Test strips, test cards, kits for detecting neutralizing antibodies to human adenovirus type 5 and preparation methods thereof

Abstract

The present invention discloses a test strip for detecting neutralizing antibodies to human adenovirus type 5. The test strip comprises a PVC base plate, and a sample pad, a fluorescent pad, a chromatographic membrane, and an absorbent pad arranged in sequence above the PVC base plate. The chromatographic membrane comprises a solid-phase nitrocellulose membrane consisting of a test line and a quality control line. The test line is coated with mouse anti-human IgG antibodies, the quality control line is coated with goat anti-rabbit IgG antibodies, and the fluorescent pad is coated with quantum dot-labeled rabbit IgG and quantum dot-labeled antigens. The quantum dot-labeled antigens are either or both of quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled human adenovirus type 5 intact inactivated virus. The present invention also discloses a method for preparing the test strip, as well as a test card and kit using the same. The present invention can be used to rapidly assess the level of neutralizing antibodies to adenovirus type 5 in a population, providing guidance for adenovirus vector therapy methods, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to a test strip for detecting neutralizing antibodies of human adenovirus type 5, a method for preparing the test strip, and a detection card and a kit using the same. Background Art

[0002] Human adenoviruses (HAdVs) are non-enveloped, double-stranded DNA viruses classified into seven species (AG). To date, approximately 100 types have been identified based on their genome sequences.

[0003] The adenovirus capsid is an icosahedral structure composed of 240 hexons, 12 pentons, and 12 fibers, along with other small proteins such as VI, VIII, IX, IIIa, and IVa2. The hexon is the primary protein that forms the 20 triangular faces of the viral capsid. The 12 tips are complexes composed of five penton subunits and three pilin proteins. Twelve pilin fibers extend from the capsid surface, with the penton proteins as their base, forming the knob at the tips. The pentons and the pilin knobs bind to viral receptors on the cell surface, playing a crucial role in viral infection.

[0004] HAdV is a highly contagious pathogen with multi-tissue tropism. Depending on the type of infection, it can cause a variety of illnesses, including acute respiratory disease (ARD), gastroenteritis, cystitis, keratoconjunctivitis, carditis, and meningoencephalitis. Susceptible populations to HAdV infection include infants and immunocompromised patients.

[0005] Adenoviruses can be engineered to delete certain genomic sequences, enabling safe and efficient use as vaccine, gene, and cancer therapy vectors for human use. Initially, human adenoviruses, particularly human adenovirus 5 (HAd5), were widely explored as gene delivery vectors. Following the outbreak of the novel coronavirus, adenovirus-based vaccine development was immediately initiated both domestically and internationally. In China, COVID-19 vaccines based on human Ad5 and Ad35 (recombinant COVID-19 vaccines using a replication-defective human adenovirus as a vector) are either commercially available or in the preclinical stage. Because these adenoviruses are widely circulated in the human population, rapid screening for pre-existing neutralizing antibodies to these antibodies is crucial when receiving treatment related to HAd5 and HAd35. Currently available assays for detecting neutralizing antibodies against adenoviruses all use ELISA, which is complex, time-consuming, and lacks typing, making it prone to false positives. Research has found that the vast majority of HAd5 neutralizing antibodies produced by humans recognize the hexon and fiber domains. The trimeric fiberknob can be used to differentiate neutralizing antibody levels against human adenovirus serotypes Ad2, Ad3, Ad5, Ad6, Ad37, Ad4, or Ad41 (a method for typing and detecting adenovirus neutralizing antibodies and a kit for typing and detecting adenovirus neutralizing antibodies). Using hexon and fiber to detect Had5 neutralizing antibodies is an ideal antigen, but rapid methods and test kits for HAd5 neutralizing antibodies are currently lacking. Summary of the Invention

[0006] One of the objectives of the present invention is to provide a test strip, a test card, and a test kit for detecting neutralizing antibodies to human adenovirus type 5, so as to solve the problems of existing detection operations being complicated, time-consuming, and prone to false positives; a second objective of the present invention is to provide a method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5, so as to improve the sensitivity and specificity of detection.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a test strip for detecting neutralizing antibodies of human adenovirus type 5, comprising a PVC base plate, and a sample pad, a fluorescent pad, a chromatographic membrane and a water-absorbing pad arranged in sequence above the PVC base plate; the chromatographic membrane is a solid-phase nitrocellulose membrane consisting of a detection line and a quality control line, the detection line is coated with mouse anti-human IgG antibodies, the quality control line is coated with goat anti-rabbit IgG antibodies, and the fluorescent pad is coated with quantum dot-labeled rabbit IgG and quantum dot-labeled antigens; the quantum dot-labeled antigens are any one or both of quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled human adenovirus type 5 complete inactivated virus.

[0009] Wherein, the human adenovirus type 5 fiber knob protein includes SEQ ID NO.1 amino acid sequence and SEQ ID NO.2 nucleic acid sequence.

[0010] Preferably, quantum dot-labeled human adenovirus type 5 fiber knob protein, quantum dot-labeled human adenovirus type 5 intact inactivated virus, and quantum dot-labeled rabbit IgG are evenly sprayed on a fluorescent pad substrate to form a fluorescent pad, wherein the fluorescent pad substrate is glass fiber; the spray volume is 4 to 6 μL / cm, and the number of spray strips is 1 to 5.

[0011] Preferably, the chromatography membrane is bonded to the PVC base plate, one side of the absorbent pad is bonded to one side of the chromatography membrane, the overlapping portion is 1 to 2 mm, and the other side of the absorbent pad is bonded to the PVC base plate; the fluorescent pad is bonded to the other side of the chromatography membrane, the overlapping portion is 1 to 2 mm, and the other side of the fluorescent pad is bonded to the PVC base plate; one side of the sample pad is bonded to the fluorescent pad, the overlapping portion is 1 to 2 mm, and the other side of the sample pad is bonded to the PVC base plate.

[0012] The present invention also discloses a method for preparing a test strip for detecting neutralizing antibodies of human adenovirus type 5, comprising the following steps:

[0013] a. Preparation of fluorescent pad

[0014] Quantum dot-labeled rabbit IgG and quantum dot-labeled antigen are prepared separately; then the mixture is sprayed on a fluorescent pad substrate to obtain a fluorescent pad; the quantum dot-labeled antigen is any one or both of quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled human adenovirus type 5 complete inactivated virus.

[0015] b. Preparation of chromatography membrane

[0016] Mouse anti-human IgG and goat anti-rabbit IgG were diluted separately and then streaked onto a nitrocellulose membrane using a streaking instrument to form a chromatographic membrane with a detection line and a quality control line.

[0017] c. Prepare test strips

[0018] The sample pad, fluorescent pad, chromatography membrane, and absorbent pad are sequentially pasted on a PVC bottom plate to obtain the test strip.

[0019] Furthermore, the quantum dot labeling method includes the following steps:

[0020] S1. Quantum Dot Activation and Coupling

[0021] Take the quantum dot suspension, disperse it in the reaction buffer, and mix it evenly; add the coupling agent EDC, mix and incubate it at room temperature for 20 to 40 minutes, centrifuge it at 10,000 to 15,000 rpm, remove the supernatant, disperse the quantum dots in the reaction buffer, and disperse them evenly by ultrasonication.

[0022] S2. Labeling Reaction

[0023] Disperse the human adenovirus type 5 fiber knob protein, human adenovirus type 5 complete inactivated virus, or rabbit IgG in the reaction buffer, quickly add the activated quantum dots to the reaction, mix well, and incubate at room temperature for 20 to 40 minutes.

[0024] S3. Post-processing

[0025] Centrifuge at 8000-10000 rpm for 5-10 minutes to remove free protein; add blocking solution, ultrasonically disperse, block at room temperature for 1.5-2 hours, and then centrifuge at 8000-10000 rpm for 5-15 minutes. After removing the supernatant, resuspend the quantum dots in quantum dot preservation solution.

[0026] Among them, the preparation method of the complete inactivated human adenovirus type 5 virus is: using 293 cells to amplify the replication-deficient Ad5 virus, separating the complete virus by CsCl density gradient centrifugation, and then placing it at 56°C for 1 hour to inactivate.

[0027] The human adenovirus type 5 fiber knob protein is expressed in vitro to form a trimer. The protein is expressed in Escherichia coli in vitro and then affinity purified by a nickel column.

[0028] Preferably, the excitation wavelength of the quantum dots is set to 365-450 nm, the emission wavelength is set to 620 nm, the particle size of the quantum dot microspheres is 80-200 nm, and the surface has carboxyl functional groups; in the labeling reaction step, the carboxyl groups on the quantum dot microspheres form peptide bonds with the amino groups on the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus or rabbit IgG, thereby connecting the quantum dots to the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus or rabbit IgG.

[0029] Preferably, the mass ratio of human adenovirus type 5 fiber knob protein to quantum dot microspheres is (1-1.5):10; the mass ratio of human adenovirus type 5 intact inactivated virus to quantum dot microspheres is (1-1.5):10.

[0030] Wherein, in step S2, the reaction buffer is a phosphate buffer, and the pH of the reaction buffer is 6.0-7.5.

[0031] Among them, quantum dot-labeled human adenovirus type 5 fiber knob protein, quantum dot-labeled human adenovirus type 5 complete inactivated virus and quantum dot-labeled rabbit IgG are mixed in a volume ratio of (0.1-5μL): (0.1-5μL): (1-3μL), then diluted to 25 uL with quantum dot preservation solution and evenly sprayed on the fluorescent pad substrate to obtain a fluorescent pad.

[0032] Furthermore, the quantum dot preservation solution includes the following components by mass fraction: 5-40% trehalose, 0.5-1.5% bovine serum albumin, 0.02-0.1% Niusha PC-300 antibacterial agent, and the remainder Tris-HCl buffer. The concentration of the Tris-HCl buffer is 20mM-200mM, and the pH of the quantum dot preservation solution is 7.2-8.3.

[0033] Preferably, the fluorescent pad substrate is first pretreated with a pretreatment liquid and then sprayed; the pretreatment liquid includes the following components by mass fraction: 1-3% trehalose, 0.5-1.5% bovine serum albumin, and the remainder Tris-HCl buffer, the concentration of the Tris-HCl buffer is 20mM-200mM, and the pH of the pretreatment liquid is 7.2-8.3.

[0034] Preferably, in process b, the mouse anti-human IgG antibody on the chromatographic membrane is diluted with coating solution at a coating concentration of 0.5-2.0 mg / mL; the goat anti-rabbit IgG antibody is diluted with coating solution at a coating concentration of 0.5-2.0 mg / mL. After dilution, they are respectively sucked into a film stripper and stripped onto a nitrocellulose membrane.

[0035] Wherein, the coating solution is a phosphate buffer solution containing 1-3% sucrose by mass.

[0036] In process c, the sample pad substrate is pretreated with a sample pad pretreatment solution to obtain a sample pad; the sample pad pretreatment solution comprises the following components by mass fraction: 0.2-1% PVP40, 0.5-1.5% sodium caseinate, 0.1-1% surfactant S9, and the remainder Tris-HCl buffer; the concentration of the Tris-HCl buffer is 50 mM to 250 mM, and the pH of the sample pad pretreatment solution is 7.2-8.3.

[0037] The present invention also discloses a detection card for detecting neutralizing antibodies to human adenovirus type 5, comprising a shell and the above-mentioned test strip for detecting neutralizing antibodies to human adenovirus type 5 packaged inside the shell. The shell is provided with a sample addition hole and an observation window. The sample addition hole is located above the sample pad, and the observation window is used to observe the detection line and the quality control line.

[0038] The present invention also discloses a kit for detecting neutralizing antibodies to human adenovirus type 5, comprising the above-mentioned detection card for detecting neutralizing antibodies to human adenovirus type 5, wherein a half-inhibitory dilution NT50 of less than 1000 is set as a neutralizing antibody-negative sample, and NT50 of ≥1000 is set as a neutralizing antibody-positive sample.

[0039] Due to the adoption of the above solution, the present invention has the following beneficial effects:

[0040] 1. The present invention can be used to rapidly evaluate the level of adenovirus type 5 neutralizing antibodies in the human population, providing guidance for adenovirus vector therapy methods, etc.

[0041] 2. The present invention coats a fluorescent pad with quantum dot-labeled rabbit IgG and a quantum dot-labeled antigen. Quantum dots are semiconductor materials whose electronic energy is quantized in three dimensions. Due to the quantum confinement effect of quantum dots, they exhibit superior fluorescent properties, including a broad and continuous excitation spectrum, a narrow and symmetrical emission spectrum, tunable color, high stability, a long fluorescence lifetime, and minimal susceptibility to external interference. Furthermore, quantum dots are resistant to chemical degradation and exhibit excellent biocompatibility. Their numerous excellent fluorescent properties make them an ideal alternative to organic fluorescent materials. Quantum dot immunochromatography technology utilizes quantum dot fluorescent probes as markers. In addition to the advantages of immunochromatography technology, such as ease of operation (simply immersion in liquid), rapid speed (all reactions can be completed within 15 minutes), stable reagents, and easy storage of the finished product (the reagent shelf life can reach two years or longer), the inherent optical properties and stability of quantum dots greatly expand the labeling range and the feasibility of simultaneous application of multiple detection index markers, significantly improving detection sensitivity.

[0042] 3. The human adenovirus type 5 fiber knob protein is expressed in vitro to form a trimer. The trimerized fiber knob can be used to distinguish the neutralizing antibody levels of human adenovirus serotypes Ad2, Ad3, Ad5, Ad6, Ad37, Ad4, or Ad41, thereby enabling the test strip prepared by the present invention to objectively reflect the total neutralizing antibody level.

[0043] 4. The sensitivity of the kit of the present invention is 98.86%, the specificity is 96.07%, and the total coincidence rate with the microneutralization test result is 97.36%, which effectively improves the sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic structural diagram of the test strip of the present invention.

[0045] Figure 2 This is a schematic diagram of the structure of the test card of the present invention

[0046] Description of main component symbols:

[0047] 1: Sample pad, 2: Fluorescent pad, 3: Detection line, 4: Quality control line, 5: Chromatography membrane, 6: Absorbent pad, 7: PVC bottom plate, 8: Housing, 9: Sample addition hole, 10: Observation window. DETAILED DESCRIPTION

[0048] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention is described in further detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example 1

[0050] like Figure 1 As shown, this embodiment discloses a test strip for detecting neutralizing antibodies against human adenovirus type 5.

[0051] The test strip for detecting neutralizing antibodies of human adenovirus type 5 comprises a PVC base plate 7, and a sample pad 1, a fluorescent pad 2, a chromatography membrane 5 and a water-absorbing pad 6 arranged in sequence above the PVC base plate 7.

[0052] Chromatographic membrane 5 is a solid-phase nitrocellulose membrane consisting of a test line 3 and a quality control line 4. Test line 3 is coated with mouse anti-human IgG antibody. Quality control line 4 is coated with goat anti-rabbit IgG antibody. Fluorescent pad 2 is coated with quantum dot-labeled rabbit IgG and quantum dot-labeled antigen. The quantum dot-labeled antigen is either or both of quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled whole inactivated human adenovirus type 5 virus.

[0053] The human adenovirus type 5 fiber knob protein includes the amino acid sequence of SEQ ID NO.1 and the nucleic acid sequence of SEQ ID NO.2, see the sequence listing for details.

[0054] Hereinafter, the human adenovirus type 5 fiber knob protein will be abbreviated as human Ad5 fiberknob protein, and the human adenovirus type 5 whole inactivated virus will be abbreviated as human Ad5 whole inactivated virus. The coating materials of each layer are shown in Table 1 below.

[0055] Table 1 Fluorescent pad and chromatography membrane coating materials

[0056]

[0057] As shown in Table 1, the fluorescent pad can be coated with either quantum dot-labeled human Ad5 fiberknob protein (Example 1) or quantum dot-labeled human Ad5 intact inactivated virus (Example 2) alone, or with a mixture of both (Example 3). Experiments demonstrated that the test strip obtained in Example 3 had a higher positive detection rate than those in Examples 1 and 2, indicating that a mixed coating can improve the detection rate and achieve better results.

[0058] In a preferred embodiment ③, quantum dot-labeled human Ad5 Fiberknob protein, quantum dot-labeled human Ad5 complete inactivated virus, and quantum dot-labeled rabbit IgG are evenly sprayed on a fluorescent pad substrate to form a fluorescent pad 2. The fluorescent pad substrate is made of glass fiber, the spray volume is 4 to 6 μL / cm, and the number of spray strips is 1 to 5.

[0059] like Figure 1 As shown, the test strip assembly steps in this embodiment are as follows:

[0060] (1) Select a PVC base plate 7 with a length of 78.5 mm and a width of 4 mm. Glue the chromatographic membrane 5 onto the PVC base plate 7, with the chromatographic membrane 5 located in the middle of the PVC base plate 7.

[0061] (2) Paste the left side of the absorbent pad 6 on the right side of the chromatography membrane 5 with an overlapping portion of 1 to 2 mm. The right side of the absorbent pad 6 is bonded to the PVC base plate 7.

[0062] (3) Paste the right side of the fluorescent pad 2 on the left side of the chromatography membrane 5 with an overlapping portion of 1 to 2 mm. The left side of the fluorescent pad 2 is bonded to the PVC base plate 7.

[0063] (4) Paste the right side of the sample pad 1 onto the left side of the fluorescent pad 2, with an overlapping portion of 1 to 2 mm. The left side of the sample pad 1 is bonded to the PVC base plate 7.

[0064] Example 2

[0065] like Figure 2 As shown, this embodiment discloses a detection card for detecting neutralizing antibodies against human adenovirus type 5.

[0066] The detection card for detecting neutralizing antibodies to human adenovirus type 5 comprises a housing 8 and the test strip for detecting neutralizing antibodies to human adenovirus type 5 in Example 1. The test strip for detecting neutralizing antibodies to human adenovirus type 5 is packaged in the housing 8 .

[0067] The housing 8 can be a plastic housing, and is provided with a sample loading hole 9 and an observation window 10. The plastic housing 8 is transparent or has an opening directly at the observation window for observing the test line 3 and the quality control line 4. The sample loading hole 9 is located above the sample pad 1 to facilitate loading of the sample onto the sample pad 1.

[0068] For easy storage, place the entire test card in an aluminum foil bag, add 0.5g of desiccant, and heat-seal.

[0069] The method of using the test card is as follows: After taking the test card out of the aluminum foil bag, place it flat on the table, take 10uL of the sample to be tested (serum, plasma or whole blood) and mix it with 70uL of sample diluent, then add it dropwise to the sample well, place it flat and let it stand for 15 minutes, and then put it into the detector to read the T / C value.

[0070] Example 3

[0071] This embodiment discloses a kit for detecting neutralizing antibodies to human adenovirus type 5, including a detection card for detecting neutralizing antibodies to human adenovirus type 5. The kit is provided with a neutralizing antibody-negative sample and a neutralizing antibody-positive sample.

[0072] The minimum detection limit of the neutralizing antibody test kit is related to the half-inhibitory dilution of the virus microneutralization test. That is, the minimum detection limit of the test kit can be 500, 1000, 1500, 2000, etc. The half-inhibitory dilution of the virus microneutralization test exceeds 1000, which indicates a high neutralizing antibody titer and affects the treatment effect. In the present invention, a half-inhibitory dilution NT50 of less than 1000 is used to indicate a negative neutralizing antibody sample, and an NT50 of ≥1000 is used to indicate a positive neutralizing antibody sample.

[0073] To facilitate the detection of the present invention, the following experiment was conducted: 190 human plasma samples were tested for neutralizing antibody levels using a human adenovirus type 5 microneutralization assay. The virus microneutralization assay uses neutralizing antibodies to block recombinant human adenovirus type 5 infection of HEK293 cells. NT50 represents the half-inhibitory dilution titer. The quantum dot detection reagent used was the kit of the present invention. T represents the test line, and C represents the control line. Table 2 below was obtained.

[0074] Table 2 Results of hAd5 neutralizing antibody detection in human plasma samples

[0075]

[0076] According to Table 2, the sensitivity, specificity and total coincidence rate were calculated as follows:

[0077] (1) Sensitivity = b / (b+d)*100% = 98.86%

[0078] (2) Specificity = c / (a+c) *100% = 96.07%

[0079] (3) Total compliance rate = (b+c) / (a+b+c+d)*100%=97.36%

[0080] Through the above experiments, it can be determined that when the cut-off value of the detection kit of the present invention is T / C=0.141, the sensitivity of the kit is 98.86%, the specificity is 96.07%, and the overall consistency rate with the microneutralization test results is 97.36%, indicating a good detection effect.

[0081] Example 4

[0082] This example discloses a method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5, and the process is described in detail as follows.

[0083] 1. Preparation of a fluorescent pad: Prepare quantum dot-labeled rabbit IgG and quantum dot-labeled antigen separately. Then, mix and spray-coat the mixture onto a fluorescent pad substrate to form a fluorescent pad. In this example, the quantum dot-labeled antigens are quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled intact inactivated human adenovirus type 5 virus.

[0084] (1) The process first involves preparing two antigens. The preparation process of the two antigens is as follows.

[0085] ①. The method for preparing the complete inactivated human adenovirus type 5 virus is as follows: Ad5 virus is amplified using TREx-293 cells, the complete virus is isolated by CsCl density gradient centrifugation, and then placed at 56°C for 1 hour for inactivation.

[0086] ②. The fiber knob protein of human adenovirus type 5 was expressed in vitro to form a trimer. It was expressed in Escherichia coli and then affinity purified using a nickel column. The preparation steps are as follows.

[0087] i) Centrifuge at 4000 rpm for 10 minutes to collect the expressed bacteria.

[0088] ii) The obtained bacterial cells were resuspended in 20 ml of PBS, centrifuged at 4000 rpm for 10 min, and the bacterial cells were collected and washed again.

[0089] iii) Centrifuge and wash twice with PBS, resuspend in 50 ml of disruption buffer, disrupt with a cell disrupter, and centrifuge at 10,000 rpm for 30 minutes to collect the supernatant.

[0090] iv) Add the supernatant to a well-equilibrated nickel column (equilibrated with 3 column volumes of ddH2O and 3 column volumes of equilibration buffer), mix thoroughly with the nickel packing, and incubate at room temperature for 1 hour.

[0091] v) Transfer to a gravity column.

[0092] vi) Wash with 10 column volumes of 20 mM, 40 mM, and 100 mM imidazole concentrations, respectively.

[0093] vii) The protein was eluted with an eluent containing 200 mM imidazole.

[0094] viii) Wash with 3 column volumes of 500 mM imidazole solution, then with 3 column volumes of ddH2O and 3 column volumes of 20% ethanol, and finally soak the nickel column in ethanol.

[0095] ix) The protein eluted in step vii was concentrated using a 10 kDa ultrafiltration tube and the protein concentration was measured.

[0096] x) Take 5 μg of protein for SDS-PAGE or Native PAGE.

[0097] (2) Rabbit IgG and two antigens were labeled with quantum dots. The steps are detailed below.

[0098] ①. Quantum dot activation and coupling

[0099] Take 100 μL of quantum dot suspension, disperse it in 200 μL of reaction buffer, and mix well; add 6 μL of coupling agent EDC (20 mg / mL), mix well and incubate at room temperature for 30 minutes, centrifuge at 12000 rpm for 10 minutes, remove the supernatant, disperse the quantum dots in 100 uL of reaction buffer, and disperse them evenly by ultrasonication.

[0100] ②. Labeling reaction

[0101] Disperse 100 μg each of human adenovirus type 5 fiber knob protein, intact inactivated human adenovirus type 5 virus, or rabbit IgG in 200 μL of reaction buffer. Rapidly add the activated quantum dots to the reaction, mix thoroughly, and incubate at room temperature for 30 minutes. The reaction buffer is phosphate buffered saline (pH 6.0-7.5).

[0102] The excitation wavelength of the quantum dots is set to 365-450 nm, the emission wavelength is set to 620 nm, the particle size of the quantum dot microspheres is set to 80-200 nm, and the surface has carboxyl functional groups. In the present invention, the particle size of the quantum dot microspheres for fluorescent immunochromatography is 100 nm ± 20 nm. In the labeling reaction step, the carboxyl groups on the quantum dot microspheres form peptide bonds with the amino groups on the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus, or the rabbit IgG, thereby connecting the quantum dots to the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus, or the rabbit IgG.

[0103] The mass ratio of human adenovirus type 5 fiber knob protein to quantum dot microspheres is (1-1.5):10, such as 1:10, 1.1:10, 1.2:10, 1.3:10, 1.4:10, or 1.5:10. For example, the amount of human adenovirus type 5 fiber knob protein used is 50-75 μg for 50 μg of quantum dot microspheres.

[0104] The mass ratio of human adenovirus type 5 intact inactivated virus to quantum dot microspheres is (1-1.5):10. For example, the mass ratio can be 1:10, 1.1:10, 1.2:10, 1.3:10, 1.4:10, or 1.5:10. For example, the amount of human Ad5 intact inactivated virus protein used is 50-75 μg relative to 50 μg of quantum dot microspheres.

[0105] ③. Post-processing

[0106] Centrifuge at 8000 rpm for 5 minutes to remove free protein. Add 200 μL of blocking solution, disperse by sonication, and block at room temperature for 2 hours. Centrifuge again at 8000 rpm for 10 minutes. Remove the supernatant and resuspend the quantum dots in 100 μL of quantum dot storage solution.

[0107] The quantum dot preservation solution contains the following components by weight: 5-40% trehalose, 0.5-1.5% bovine serum albumin (BSA), 0.02-0.1% Niusha PC-300 antibacterial agent, and the remainder Tris-HCl buffer. The concentration of Tris-HCl buffer is 20mM to 200mM. The pH of the quantum dot preservation solution is 7.2-8.3. This is shown in Table 3 below.

[0108] Table 3 Quantum dot storage solution components

[0109] Components Trehalose BSA Niusha PC-300 antibacterial agent Tris-HCl buffer (20mM~200mM) Example ① 5% 1.5% 0.02% 93.48% Example ② 20% 1% 0.04% 78.6% Example 3 35% 0.8% 0.1% 64.1%

[0110] (3) Mixed spraying

[0111] Mix quantum dot-labeled human adenovirus type 5 fiber knob protein, quantum dot-labeled human adenovirus type 5 intact inactivated virus, and quantum dot-labeled rabbit IgG at a volume ratio of 1.5 μL:1.5 μL:2 μL. Dilute to 25 μL with quantum dot storage solution and evenly spray onto the fluorescent pad substrate to form a fluorescent pad. Dry the fluorescent pad at 37°C for 16-18 hours before use.

[0112] The fluorescent pad substrate was pretreated with a pretreatment solution before spraying. The pretreatment solution consisted of the following components by weight: 1-3% trehalose, 0.5-1.5% bovine serum albumin, and the remainder Tris-HCl buffer. The concentration of the Tris-HCl buffer was 20mM to 200mM, and the pH of the pretreatment solution was 7.2 to 8.3. This is shown in Table 4 below.

[0113] Table 4 Components of the pretreatment solution for fluorescent pad substrate

[0114] Components Trehalose BSA Tris-HCl buffer (20mM~200mM) Example ① 1% 1.5% 97.5% Example ② 2% 1% 97% Example 3 3% 0.5% 96.5%

[0115] 2. Preparation of Chromatography Membrane

[0116] Mouse anti-human IgG and goat anti-rabbit IgG were diluted separately and then streaked onto a nitrocellulose membrane using a streaking instrument to form a chromatographic membrane with a detection line and a quality control line.

[0117] Dilute with coating solution, which consists of phosphate buffer containing 1-3% sucrose (pH 7.2). The coating concentration for mouse anti-human IgG is 0.5-2.0 mg / mL, preferably 1.5 mg / mL. The coating concentration for goat anti-rabbit IgG is 0.5-2.0 mg / mL, preferably 1.5 mg / mL. After dilution, draw the solution into a film stripper at a spray volume of 1 μL / cm and stripe onto a nitrocellulose membrane.

[0118] 3. Prepare test strips

[0119] Sample pad preparation: The sample pad substrate was pretreated with a sample pad pretreatment solution to obtain the sample pad. The sample pad pretreatment solution contained the following components by weight: 0.2-1% PVP40, 0.5-1.5% sodium caseinate, 0.1-1% surfactant S9, and the balance Tris-HCl buffer. The concentration of the Tris-HCl buffer was 50 mM to 250 mM, and the pH of the sample pad pretreatment solution was 7.2-8.3. This is shown in Table 5 below.

[0120] Table 5 Sample pad pretreatment solution components

[0121] Components PVP40 Sodium caseinate Surfactant S9 Tris-HCl buffer (50mM~250mM) Example ① 0.5% 1% 0.6% 97.9% Example ② 1% 0.5% 0.8% 97.7% Example 3 0.8% 1.5% 1% 96.7%

[0122] The sample pad, fluorescent pad, chromatography membrane, and absorbent pad were sequentially attached to a PVC base to obtain the test strip of Example 1 of the present invention. The sample pad, fluorescent pad, chromatography membrane, and absorbent pad were sequentially attached to the PVC base, with the sample pad overlapping the fluorescent pad by 1.5 mm; the fluorescent pad overlapping the chromatography membrane by 1.5 mm; and the absorbent pad overlapping the chromatography membrane by 1.5 mm.

[0123] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention. <110> Xiamen Fuchen Biotech Co., Ltd., Xiamen Joint Respiratory Health Research Institute <120> Test strips, test cards, kits for detecting neutralizing antibodies to human adenovirus type 5 and preparation methods thereof <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 195 <212> PRT <213> Human adenovirus type 5 <400> 1 Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr Leu Trp 1 5 10 15 Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu Lys Asp 20 25 30 Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile Leu Ala 35 40 45 Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile Ser Gly 50 55 60 Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn Gly Val 65 70 75 80 Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn 85 90 95 Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly Phe Met 100 105 110 Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala Lys Ser 115 120 125 Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys Pro Val 130 135 140 Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp Thr Thr 145 150 155 160 Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly His Asn 165 170 175 Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser Tyr Ile 180 185 190 For Gln Glu 195 <210> 2 <211> 588 <212> DNA <213> Page 5 <400> 2 ggtgccatta cagtaggaaa caaaaataat gataagctaa ctttgtggac cacaccagct 60 ccatctccta actgtagact aaatgcagag aaagatgcta aactcacttt ggtcttaaca 120 aaatgtggca gtcaaatact tgctacagtt tcagttttgg ctgttaaagg cagtttggct 180 ccaatatctg gaacagttca aagtgctcat cttattataa gatttgacga aaatggagtg 240 ctactaaaca attccttcct ggacccagaa tattggaact ttagaaatgg agatcttact 300 gaaggcacag cctatacaaa cgctgttgga tttatgccta acctatcagc ttatccaaaa 360 tctcacggta aaactgccaa aagtaacatt gtcagtcaag tttacttaaa cggagacaaa 420 actaaacctg taacactaac cattacacta aacggtacac aggaaacagg agacacaact 480 ccaagtgcat actctatgtc attttcatgg gactggtctg gccacaacta cattaatgaa 540 atatttgcca catcctctta cactttttca tacattgccc aagaataa 588

Claims

1. A test strip for detecting neutralizing antibodies against human adenovirus type 5, characterized in that: It includes a PVC base plate, and a sample pad, a fluorescent pad, a chromatography membrane and a water absorbent pad arranged in sequence above the PVC base plate; The chromatographic membrane is a solid phase nitrocellulose membrane consisting of a detection line and a quality control line, the detection line is coated with mouse anti-human IgG antibody, the quality control line is coated with goat anti-rabbit IgG antibody, and the fluorescent pad is coated with quantum dot-labeled rabbit IgG and quantum dot-labeled antigen; The quantum dot-labeled antigens are quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled human adenovirus type 5 complete inactivated virus.

2. The test strip for detecting neutralizing antibodies to human adenovirus type 5 according to claim 1, wherein: The human adenovirus type 5 fiber knob protein includes the amino acid sequence of SEQ ID NO.1 and the nucleic acid sequence of SEQ ID NO.2; quantum dot-labeled human adenovirus type 5 fiber knob protein, quantum dot-labeled human adenovirus type 5 complete inactivated virus and quantum dot-labeled rabbit IgG are evenly sprayed on a fluorescent pad substrate to form a fluorescent pad, and the fluorescent pad substrate is glass fiber; the spray volume is 4 to 6 μL / cm, and the number of spray strips is 1 to 5; the chromatography membrane is bonded to a PVC base plate, one side of the absorbent pad is adhered to one side of the chromatography membrane, the overlapping portion is 1 to 2 mm, and the other side of the absorbent pad is adhered to the PVC base plate; the fluorescent pad is adhered to the other side of the chromatography membrane, the overlapping portion is 1 to 2 mm, and the other side of the fluorescent pad is adhered to the PVC base plate; one side of the sample pad is adhered to the fluorescent pad, the overlapping portion is 1 to 2 mm, and the other side of the sample pad is adhered to the PVC base plate.

3. A method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5, characterized in that: The following processes are included: a. Preparation of fluorescent pad preparing quantum dot-labeled rabbit IgG and quantum dot-labeled antigen respectively; and then mixing and spraying the mixture onto a fluorescent pad substrate to obtain a fluorescent pad; The quantum dot-labeled antigens are quantum dot-labeled human adenovirus type 5 fiber knob protein and quantum dot-labeled human adenovirus type 5 intact inactivated virus; b. Preparation of chromatography membrane Mouse anti-human IgG and goat anti-rabbit IgG were diluted separately and then streaked onto a nitrocellulose membrane using a streaking instrument to form a chromatographic membrane with a detection line and a quality control line; c. Prepare test strips The sample pad, fluorescent pad, chromatography membrane, and absorbent pad are sequentially pasted on a PVC bottom plate to obtain the test strip.

4. The method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5 according to claim 3, wherein: The method of quantum dot labeling comprises the following steps: S1. Quantum Dot Activation and Coupling Take the quantum dot suspension, disperse it in the reaction buffer, and mix it evenly; add the coupling agent EDC, mix and incubate it at room temperature for 20 to 40 minutes, centrifuge it at 10,000 to 15,000 rpm, remove the supernatant, and disperse the quantum dots in the reaction buffer and disperse them evenly by ultrasonication; S2. Labeling Reaction Disperse the human adenovirus type 5 fiber knob protein, human adenovirus type 5 intact inactivated virus, or rabbit IgG in the reaction buffer, quickly add the activated quantum dots to the reaction, mix well, and incubate at room temperature for 20 to 40 minutes. S3. Post-processing Centrifuge at 8000-10000 rpm for 5-10 minutes to remove free protein; add blocking solution, ultrasonically disperse, block at room temperature for 1.5-2 hours, and then centrifuge at 8000-10000 rpm for 5-15 minutes. After removing the supernatant, resuspend the quantum dots in quantum dot preservation solution.

5. The method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5 according to claim 4, wherein: The method for preparing the complete inactivated human adenovirus type 5 virus comprises: amplifying the Ad5 virus using TREx-293 cells, isolating the complete virus by CsCl density gradient centrifugation, and then inactivating the virus at 56°C for 1 hour; the human adenovirus type 5 fiberknob protein is expressed in vitro to form a trimer, expressed in Escherichia coli in vitro, and then affinity purified using a nickel column; The excitation wavelength of the quantum dots is set to 365-450 nm, the emission wavelength is set to 620 nm, the particle size of the quantum dot microspheres is set to 80-200 nm, and the surface has carboxyl functional groups; in the labeling reaction step, the carboxyl groups on the quantum dot microspheres are formed with the amino groups on the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus or rabbit IgG to form peptide bonds, thereby connecting the quantum dots to the human adenovirus type 5 fiber knob protein, the human adenovirus type 5 complete inactivated virus or rabbit IgG.

6. The method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5 according to claim 5, wherein: The mass ratio of human adenovirus type 5 fiber knob protein to quantum dot microspheres is (1-1.5):10; the mass ratio of human adenovirus type 5 intact inactivated virus to quantum dot microspheres is (1-1.5):10; in step S2, the reaction buffer is a phosphate buffer, and the pH of the reaction buffer is 6.0-7.5; In step S3, the quantum dot preservation solution includes the following components by mass fraction: 5-40% trehalose, 0.5-1.5% bovine serum albumin, 0.02-0.1% Niusha PC-300 antibacterial agent, and the remainder Tris-HCl buffer. The concentration of the Tris-HCl buffer is 20mM-200mM, and the pH of the quantum dot preservation solution is 7.2-8.

3.

7. The method for preparing a test strip for detecting neutralizing antibodies to human adenovirus type 5 according to claim 3, wherein: Quantum dot-labeled human adenovirus type 5 fiber knob protein, quantum dot-labeled human adenovirus type 5 intact inactivated virus, and quantum dot-labeled rabbit IgG were mixed in a volume ratio of (0.1-5 μL): (0.1-5 μL): (1-3 μL), then diluted to 25 μL with quantum dot preservation solution, and evenly sprayed on the fluorescent pad substrate to obtain a fluorescent pad.

8. The method for preparing a test strip for detecting neutralizing antibodies against human adenovirus type 5 according to claim 7, wherein: First, the fluorescent pad substrate is pretreated with a pretreatment liquid and then sprayed; The pretreatment solution comprises the following components by mass fraction: 1-3% trehalose, 0.5-1.5% bovine serum albumin, and the balance Tris-HCl buffer, wherein the concentration of the Tris-HCl buffer is 20mM-200mM, and the pH of the pretreatment solution is 7.2-8.3; In process b, the mouse anti-human IgG antibody on the chromatography membrane is diluted with a coating solution at a coating concentration of 0.5-2.0 mg / mL; the goat anti-rabbit IgG antibody is diluted with a coating solution at a coating concentration of 0.5-2.0 mg / mL. After dilution, they are respectively sucked into a membrane stripper and stripped onto a nitrocellulose membrane; the coating solution is a phosphate buffer containing 1-3% by mass sucrose; In process c, the sample pad substrate is pretreated with a sample pad pretreatment solution to obtain a sample pad; the sample pad pretreatment solution includes the following components by mass fraction: PVP40 0.2-1%, sodium caseinate 0.5-1.5%, surfactant S9 0.1-1%, and the balance Tris-HCl buffer. The concentration of the Tris-HCl buffer is 50 mM to 250 mM, and the pH of the sample pad pretreatment solution is 7.2-8.

3.

9. A test card for detecting neutralizing antibodies to human adenovirus type 5, characterized by: The invention comprises a shell and a test strip for detecting neutralizing antibodies of human adenovirus type 5 as claimed in claim 1 or 2 packaged inside the shell, wherein the shell is provided with a sample addition hole and an observation window, the sample addition hole is located above the sample pad, and the observation window is used to observe the test line and the quality control line.

10. A kit for detecting neutralizing antibodies against human adenovirus type 5, characterized in that: The invention comprises a detection card for detecting neutralizing antibodies of human adenovirus type 5 as described in claim 9, wherein a half-inhibition dilution NT50 < 1000 is set as a neutralizing antibody negative sample, and NT50 ≥ 1000 is set as a neutralizing antibody positive sample.

Images