A method, kit and application of rapid viral antigen detection signal amplification

Abstract

The present invention belongs to the technical field of analysis and detection, and specifically relates to a method for rapid detection of viral antigens, a test kit and its application. In order to solve the problem of low detection sensitivity commonly found in antigen detection kits, the present invention is a four-test card in a flat circle, which surrounds a shared unique detection hole and is, in turn, a sample card, a first series antibody card, a second series antibody card and a third series antibody card. The sample test card includes a sample pad, a conjugation pad, an NC membrane and a water-absorbing pad. The layout of the first, second and third series antibody cards is similar, including a wash pad and a conjugation pad, and behind the test card on the NC membrane is the quality control line of each card.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection, and specifically relates to a method for rapid detection of viral antigens, a kit and an application thereof. Background Art

[0002] Existing antigen detection kits for the new coronavirus and other diseases generally have the problem of low detection sensitivity and can only be used in conjunction with nucleic acid detection. After the infectious virus particles are cleared from the patient's body, the viral nucleic acid can still be retained for a period of time. Antigen detection can more accurately identify infectious virus particles than nucleic acid detection, and there is an urgent need for highly sensitive viral antigen rapid detection kits. The present invention designs a cascade amplification system for antigen detection signals based on the tandem antigen-antibody interaction of antibody fusion proteins, and realizes highly sensitive antigen detection by amplifying the detection signal, which is expected to improve the current problem of low sensitivity of viral antigen detection. Summary of the invention

[0003] In view of the above problems, the present invention provides a method, a kit and an application for rapid detection of viral antigen signals.

[0004] In order to achieve the above object, the present invention adopts the following technical solutions:

[0005] A virus antigen rapid detection kit, comprising an NC membrane and a sample detection card arranged around the NC membrane, a first series antibody card, a second series antibody card and a third series antibody card;

[0006] The sample detection card is composed of a sample pad, a binding pad, an NC membrane and a water absorption pad arranged in sequence, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are all composed of a washing pad, a binding pad, an NC membrane and a water absorption pad, the thickness of the NC membrane of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card is lower than the binding pad and the water absorption pad on both sides thereof, a washing bubble is arranged on the sample pad, a detection hole and a quality control line are arranged on the NC membrane, the detection hole is close to the binding pad, the quality control line is close to the water absorption pad, and a washing bubble is arranged on the washing pad;

[0007] The sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are on the same plane, and the detection holes of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card overlap and share one detection hole.

[0008] Furthermore, the conjugate pad of the sample detection card is coated with a viral antibody of fusion protein A, the conjugate pad of the first tandem antibody card is coated with a protein A antibody of fusion protein B, the conjugate pad of the second tandem antibody card is coated with a protein B antibody of fusion protein C, and the conjugate pad of the third tandem antibody card is coated with a protein C antibody, wherein the antibodies are all colloidal gold labeled, and proteins A, B, and C are all exogenous proteins of the human body, which neither interact with viral components or their antibodies, nor interact with components of the upper respiratory tract mucosa of the human body.

[0009] A method for rapid detection of viral antigens and amplification of signals, comprising the following steps:

[0010] (1) The sample is fully lysed in the lysis solution, the sample test card is placed in a vertical direction, and then added to the sample well. Under the action of paper chromatography, it combines with the virus antibody of fusion protein A on the binding pad of the sample test card, runs to the detection well of the sample test card, and combines with the virus antibody (T line antibody) embedded there to develop color. The remaining antibody migrates to the quality control line and combines with the secondary antibody (C line antibody) embedded there to develop color; at this time, due to the low antigen content in the sample, the signal in the detection well is not strong, and it cannot even be recognized by the naked eye;

[0011] (2) placing the first tandem antibody card in a vertical direction, squeezing the wash bubble on the first tandem antibody card, and using the wash chromatography effect to transfer the protein A antibody of the fusion protein B on the binding pad of the first tandem antibody card to the NC membrane, and combining with the protein A of the fusion virus antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the first tandem antibody card;

[0012] (3) placing the second tandem antibody card in a vertical direction, squeezing the wash bubble on the second tandem antibody card, and using the wash chromatography effect to transfer the protein B antibody of fusion protein C on the binding pad of the second tandem antibody card to the NC membrane, and combining with the protein B of fusion protein A antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the second tandem antibody card;

[0013] (4) The third tandem antibody card is placed in a vertical direction, and the wash bubble on the third tandem antibody card is squeezed. With the help of the wash liquid chromatography, the protein C antibody on the binding pad of the third tandem antibody card is transferred to the NC membrane, and combined with the protein C of the fusion protein B antibody in the detection well, and the remaining antibody is transferred to the quality control line corresponding to the third tandem antibody card.

[0014] The signal amplification method of this kit is as follows Figure 3As shown: In the detection hole on the NC membrane, the embedded virus antibody binds to the COVID-19 antigen-antibody (fusion protein A) complex, and then sequentially binds to the colloidal gold-labeled protein A antibody (fusion protein B), protein B antibody (fusion protein C), and protein C antibody, completing the cascade amplification effect of the colloidal gold detection signal. Even when the antigen content is very low, it can be sensitively detected through the amplified signal.

[0015] An application of the viral antigen rapid detection kit is applied to the detection of the new coronavirus.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] Compared with traditional viral antigen detection technology, this kit has a higher antigen detection sensitivity, that is, a lower minimum detection limit. The minimum detection limit of traditional antigen detection kits is about 1-10 ng / ml, which is approximately equivalent to 20,000-50,000 copies / ml of viral nucleic acid detection sensitivity. The minimum detection limit of this kit can reach 4.4 fg / mL, which is equivalent to 172 copies / mL of viral nucleic acid detection sensitivity, which is lower than the minimum detection limit of general fluorescent PCR method for virus detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the kit of the present invention;

[0019] Figure 2 The internal structure analysis diagram of the kit of the present invention;

[0020] Figure 3 This is the signal amplification principle of the kit of the present invention. DETAILED DESCRIPTION

[0021] Example 1

[0022] like Figure 1 and Figure 2 As shown, a rapid virus antigen detection kit of this embodiment includes an NC membrane and a sample detection card arranged around the NC membrane, a first tandem antibody card, a second tandem antibody card and a third tandem antibody card;

[0023] The sample detection card is composed of a sample pad, a binding pad, an NC membrane and a water absorption pad arranged in sequence, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are all composed of a washing pad, a binding pad, an NC membrane and a water absorption pad, the thickness of the NC membrane of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card is lower than the binding pad and the water absorption pad on both sides thereof, a washing bubble is arranged on the sample pad, a detection hole and a quality control line are arranged on the NC membrane, the detection hole is close to the binding pad, the quality control line is close to the water absorption pad, and a washing bubble is arranged on the washing pad;

[0024] The sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are on the same plane, and the detection holes of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card overlap.

[0025] Taking the detection of the new coronavirus as an example, the binding pad of the sample detection card is coated with a virus antibody of fusion protein A, the binding pad of the first tandem antibody card is coated with a protein A antibody of fusion protein B, the binding pad of the second tandem antibody card is coated with a protein B antibody of fusion protein C, and the binding pad of the third tandem antibody card is coated with a protein C antibody, wherein the antibodies are all colloidal gold labeled, and proteins A, B, and C are all exogenous proteins of the human body, which neither interact with viral components or their antibodies, nor interact with components of the upper respiratory tract mucosa of the human body. Protein A: alcohol-soluble protein, protein B: alpha gliadin, protein C: glutenin Glu-B1.

[0026] A method for rapid detection of viral antigen signals based on the kit comprises the following steps:

[0027] (1) The sample is fully lysed in the lysis solution, the sample test card is placed in a vertical direction, and then added to the sample well. Under the action of paper chromatography, it binds to the virus antibody of fusion protein A on the binding pad of the sample test card, runs to the detection well of the sample test card, and combines with the virus antibody embedded there to develop color. The remaining antibody migrates to the quality control line and combines with the secondary antibody embedded there to develop color;

[0028] (2) placing the first tandem antibody card in a vertical direction, squeezing the wash bubble on the first tandem antibody card, and using the wash chromatography effect to transfer the protein A antibody of the fusion protein B on the binding pad of the first tandem antibody card to the NC membrane, and combining with the protein A of the fusion virus antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the first tandem antibody card;

[0029] (3) placing the second tandem antibody card in a vertical direction, squeezing the wash bubble on the second tandem antibody card, and using the wash chromatography effect to transfer the protein B antibody of fusion protein C on the binding pad of the second tandem antibody card to the NC membrane, and combining with the protein B of fusion protein A antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the second tandem antibody card;

[0030] (4) The third tandem antibody card is placed in a vertical direction, and the wash bubble on the third tandem antibody card is squeezed. With the help of the wash liquid chromatography, the protein C antibody on the binding pad of the third tandem antibody card is transferred to the NC membrane, and combined with the protein C of the fusion protein B antibody in the detection well, and the remaining antibody is transferred to the quality control line corresponding to the third tandem antibody card.

[0031] The minimum detection limit of this method is compared with that of the traditional method, as shown in Table 1:

[0032] method Minimum detection limit Equivalent to the detection limit of viral nucleic acid Traditional methods 5.1±3.8ng / ml 38000±13000 copies / ml This method 4.4±0.5fg / ml 172±20 copies / mL

[0033] As can be seen from Table 1, the minimum detection limit of traditional antigen detection kits is about 1-10 ng / ml, which is approximately equivalent to 20,000-50,000 copies / ml of viral nucleic acid detection sensitivity. The minimum detection limit of this kit can reach 4.4 fg / mL, which is equivalent to 172 copies / ml of viral nucleic acid detection sensitivity, which is lower than the minimum detection limit of general fluorescent PCR method for virus detection.

Claims

1. A rapid viral antigen detection kit, characterized in that: It includes an NC membrane and a sample detection card arranged around the NC membrane, a first series antibody card, a second series antibody card and a third series antibody card; The sample detection card is composed of a sample pad, a binding pad, an NC membrane and a water absorption pad arranged in sequence, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are all composed of a washing pad, a binding pad, an NC membrane and a water absorption pad, the thickness of the NC membrane of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card is lower than the binding pad and the water absorption pad on both sides thereof, a washing bubble is arranged on the sample pad, a detection hole and a quality control line are arranged on the NC membrane, the detection hole is close to the binding pad, the quality control line is close to the water absorption pad, and a washing bubble is arranged on the washing pad; The sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card are on the same plane, and the detection holes of the sample detection card, the first tandem antibody card, the second tandem antibody card and the third tandem antibody card overlap; The conjugate pad of the sample detection card is coated with a virus antibody of fusion protein A, the conjugate pad of the first tandem antibody card is coated with a protein A antibody of fusion protein B, the conjugate pad of the second tandem antibody card is coated with a protein B antibody of fusion protein C, and the conjugate pad of the third tandem antibody card is coated with a protein C antibody, wherein the antibodies are all colloidal gold labeled, and proteins A, B, and C are all exogenous proteins of the human body, which neither interact with viral components or their antibodies, nor interact with components of the upper respiratory tract mucosa of the human body.

2. A rapid detection signal amplification method based on the kit according to claim 1, characterized in that: The following steps are involved: (1) The sample is fully lysed in the lysis buffer, the sample test card is placed in a vertical direction, and then added to the sample well. Under the action of paper chromatography, it binds to the virus antibody of fusion protein A on the binding pad of the sample test card, runs to the detection well of the sample test card, and combines with the virus antibody embedded there to develop color. The remaining antibody migrates to the quality control line and combines with the secondary antibody embedded there to develop color; (2) Place the first tandem antibody card in a vertical direction, squeeze the wash bubble on the first tandem antibody card, and with the help of the wash chromatography, transfer the protein A antibody of the fusion protein B on the binding pad of the first tandem antibody card to the NC membrane, bind to the protein A of the fusion virus antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the first tandem antibody card; (3) Place the second tandem antibody card in a vertical direction, squeeze the wash bubble on the second tandem antibody card, and with the help of the wash chromatography, transfer the protein B antibody of fusion protein C on the binding pad of the second tandem antibody card to the NC membrane, bind to the protein B of fusion protein A antibody in the detection well, and the remaining antibody transfers to the corresponding quality control line of the second tandem antibody card; (4) Place the third tandem antibody card in a vertical direction, squeeze the wash bubble on the third tandem antibody card, and with the help of the wash chromatography, transfer the protein C antibody on the binding pad of the third tandem antibody card to the NC membrane, bind to the protein C of the fusion protein B antibody in the detection well, and the remaining antibody transfers to the quality control line corresponding to the third tandem antibody card.

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