Ultra-sensitive protein chip and kit for detecting hepatitis B virus surface antigen

Abstract

The invention relates to an ultra-sensitive protein chip and a kit for detecting hepatitis virus surface antigen, which belong to a small molecular marker detection technology, and are characterized in that the ultra-sensitive protein chip for detecting hepatitis B surface antigen is provided with at least one sub-detection area, at least one detection spot is arranged in each sub-detection region, and capture molecules which are not overlapped with each other, are dispersed and tiled and have the coating density of 1.5 * 10 < 11 > per square millimeter are arranged in the detection spots; the capture molecule is a hepatitis B surface antigen mouse monoclonal antibody. According to the ultra-sensitive protein chip for detecting the hepatitis virus surface antigen, the sensitivity is improved by 1000 times compared with that of a traditional detection method, the femtogram-level hepatitis B surface antigen in a sample can be detected, the sensitivity of hepatitis B surface antigen detection is improved, and early and correct diagnosis of hepatitis B can be improved.

Description

Technical Field

[0001] The present invention relates to a hepatitis B virus detection technology, and in particular to an ultra-sensitive protein chip and a kit for detecting hepatitis B virus surface antigen (HbsAg). Background Art

[0002] Hepatitis B virus surface antigen (HbsAg) is the outer shell protein of hepatitis B virus (HBV) and is one of the five serum immune marker tests for HBV. HbsAg itself is only antigenic and not contagious. A positive HbsAg indicates the presence of HBV or integrated HBV DNA fragments in the body, and a negative HbsAg indicates that the person is not currently infected with the hepatitis B virus.

[0003] In latent HBV infection and certain stages of chronic HBV infection, when HBV DNA is present at very low levels in the body, the level of HBsAg may be very low, close to the detection limit of traditional detection methods. Due to the low sensitivity of conventional detection methods, conventional HBsAg detection methods cannot accurately detect it. Summary of the invention

[0004] Based on the needs of the above fields, the present invention provides an ultra-sensitive hepatitis B surface antigen protein chip and detection kit, which is an ultra-sensitive hepatitis B surface antigen detection method with a sensitivity 1000 times higher than that of traditional detection methods. It can detect femtogram-level hepatitis B surface antigen in samples, improve the sensitivity of hepatitis B surface antigen detection, and help improve the correct diagnosis of early hepatitis B and HBV latent infection. The technical solution for which the present invention is requested to be protected is as follows:

[0005] An ultrasensitive protein chip for detecting hepatitis B surface antigen, characterized in that the protein chip has at least one sub-detection area, each sub-detection area has at least one detection spot, the detection spots are non-overlapping and dispersed, and the coating density is 1.5x10 per square millimeter 11 The capture molecule is a mouse monoclonal antibody against hepatitis B surface antigen.

[0006] Preferably, the ultrasensitive protein chip is characterized in that the diameter of the detection spot is 3 to 5 mm.

[0007] 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.

[0008] Preferably, the ultrasensitive protein chip is characterized by having 20-30 sub-detection areas.

[0009] 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.

[0010] Preferably, the ultrasensitive protein chip is characterized in that the hepatitis B surface antigen monoclonal antibodies on the detection spots between the sub-detection areas are completely identical to each other; or there are at least two sub-detection areas, where the hepatitis B surface antigen monoclonal antibodies on the detection spots are different from each other.

[0011] Another aspect of the present invention is a kit for detecting hepatitis B surface antigen, characterized in that it comprises at least one of the above-mentioned protein chips, as well as HRP-labeled hepatitis B surface antigen polyclonal antibodies, 2% BSA, 0.05% PBS-Tween solution and HRP luminescent substrate solution.

[0012] 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:

[0013] (1) obtaining a slide having at least one sub-detection area,

[0014] (2) In each sub-detection area, the hepatitis B surface antigen 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 1 capture molecule; the capture molecule is a monoclonal antibody against the hepatitis B surface antigen, and then dried to form the detection spot.

[0015] The hepatitis B surface antigen protein chip of the present invention can detect hepatitis B surface antigen as low as 5 to 50 fg / ml quickly and at low cost, providing a powerful detection means for the diagnosis of hepatitis B, determination of disease staging and other research.

[0016] In order to improve the sensitivity, the present invention repeatedly makes precise adjustments to the following items: strictly and precisely adjust the pH value of the captured protein according to the properties of the hepatitis B surface antigen monoclonal antibody; strictly calculate the required number according to the molecular weight of the captured protein to determine the capture density of the captured molecules; 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.

[0017] The capture antibody of the invention is a mouse monoclonal antibody against the surface antigen of hepatitis B, and the HRP detection antibody is a rabbit polyclonal antibody against the surface antigen of hepatitis B.

[0018] 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 certain spacing, the present invention determines that 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.

[0019] 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.

[0020] 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 dispersion 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 hepatitis B surface antigen molecule and the captured 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 requirements.

[0021] The protein chip provided by the present invention has all hepatitis B surface antigen monoclonal antibody molecules dispersedly arranged in the detection spot without affecting each other. When HRP-labeled rabbit polyclonal antibody is added during detection, there are as few as one or several hepatitis B surface antigen protein molecules in the sample to be tested. According to the theory of Poisson distribution, each hepatitis B surface antigen monoclonal antibody molecule on the detection spot will capture one hepatitis B surface antigen or not capture it. The captured hepatitis B surface antigen molecule combines with the HRP-labeled rabbit polyclonal antibody complex, catalyzing the substrate to generate a luminescent signal, and the detection signal can be captured by a CCD camera. Since each captured hepatitis B surface antigen will generate a signal unit, the corresponding hepatitis B surface antigen molecule concentration can be calculated according to the intensity of the luminescent signal on each detection spot.

[0022] The present invention provides a simple, easy, economical, rapid, and reliable protein chip method for detecting hepatitis B surface antigen small molecules. 24 samples can be detected at a time and the test results can be issued within 40 minutes. The hepatitis B surface antigen molecules in clinical samples can be qualitatively and quantitatively detected with accurate results and high sensitivity.

[0023] The innovation of the present invention lies in the change of the preparation process, that is, the coating method of capturing protein on the chip, which greatly improves the sensitivity, and is therefore not limited to making a protein chip for detecting hepatitis B surface antigen.

[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 hepatitis B surface antigen of the present invention;

[0027] Figure 2 . Schematic diagram of the detection principle of the ultrasensitive protein chip for hepatitis B surface antigen of the present invention;

[0028] Figure 3 . Scanning diagram of the detection result of the ultrasensitive protein chip of hepatitis B surface antigen of the present invention for detecting the hepatitis B surface antigen standard;

[0029] Figure 4 .Scanning diagram of the detection results of the ultrasensitive protein chip of hepatitis B surface antigen of the present invention for detecting the hepatitis B surface antigen standard. DETAILED DESCRIPTION

[0030] 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.

[0031] like Figure 1 As shown, the present invention provides an ultra-sensitive protein chip for detecting hepatitis B surface antigen, 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 mouse monoclonal antibody against hepatitis B surface antigen.

[0032] Preferably, the ultrasensitive protein chip is characterized in that the diameter of the detection spot is 3-5 mm. Preferably, the ultrasensitive protein chip is characterized in that each sub-detection 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. Preferably, the ultrasensitive protein chip is characterized in that it has 20-30 sub-detection areas. Preferably, the ultrasensitive protein chip is characterized in that adjacent sub-detection areas are coated with Teflon to separate the detection sub-areas and prevent cross-contamination between samples.

[0033] Preferably, the ultrasensitive protein chip is characterized in that the hepatitis B surface antigen monoclonal antibodies on the detection spots between the sub-detection areas are completely identical to each other; or there are at least two sub-detection areas, where the hepatitis B surface antigen monoclonal antibodies on the detection spots are different from each other.

[0034] Example 1. Preparation of ultrasensitive hepatitis B surface antigen protein chip

[0035] Reagents used:

[0036] Antibody 1. Product name: Mouse anti-hepatitis B virus surface antigen (Ad / Ay) antibody [HB24], purchased from Abcam, catalog number ab54247;

[0037] Hepatitis B surface antigen protein standard: Product name: Recombinant hepatitis B virus Hepatitis B Surface Antigen AD protein, purchased from Abcam, catalog number: ab193473.

[0038] Antibody 2. Product name: HRP-labeled polyclonal rabbit anti-hepatitis B virus surface antigen (Ad / Ay) antibody, purchased from Abcam, catalog number ab20878;

[0039] Bovine serum albumin fraction 5, Bovine Serum Albumin Fraction V, purchased from: Sigma-Aldrich (Shanghai) Trading Co. Ltd; Catalog No.: 810533

[0040] 1XPBS formula: sodium chloride (NaCl) 8g, potassium chloride (KCl) 0.2g, disodium hydrogen phosphate (Na2HPO4) 1.44g, potassium dihydrogen phosphate (KH2PO4) 0.24g, adjust pH 7.4, constant volume 1L

[0041] PBST formula: 1L PBS, + 1ml Tween-20;

[0042] 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 .

[0043] Chip preparation process: The capture protein used is: hepatitis B virus surface antigen mouse 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. Figure 1 As shown on the right, the hepatitis B virus surface antigen mouse monoclonal antibody protein molecules are dispersed in an array within the detection spot without overlapping each other. Example 2. Sensitivity test of the ultrasensitive hepatitis B virus surface antigen protein chip

[0044] The test samples were: dilutions of different concentrations of hepatitis B surface antigen protein standard, the diluent was 1X PBS; the blank control was 1X PBS.

[0045] Here are the steps:

[0046] 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 hepatitis B virus surface antigen that may be contained in the sample to bind to the hepatitis B virus surface antigen mouse monoclonal antibody on the chip;

[0047] 3. Wash the chip with 0.05% PBS-Tween for 5 seconds each time, 5 times to remove non-specific binding;

[0048] 4. Add HRP detection antibody to form a complex of mouse monoclonal antibody to hepatitis B virus surface antigen-hepatitis B virus surface antigen-HRP labeled hepatitis B virus surface antigen rabbit polyclonal antibody on the chip. Figure 2 shown.

[0049] 4. Wash the chip with 0.05% PBS-Tween for 5 seconds each time, 5 times to remove non-specific binding;

[0050] 5. Add horseradish peroxidase luminescent substrate.

[0051] Expected results: If the sample contains hepatitis B virus surface antigen, the test spot will show a luminescent signal; if the sample does not contain hepatitis B virus surface antigen, the test spot will have no signal.

[0052] Figure 3The figure shows the result of a detection experiment. The numbers on the left indicate the concentration of the standard dilution added in each row. There are 4 replicates for each concentration, and the bottom row is the control.

[0053] It can be seen that as the concentration of the standard is continuously diluted, the signal intensity generally shows a gradual decrease trend. The concentration of the standard dilution solution is as low as 5×10 -10 The detection signal at 500 fg / ml was significantly higher than that of the blank control; the concentration of the standard dilution was as low as 5×10 -11 The detection signal at 50 fg / ml was still higher than that of the blank control, and the sensitivity of this detection reached 50 fg / ml.

[0054] Figure 4 The results of another test are shown, in which the three test spots on the left of each row are 3 repetitions of the standard with the same dilution concentration, and the one test spot on the right is a blank control. The numbers on the left indicate the concentration of the standard dilution added to each row.

[0055] It can be seen that as the concentration of the standard is continuously diluted, the signal intensity generally shows a gradual downward trend. The detection signal of the 50fg / ml standard dilution is significantly higher than that of the blank control; the detection signal of the 5fg / ml standard dilution is higher than that of the blank control, that is, the ultrasensitive protein chip provided by the present invention can detect the hepatitis B virus surface antigen with a sensitivity of up to 5fg / ml.

[0056] The results of the two tests showed that the sensitivity of the chip provided by the present invention in detecting hepatitis B virus surface antigen reached 5 to 50 fg / ml.

Claims

1. An ultrasensitive protein chip for detecting hepatitis B virus surface antigen, 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 1.5x per square millimeter. 11 The capture molecule is a mouse monoclonal antibody against the surface antigen of hepatitis B virus.

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: The mouse monoclonal antibodies against the surface antigen of hepatitis B virus on the detection spots between the sub-detection areas are completely identical.

8. The ultrasensitive protein chip according to claim 1, characterized in that: There are at least two sub-detection zones between which the HBV surface antigen mouse monoclonal antibodies detected on the spots are different from each other.

9. A kit for detecting hepatitis B virus surface antigen, characterized in that: The invention comprises at least one ultrasensitive protein chip according to any one of claims 1 to 8, as well as HRP-labeled rabbit polyclonal antibody, PBS-Tween solution containing 0.05% BSA 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 hepatitis B virus surface antigen mouse monoclonal antibody protein molecules are evenly dispersed and sprayed to form a non-overlapping array, with a dispersion of 1.5×10 per square millimeter. 11 The coating density of capture molecules is 300; the capture molecules are hepatitis B virus surface antigen mouse monoclonal antibody protein molecules and then dried to form the detection spots.

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