a back plate (1). From left to right on the back plate (1), a sample treatment pad (2), a substrate labeling pad (3), a coating pad (4), and a blotting paper (5) are adhered in sequence by an adhesive; The sample treatment pad (2) is made by diluting concanavalin A to a 0.1 mg / ml solution with 0.01 M PB, then soaking glass fiber paper, and drying it at 37°C after being completely soaked to make the sample treatment pad; The substrate labeling pad (3) is made by labeling a monoclonal antibody of CRP with acridinesulfonamide, diluting it to 0.08 mg / ml with a release buffer (containing 20% sucrose, 5% trehalose, 0.5% N,O-bis(trimethylsilyl)acetamide (BSA), 0.1% sodium azide), and then spraying it onto a glass fiber membrane, and then drying it at 37°C to make the substrate labeling pad; The coating pad (4) is made by diluting a coated monoclonal antibody of CRP to 0.1 mg / ml with a coating diluent: 0.1 M CB, 0.138 M NaCl, 0.005 M EDTA-Na-2H2O, pH 7.5, and then spraying it onto a nitrocellulose membrane to form a thick line, and then drying it at 37°C to make the coating pad; The detection card shell includes a bottom shell (6) and an upper cover (7). The bottom shell (6) is installed on the lower surface of the back plate (1), and the upper cover (7) is placed above the dry chromatography test strip; The upper cover (7) is provided with a sample addition hole (8) at the position of the sample treatment pad (2) and a detection hole (9) at the position of the coating pad (4); The starting liquid is prepared by taking purified water and containing 0.15 M sodium hydroxide, 0.5% urea peroxide, and 1% octyltrimethylammonium chloride.

2. The detection system of a dry chemical luminescence method according to claim 1, characterized in that, The detection instrument includes a detection module and a calculation module.

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