Ultra-sensitive protein chip and kit for detecting influenza A H3 (H3N2) hemagglutinin protein

Abstract

The invention relates to an ultra-sensitive protein chip and a kit for detecting influenza A H3 (H3N2) hemagglutinin protein, which belong to a small molecular marker detection technology and are characterized in that the protein chip is provided with at least one sub-detection area, each sub-detection area is provided with at least one detection spot, and the detection spots are provided with non-overlapping, dispersed and tiled and non-overlapping detection spots. The coating density is 1.5 * 10 < 11 > capture molecules per square millimeter; the capture molecule is a testosterone monoclonal antibody. The testosterone protein chip disclosed by the invention can be used for rapidly detecting influenza A H3 (H3N2) hemagglutinin protein as low as 125fg / ml at low cost, and is beneficial to improving the early and correct diagnosis of influenza A.

Description

Technical Field

[0001] The present invention relates to influenza A H3 detection technology, and in particular to an ultra-sensitive protein chip and a kit for detecting influenza A H3 (H3N2) hemagglutinin / HA protein. Background Art

[0002] Influenza A virus is a common influenza virus. It is a spherical virus coated with lipid molecules. Two proteins, hemagglutinin (HA protein) and neuraminidase (NA protein), are distributed on the surface of the virus. When the influenza virus enters the host cell, the function of the HA protein is to connect with the sialic acid on the cell surface, so that the virus adheres to the host surface and enters the host body through endocytosis; after the new virus is reproduced, the NA protein is responsible for releasing the new virus from the host cell.

[0003] According to the different structures and genetic characteristics of the surface antigens hemagglutinin (HA) and neuraminidase (NA) of influenza A virus, there are many subtypes. So far, 18 subtypes of hemagglutinin (H1-H18) and 11 subtypes of neuraminidase (N1-N11) have been discovered. Among them, only H1N1, H2N2, and H3N2 mainly infect humans. H1N1 and H3N2 are the main subtypes that cause seasonal influenza. At present, viral antigen detection mostly uses colloidal gold method and immunofluorescence method, and positive detection supports diagnosis. Due to the low sensitivity of the above antigen detection methods, negative viral antigens cannot rule out influenza.

[0004] The present invention is an ultra-sensitive influenza A H3 (H3N2) hemagglutinin protein detection method, which can detect femtogram-level hemagglutinin protein in a sample, improves the sensitivity of influenza A antigen detection, and helps to improve the early and correct diagnosis of influenza A. Summary of the invention

[0005] Based on the needs of the above fields, the present invention provides an ultra-sensitive influenza A H3 (H3N2) hemagglutinin protein chip and detection kit, and the technical solution is as follows:

[0006] An ultrasensitive protein chip for detecting influenza A H3 (H3N2) hemagglutinin protein, characterized in that the protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spots are non-overlapping and dispersed, with a coating density of 1.5x10 per square millimeter. 11 The capture molecule is a monoclonal antibody against influenza A H3 (H3N2) hemagglutinin protein.

[0007] Preferably, the ultrasensitive protein chip is characterized in that the diameter of the detection spot is 3 to 5 mm.

[0008] Preferably, the ultrasensitive protein chip is characterized in that each sub-detection area is a detection spot, which is a regular or irregular pattern with an area of ​​9-25 mm, preferably a circle with a diameter of 4 mm.

[0009] Preferably, the ultrasensitive protein chip is characterized by having 20-30 sub-detection areas.

[0010] Preferably, the ultrasensitive protein chip is characterized in that a Teflon coating is provided between adjacent sub-detection areas to separate the sub-detection areas and prevent cross contamination between samples.

[0011] Preferably, the ultrasensitive protein chip is characterized in that, between the sub-detection areas, the monoclonal antibodies for detecting influenza A H3 (H3N2) hemagglutinin protein on the detection spots are completely identical to each other; or between at least two sub-detection areas, the monoclonal antibodies for detecting influenza A H3 (H3N2) hemagglutinin protein on the detection spots are different from each other.

[0012] Another aspect of the present invention is a kit for detecting influenza A H3 (H3N2) hemagglutinin protein, characterized in that it comprises at least one of the above protein chips, as well as HRP-labeled influenza A H3 (H3N2) hemagglutinin protein monoclonal antibodies, 2% BSA, 0.05% PBS-Tween solution and HRP luminescent substrate solution.

[0013] In another aspect of the present invention, a method for preparing any of the above protein chips is provided, characterized in that it comprises the following steps:

[0014] (1) obtaining a slide having at least one sub-detection area,

[0015] (2) In each sub-detection area, the influenza A H3 (H3N2) hemagglutinin protein monoclonal antibody protein molecules are evenly dispersed and sprayed to form a non-overlapping, dispersed and flat layer, 1.5x10 per square millimeter 11 The coating density of the capture molecules is 100; the capture molecules are monoclonal antibodies against influenza A H3 (H3N2) hemagglutinin protein, and then dried to form the detection spots.

[0016] The influenza A H3 (H3N2) hemagglutinin protein chip of the present invention can detect influenza A H3 (H3N2) hemagglutinin protein as low as 125 fg / ml quickly and at low cost, providing a powerful detection means for the diagnosis of influenza A H3 (H3N2), determination of disease staging and other research.

[0017] In order to improve the sensitivity, the present invention makes precise adjustments to the following items for many times: strictly and precisely adjust the pH value of the captured protein according to the properties of the monoclonal antibody of influenza A H3 (H3N2) hemagglutinin protein; strictly calculate the required number according to the molecular weight of the captured protein to determine the capture density of the captured molecule; precisely adjust the required number of detection antibodies according to the molecular weight of the detection antibodies; adjust the pH value, temperature, and washing time of the buffer solution; adjust the incubation time and temperature; and adjust various parameters required for signal scanning.

[0018] The capture antibody and HRP detection antibody of the present invention are influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibodies.

[0019] During the research and development, it was found that the independent dispersion arrangement of the capture protein molecules plays a decisive role in improving the detection sensitivity. Through intensive analysis and calculation, the present invention has concluded that the detection hole with a diameter of 4 mm has an area of ​​12.56 square millimeters, which can be tiled in a single layer of 3x10 11 In order to allow the captured protein molecules on the detection spot to be dispersed with a spacing between each other, the present invention determines that the spacing between the captured protein molecules on the detection spot is 12.56 mm. 2 1.5x10 11 The chip of the present invention can capture the reaction signals between single protein molecules, and even if the sample to be tested contains only one target protein molecule, the corresponding signal can still be detected.

[0020] The capture protein coating and fixation method of the present invention can use a GeSim ultra-micro sample spot meter (NanoPlotter NP 2.1 / 2.1E) to evenly disperse and fix the capture protein molecules on an aldehyde-based glass slide according to a preset pattern and size, or other instruments and equipment can be used to evenly disperse and coat the capture protein on the glass slide to ensure that the capture protein molecules on the glass slide are evenly dispersed in an array and the capture protein molecules do not affect each other.

[0021] The present invention claims protection for the capture protein dispersed coating fixed on the glass slide coating parameters (i.e. 1.5x10 per square millimeter 11 The dispersed fixation method improves the detection sensitivity. No matter what method is used to disperse and fix the captured protein on the glass slide, the reaction between a single influenza A H3 (H3N2) hemagglutinin protein molecule and the capture antibody can be detected, thereby improving the detection sensitivity. The capture protein fixation method that reaches the femtogram level is within the scope of protection of the technical claim.

[0022] The protein chip provided by the present invention has all influenza A H3 (H3N2) hemagglutinin protein monoclonal antibody molecules dispersedly arranged in the detection spot without affecting each other. When HRP-labeled mouse monoclonal antibody is added during detection, the sample to be tested has as low as one or several influenza A H3 (H3N2) hemagglutinin protein molecules. According to the theory of Poisson distribution, each influenza A H3 (H3N2) hemagglutinin protein monoclonal antibody molecule on the detection spot will capture one influenza A H3 (H3N2) hemagglutinin protein or not capture it. The captured influenza A H3 (H3N2) hemagglutinin protein molecule is combined with the HRP-labeled mouse monoclonal antibody complex, catalyzing the substrate to generate a luminescent signal, and the detection signal can be captured by a CCD camera. Since each captured influenza A H3 (H3N2) hemagglutinin protein small molecule will generate a signal unit, the corresponding influenza A H3 (H3N2) hemagglutinin protein molecule concentration can be calculated according to the intensity of the luminescent signal on each detection spot.

[0023] The present invention provides a simple, easy, economical, rapid, and reliable protein chip method for detecting influenza A H3 (H3N2) hemagglutinin protein small molecules. 24 samples can be detected at one time, and the test results can be issued within 40 minutes. The method can qualitatively and quantitatively detect influenza A H3 (H3N2) hemagglutinin protein molecules in clinical samples, and the results are accurate and highly sensitive, which is helpful for the early diagnosis and prevention and control of influenza A.

[0024] Other advantages of the protein array and kits provided in this study:

[0025] It allows for the simultaneous testing of multiple samples, or multiple repetitions of the same sample, or samples taken at different time points to obtain dynamic values, or simultaneous testing of samples from different patients, to achieve high-throughput and high-sensitivity testing, overall reducing testing costs and improving testing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 . A schematic plan view of the ultrasensitive protein chip of influenza A H3 (H3N2) hemagglutinin protein of the present invention;

[0027] Figure 2 . Schematic diagram of the detection principle of the ultrasensitive protein chip of influenza A H3 (H3N2) hemagglutinin protein of the present invention;

[0028] Figure 3 . Scanning diagram of the detection results of the influenza A H3 (H3N2) hemagglutinin protein standard detected by the ultrasensitive protein chip of the present invention; wherein 1, 125fg / ml; 2, 12.5fg / ml; 3, 1.23fg / ml; 4, diluent blank control DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific embodiments, but the scope of the present invention is not limited thereto. Unless otherwise specified, the operations used in the following examples are all conventional methods, and the reagents used can all be commercially available.

[0030] like Figure 1 As shown, the present invention provides an ultrasensitive protein chip for detecting influenza A H3 (H3N2) hemagglutinin protein, characterized in that the protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spots are non-overlapping and dispersed, with a coating density of 1.5x10 per square millimeter. 11 The capture molecule is a rabbit monoclonal antibody against influenza A H3 (H3N2) hemagglutinin protein.

[0031] Preferably, the ultrasensitive protein chip is characterized in that the diameter of the detection spot is 3-5 mm.

[0032] Preferably, the ultrasensitive protein chip is characterized in that each sub-detection area is a detection spot, which is a regular or irregular pattern with an area of ​​9-25 mm, preferably a circle with a diameter of 4 mm.

[0033] Preferably, the ultrasensitive protein chip is characterized by having 20-30 sub-detection areas.

[0034] Preferably, the ultrasensitive protein chip is characterized in that a Teflon coating is provided between adjacent sub-detection areas to separate the detection sub-areas and prevent cross contamination between samples.

[0035] Preferably, the ultrasensitive protein chip is characterized in that, between the sub-detection areas, the monoclonal antibodies against influenza A H3 (H3N2) hemagglutinin protein on the detection spots are completely identical to each other; or between at least two sub-detection areas, the monoclonal antibodies against influenza A H3 (H3N2) hemagglutinin protein on the detection spots are different from each other.

[0036] Example 1. Preparation of ultrasensitive influenza A H3 (H3N2) hemagglutinin protein chip

[0037] Reagents used:

[0038] Antibody 1. Product name: Influenza AH3N2 (A / Brisbane / 10 / 2007) Hemagglutinin / HA Antibody, Rabbit MAb, purchased from Beijing Sino Biological Technology Co., Ltd. (China), Catalog No. 11056-R006;

[0039] Standard: Influenza AH3N2 (A / Brisbane / 10 / 2007) Hemagglutinin / HAProtein (His Tag), purchased from Beijing Sino Biological Technology Co., Ltd. (China). Catalog No. 11056-V08H;

[0040] Antibody 2. Product name: HRP-labeled influenza A H3 (H3N2) hemagglutinin protein mouse monoclonal antibody, Influenza AH3N2 (A / Brisbane / 10 / 2007) Hemagglutinin / HAAntibody (HRP), Mouse MAb was purchased from Beijing Sino Biological Technology Co., Ltd. (China), catalog number 11056-MM01-H;

[0041] Bovine Serum Albumin Fraction V, purchased from: Sigma-Aldrich (Shanghai) Trading Co. Ltd. Catalog number: 810533

[0042] PBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH to 7.4, constant volume 1L

[0043] PBST formula: 1L PBS, + 1ml Tween-20;

[0044] The chip substrate is a diagnostic glass slide purchased from Japan. Each chip contains 24 detection holes (sub-detection areas) with a diameter of 4 mm. Each detection hole is a detection spot. In the sub-detection area, the detection spots evenly cover the detection holes. Figure 1 .

[0045] Chip preparation process: The capture protein used is: influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibody. The capture protein molecules are evenly dispersed and fixed on the aldehyde-based glass slide according to the preset pattern and size using the GeSim ultra-micro sample spotter (NanoPlotter NP 2.1 / 2.1E). The parameters are set so that the coating density is 1.5x10 per square millimeter. 11 capture molecules, so that the detection spot Figure 1 As shown on the right, the protein molecules of rabbit monoclonal antibody to influenza A H3 (H3N2) hemagglutinin protein are dispersed and arranged without overlapping.

[0046] Example 2. Sensitivity verification of ultrasensitive influenza A H3 (H3N2) hemagglutinin protein chip

[0047] The test samples were: 125fg / ml standard, 12.5fg / ml standard, 1.23fg / ml standard, and diluent blank control (the diluent was 1XPBS).

[0048] Repeat 10 times

[0049] Testing steps:

[0050] 1. On the chip prepared in Example 1, a test sample (8ul) was added to each sub-detection area and stored at room temperature for 20 minutes to allow the influenza A H3 (H3N2) hemagglutinin protein that may be contained in the sample to bind to the influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibody on the chip;

[0051] 3. Wash the chip with 0.05% PBS-Tween for 5 seconds each time, 5 times to remove non-specific binding;

[0052] 4. Add HRP detection antibody to form influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibody-influenza A H3 (H3N2) hemagglutinin protein-HRP labeled influenza A H3 (H3N2) hemagglutinin protein mouse monoclonal antibody complex on the chip. Figure 2 shown.

[0053] 4. Wash the chip with 0.05% PBS-Tween for 5 seconds each time, 5 times to remove non-specific binding;

[0054] 5. Add horseradish peroxidase luminescent substrate.

[0055] Expected results: If the sample contains influenza A H3 (H3N2) hemagglutinin protein, the test spot will show a luminescent signal; if the sample does not contain influenza A H3 (H3N2) hemagglutinin protein, the test spot will have no signal.

[0056] The actual test results are scanned as follows Figure 3 As shown, the detection signal of the 125 fg / ml standard is significantly higher than that of the diluent blank control, that is, the detection sensitivity of the chip provided by the present invention to influenza A H3 (H3N2) hemagglutinin protein is higher than 125 fg / ml.

Claims

1. An ultrasensitive protein chip for detecting influenza A H3 (H3N2) hemagglutinin protein, characterized in that: The protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, and the detection spots are non-overlapping and dispersed, with a coating density of 1.5×10 per square millimeter. 11 The capture molecule is a rabbit monoclonal antibody against influenza A H3 (H3N2) hemagglutinin protein.

2. The ultrasensitive protein chip according to claim 1, characterized in that: The diameter of the detection spot is 3-5 mm.

3. The ultrasensitive protein chip according to claim 1, characterized in that: Each sub-detection area is a detection spot, which is a regular or irregular pattern with an area of ​​9-25 mm.

4. The ultrasensitive protein chip according to claim 1, characterized in that: Each sub-detection area is a circle with a diameter of 4 mm.

5. The ultrasensitive protein chip according to claim 1, characterized in that: There are 20-30 sub-detection areas.

6. The ultrasensitive protein chip according to claim 1, characterized in that: There is a Teflon coating between adjacent sub-detection areas to separate the sub-detection areas and prevent cross contamination between samples.

7. The ultrasensitive protein chip according to claim 1, characterized in that: Between the sub-detection areas, the influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibodies on the detection spots therein are completely identical to each other.

8. The ultrasensitive protein chip according to claim 1, characterized in that: At least between two of the sub-detection zones, the influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibodies on the detection spots are different from each other.

9. A kit for detecting influenza A H3 (H3N2) hemagglutinin protein, characterized in that: The invention comprises at least one protein chip as mentioned above, as well as HRP-labeled influenza A H3 (H3N2) hemagglutinin protein monoclonal antibody, 2% BSA, 0.05% PBS-Tween solution and HRP luminescent substrate solution.

10. The method for preparing the ultrasensitive protein chip according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) obtaining a slide having at least one sub-detection area, (2) In each sub-detection area, the influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibody protein molecules are evenly sprayed to form a non-overlapping, dispersed and flat layer with a density of 1.5×10 per square millimeter. 11 The coating density of capture molecules is 1 capture molecule; the capture molecule is influenza A H3 (H3N2) hemagglutinin protein rabbit monoclonal antibody protein molecule and then dried to form the detection spot.

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