A latex test strip for detecting neutralizing antibodies of the novel coronavirus that can be traced back to the titer of the neutralization test of the novel coronavirus

Abstract

The invention discloses a novel coronavirus neutralizing antibody latex test strip traceable to the titer of the novel coronavirus neutralization test, which contains a sample pad, a conjugation pad, a reaction membrane and a blotting paper sequentially joined on a base plate, wherein the conjugation pad is coated with latex microspheres coupled to RBD protein 1 and sheep anti-chicken IgY respectively; a detection limit and a quality control line are provided on the reaction membrane, the detection limit is coated with RBD protein 2, and the quality control line is coated with chicken IgY antibody. Compared with cVNT, it has the characteristics of low cost, fast detection, sensitivity, instant, no need for professional operation, etc., and can be completed in a secondary biosafety laboratory; and compared with conventional latex test strips, it has a semi-quantitative advantage; it can effectively monitor the vaccination reaction in clinical research and serve as an evaluation index of vaccine effect; improve the novel coronavirus detection rate, make up for the missed detection caused by the false negative of nucleic acid detection; detect the neutralizing antibody content in the body of the novel coronavirus infected / recovered person to determine whether there is a risk of reinfection.

Description

Technical Field

[0001] The present invention relates to the field of virus detection technology, and in particular to a novel coronavirus neutralization antibody latex test strip that can be traced back to the novel coronavirus neutralization test titer. Background Art

[0002] The new coronavirus (SARS-CoV-2) is a new type of β-coronavirus with a diameter of 60 to 140 nm and a crown-like shape under an electron microscope. The receptor binding domain (RBD) of the S protein (spike protein) of SARS-CoV-2 can bind to the human ACE2 protein, allowing the virus to enter cells and replicate. At the same time, RBD is also the main target of neutralizing antibodies. Neutralizing antibodies (NAbs) of the new coronavirus are antibodies produced in the bodies of patients with new coronavirus pneumonia and those who have received the new coronavirus vaccine. They can bind to the RBD of the S protein to block the binding of the virus to human ACE2, showing direct neutralizing activity. Therefore, the detection of neutralizing antibodies can determine whether the human body has the ability to resist infection with this virus, and can effectively monitor vaccination responses and evaluation indicators of vaccine effectiveness in clinical studies.

[0003] The live virus neutralization test (cVNT) is the laboratory gold standard method for neutralizing antibody detection. It can detect the neutralizing antibody titer in samples, but it needs to be tested in a Level 3 biosafety laboratory. Due to the limitations of experimental conditions, it is difficult to carry out routinely, and the operation process is cumbersome and time-consuming. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a latex test strip for detecting neutralizing antibodies of the novel coronavirus that can be traced back to the titer of the novel coronavirus neutralization test.

[0005] The first object of the present invention is to provide a latex test strip for detecting neutralizing antibodies of the novel coronavirus that can be traced back to the titer of the neutralization test of the novel coronavirus.

[0006] The second object of the present invention is to provide the use of the novel coronavirus neutralizing antibody latex test strip in the preparation of a novel coronavirus neutralizing antibody kit.

[0007] The third object of the present invention is to provide a method for interpreting the results of the novel coronavirus neutralizing antibody latex test strip.

[0008] In order to achieve the above object, the present invention is implemented by the following scheme:

[0009] The present invention prepares a new crown neutralizing antibody double antigen latex test strip, and its detection principle is as follows Figure 1As shown, the test strip is composed of a sample pad, a conjugation pad, a reaction membrane and a water-absorbing paper which are sequentially connected to a PVC bottom plate. The RBD protein is sprayed on the conjugation pad using a double antigen sandwich immunoassay; and the RBD protein is coated on the test line (T line) on the reaction membrane.

[0010] If the blood sample tested contains neutralizing antibodies to the new coronavirus, the neutralizing antibodies in the sample can bind to the latex-labeled RBD protein to form an immune complex. Under the action of chromatography, the complex moves forward along the test strip and reacts with the coated RBD protein when passing the test line (T line) to form an immune complex and present a red strip. After the test result is judged to be positive, the test sample can be further diluted to estimate the range of neutralizing antibody titers.

[0011] In this test method, latex-labeled sheep anti-chicken IgY is used as a quality control system to play a quality control and control role. If no red stripe appears at the quality control line (C line) during the test, it means that the measured value is invalid and the test should be repeated.

[0012] The present invention provides a latex microsphere test strip for detecting dual antigens of neutralizing antibodies of the novel coronavirus, wherein the latex microsphere test strip comprises a sample pad, a binding pad, a reaction membrane and absorbent paper sequentially connected to a bottom plate.

[0013] Wherein, the conjugate pad is coated with latex microspheres coupled with RBD protein and goat anti-chicken IgY;

[0014] A detection line and a quality control line are arranged on the reaction membrane, wherein the detection line is coated with RBD protein, and the quality control line is coated with chicken IgY antibody.

[0015] Preferably, the RBD protein coated on the detection line is: Feipeng Bio, product number: NCOV-PS-AG27, and the RBD protein coupled to the latex microspheres is: Baixi Bio, product number: COV-RBD2.

[0016] Preferably, the latex microspheres are red latex microspheres, and more preferably, the latex microspheres are red carboxyl latex microspheres.

[0017] Preferably, the amount ratio of RBD coupled to red carboxyl latex microspheres and goat anti-chicken IgY coupled to red carboxyl latex microspheres is 120:25, calculated based on the mass of RBD protein and goat anti-chicken IgY.

[0018] Preferably, the latex microspheres coupled to the RBD protein and the latex microspheres coupled to the goat anti-chicken IgY coated on the conjugate pad are calculated based on the mass of the RBD protein and the goat anti-chicken IgY, and the conjugate pad of 10 mm × 30 cm is coated with 8.64 μg / sheet of RBD protein and 1.8 μg / sheet of goat anti-chicken IgY, and the spray volume is 6 μL / cm (specifically, the latex microspheres coupled to the RBD protein coated on the conjugate pad are calculated based on the mass of the RBD protein, and the concentration is 30 μg / 500 μL, and the latex microspheres coated on the conjugate pad are 100 μg / sheet of RBD protein and 1.8 μg / sheet of goat anti-chicken IgY. The concentration of the microsphere-coupled goat anti-chicken IgY is 25 μg / 500 μL, calculated based on the mass of goat anti-chicken IgY. In the marker mixture, the concentration of RBD protein is 24 μg / 500 μL, and the concentration of goat anti-chicken IgY is 5 μg / 500 μL. Each 10 mm × 30 cm conjugate pad is coated with 180 μL of marker mixture, and at 6 μL / cm, 8.64 μg / sheet of RBD protein and 1.8 μg / sheet of goat anti-chicken IgY are coated); the detection line is coated with 1 mg / mL RBD protein, and the spray volume is 1 μL / cm; the quality control line is coated with 1 mg / mL chicken IgY, and the spray volume is 1 μL / cm.

[0019] Preferably, the latex microspheres coupled with RBD protein and goat anti-chicken IgY are latex microspheres coupled with RBD protein and latex microspheres coupled with goat anti-chicken IgY.

[0020] Preferably, the preparation method of the latex microspheres coupled to the RBD protein is: using EDC and suflo-NHS to activate the latex microspheres, mixing them with the RBD protein, covalently coupling, and adding a blocking buffer for blocking after sufficient reaction; the preparation method of the latex microspheres coupled to the goat anti-chicken IgY is: using EDC and suflo-NHS to activate the latex microspheres, mixing them with the goat anti-chicken IgY, covalently coupling, and adding a blocking buffer for blocking after sufficient reaction.

[0021] Preferably, the sample pad is soaked in a sample pad buffer and then dried. The sample pad buffer has the following formula: 0.1% (mass fraction) NaN 3 , 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa (sodium caseinate), 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, and 0.5% (mass fraction) surfactant S9 (propylene oxide-ethylene oxide-ethylene diamine) were dissolved in 10 mM pH 8.0 Tris-HCl buffer.

[0022] Preferably, the conjugate pad is a polyester film material.

[0023] Preferably, the reaction membrane is a nitrocellulose membrane material.

[0024] Preferably, the latex microspheres have a diameter of 400 nm.

[0025] The use of any of the above-mentioned latex microsphere test strips in the preparation of a COVID-19 neutralization test potency test kit.

[0026] Preferably, if the test result is positive, the sample to be tested is further diluted to obtain a range of neutralizing antibody titers for the sample to be tested.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] A kind of latex test paper strip for detecting neutralizing antibodies of the novel coronavirus, which is traced back to the titer of the neutralization test of the novel coronavirus, prepared by the present invention, uses a nitrocellulose membrane as a solid phase carrier, labels antigens or antibodies by red latex signal substances, and forms red strips based on immune complexes to detect and analyze samples to be tested. Compared with the live virus neutralization test (cVNT), it has the characteristics of low cost, rapid detection, sensitivity, instant detection, and no need for professional operation, and can be completed in a secondary biosafety laboratory. And after the result is judged to be positive, it can be further diluted to obtain a neutralizing antibody titer range, which has a semi-quantitative advantage compared to conventional latex test strips, and has a good replacement effect when the virus neutralization test (cVNT) is difficult to popularize. The test strip can effectively monitor the vaccination response in clinical research and serve as an evaluation index of vaccine effect; improve the detection rate of the novel coronavirus, make up for the missed detection caused by the false negative of nucleic acid detection; detect the neutralizing antibody content in the body of the recovered novel coronavirus patient to determine whether there is a risk of reinfection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the novel coronavirus neutralizing antibody dual antigen latex test strip of the present invention.

[0030] Figure 2 This is the test result of the coronavirus neutralizing antibody dual antigen latex test strip of the present invention detecting the blood sample of the vaccine recipient. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example 1 Preparation method of a latex microsphere test strip for double antigen detection of new coronavirus neutralizing antibodies

[0033] 1. Experimental Methods

[0034] 1. Preparation of sample pad

[0035] A glass fiber with a size of 25 cm × 30 cm was soaked in a sample pad buffer for 30 minutes, taken out, dried at room temperature for 16 to 18 hours, and then cut into a sample pad with a size of 2 cm × 30 cm.

[0036] The sample pad buffer formula is: 0.1% (mass fraction) NaN 3 , 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, and 0.5% (mass fraction) surfactant S9 were dissolved in 10 mM pH 8.0 Tris-HCl buffer.

[0037] 2. Preparation of latex microsphere markers and conjugate pads

[0038] 1) Preparation of red carboxyl latex microspheres labeled with RBD protein 1: 50 μL of red carboxyl latex microspheres (diameter 400 nm) were added to a 2 mL EP tube, and then 950 μL of 0.1 M pH 6.0 MES was added and mixed; then 10 μL of 50 mg / mL NHS (N-hydroxysuccinimide) and 10 μL of 10 mg / mL EDC (carbodiimide) were added, and the mixture was activated at room temperature for 30 min. After the activation reaction, the mixture was centrifuged at 15000 rpm and 4°C for 30 min, the supernatant was discarded, 1 mL of MES was added, the latex microspheres were resuspended by ultrasound, and then 30 μg of RBD protein 1 (Baixi Biotechnology, catalog number: COV-RBD2 2 mg / mL) was added, and the mixture was reacted at room temperature for 3 h; then 10 μL Blocked with 10% BSA, mixed and reacted at room temperature for 30 minutes; centrifuged at 15000 rpm and 4°C for 30 minutes, collected the precipitate after centrifugation and re-dissolved to 500 μL with re-dissolution solution (formula: 0.1 M Gly-NaOH at pH 8.5: 2% (mass fraction) BSA: 8% (mass fraction) sucrose: 1% (mass fraction) D-trehalose: 0.5% (mass fraction) sodium caseinate: 1% (mass fraction) PEG-4000) to obtain RBD protein 1 labeled with red carboxyl latex microspheres.

[0039] 2) Preparation of red carboxyl latex microspheres labeled with goat anti-chicken IgY (Hangzhou Qitai, catalog number: GCY04): 50 μL of red carboxyl latex microspheres were added to a 2 mL EP tube, and then 950 μL of 0.1 M pH 6.0 MES was added and mixed; then 10 μL of 50 mg / mL NHS (N-hydroxysuccinimide) and 10 μL of 10 mg / mL EDC (carbodiimide) were added, mixed and activated at room temperature for 30 min, after activation, centrifuged at 15000 rpm, 4 ° C for 30 min, the supernatant was discarded, 1 mL of MES was added, the latex microspheres were resuspended by ultrasound, and then 25 μg of goat anti-chicken IgY (2 mg / mL) was added, mixed and reacted at room temperature for 3 h; then 10 μL Block with 10% BSA (bovine serum albumin), mix well and react at room temperature for 30 minutes; centrifuge at 15000rpm, 4℃ for 30 minutes, collect the precipitate after centrifugation and re-dissolve it with re-dissolution solution (formula: 0.1M Gly-NaOH at pH 8.5: 2% (mass fraction) BSA: 8% (mass fraction) sucrose: 1% (mass fraction) D-trehalose: 0.5% (mass fraction) sodium caseinate: 1% (mass fraction) PEG-4000) to obtain latex-labeled sheep anti-chicken IgY.

[0040] 3) The red carboxyl latex microspheres labeled RBD protein 1 solution and the red carboxyl latex microspheres coupled to goat anti-chicken IgY solution were mixed in a volume ratio of 4:1 to obtain a marker mixture.

[0041] 4) Spray the mixed marker mixture onto a 10 mm × 30 cm polyester film using a gold spray film scratcher at a spray volume of 6 μL / cm2 (180 μL / sheet). Dry at 37°C for 18 to 24 hours to obtain a conjugate pad for later use.

[0042] 3. Preparation of reaction membrane

[0043] 1) Coating reaction membrane.

[0044] Cut it to the required length and stick it on the base plate, then cut the film:

[0045] Quality control line (C line): draw a line on the nitrocellulose membrane. The distance between the C line and the lower edge of the nitrocellulose membrane is 0.5 cm, and the distance between the C line and the upper edge is 2 cm. Dilute the chicken IgY antibody to 1 mg / mL with coating buffer for coating. The drawing parameter is 1 μL / cm.

[0046] Detection line (T line): The distance between the T line and the upper edge of the nitrocellulose membrane is 2 cm, and the distance from the upper edge is 0.5 cm. RBD protein 2 (Feipeng Bio, catalog number: NCOV-PS-AG27) was diluted to 1 mg / mL with coating buffer for coating, and the streaking parameter was 1 μL / cm;

[0047] The coating buffer is: 1% (mass fraction) sucrose, 0.015M phosphate buffer pH 7.4, 3.2233g / L Na 2 HPO 4 12H 2 O, 0.1560 g / L NaH 2 PO4·2H 2 O, 0.10% (mass fraction) NaN 3 , 2.00% (mass fraction) NaCl, 0.50% (mass fraction) PEG20000.

[0048] 2) Drying

[0049] Place in a 37°C oven and dry for 6 to 12 hours.

[0050] 4. Assembly

[0051] The arrangement order of the components on the base plate is absorbent paper - reaction membrane (nitrocellulose membrane) - binding pad - sample pad. The assembly is carried out on the reaction membrane that has been affixed and the membrane has been scratched: 2) Pasting of absorbent paper: Lay the base plate flat on the work surface; peel off the protective film on the upper edge of the base plate where the absorbent paper is pasted, and adhere the absorbent paper on it, with the absorbent paper covering the reaction membrane (nitrocellulose membrane) by 2 mm.

[0052] 3) Pasting of the binding pad: peel off the protective film at the edge of the nitric acid reaction membrane (nitrocellulose membrane) where the binding pad is pasted, and adhere the binding pad on it using the same method as absorbent paper. The binding pad covers the reaction membrane (nitrocellulose membrane) by 2 mm.

[0053] 4) Pasting of sample pad: Paste the sample pad to the bottom of the conjugate pad in the same way as the absorbent pad. The sample pad covers the conjugate pad by 2 mm.

[0054] 5) Test strip cutting and card loading: Put the pasted base plate into the strip cutting machine, cut it into 4mm wide test strips, and then load it into the plastic card shell.

[0055] Example 2 A latex microsphere test strip for dual antigen detection of novel coronavirus neutralizing antibodies

[0056] 1. Composition

[0057] like Figure 1 As shown, the paper strip includes a sample pad, a conjugate pad, a reaction membrane (nitrocellulose membrane) and absorbent paper which are sequentially bonded to the bottom plate.

[0058] The preparation method of the conjugate pad is as in Example 1, and the conjugate pad is coated with latex microspheres coupled with RBD protein 1 and latex microspheres coupled with sheep anti-chicken IgY.

[0059] Sample pad: glass fiber with a specification of 25 cm × 30 cm, soaked in sample pad buffer and then dried. The sample pad buffer formula is as follows: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in 10 mM pH 8.0 Tris-HCl buffer;

[0060] The reaction membrane (nitrocellulose membrane) is 25 mm wide and is provided with a detection line and a quality control line. The detection line is coated with RBD2, and the quality control line is coated with chicken IgY antibody. The preparation method is the same as in Example 1.

[0061] The paper strip is packaged in a shell to form a test card, on which a sample injection hole and an observation hole window are sequentially arranged, wherein the test line and the quality control line correspond to the position of the observation hole window, and the sample injection hole is arranged above the sample pad.

[0062] 3. Usage

[0063] 1. Return the test strips and the sample to be tested to room temperature;

[0064] 2. Add the used sample diluent (3.2233 g / L Na 2 HPO 4 12H 2 O, 0.1560 g / L NaH 2 PO4·2H 2 O, 0.10% (mass fraction) NaN 3 , 2.00% (mass fraction) NaCl, 0.50% (mass fraction) Tween-20, 0.50% (mass fraction) PEG20000, pure water as the balance. ) 100 μL of the 40-fold diluted serum sample to be tested, react for 15 minutes, and observe and record the results.

[0065] 3. Interpretation of results: After 15 minutes of reaction, observe with the naked eye whether a red strip is formed on the test line to determine the result; the result displayed after 30 minutes is invalid.

[0066] 4. If you need to further determine the titer of the antibody in the sample, you can dilute the diluted serum in multiples, that is, dilute the sample diluted in step 1 by 2 times, 4 times, and 8 times with sample diluent (that is, the sample is diluted 80, 160, and 320 times), repeat steps 2 and 3, and refer to [Explanation of test results] to semi-quantitatively determine the titer of the antibody in the sample.

[0067] IV. Result determination and interpretation

[0068] Negative: There is no red band on the test line, but a red band appears on the control line

[0069] Positive: red stripes appear on both the test line and the control line

[0070] Invalid result: If no red strip appears on the quality control line, the test result is invalid and retesting is recommended.

[0071] Interpretation of test results

[0072] If the test result is negative after the initial dilution of the sample, the titer of the antibody in the sample is less than 1:4, and if it is positive, the titer of the antibody is greater than or equal to 1:4. If further evaluation of the antibody titer in the sample is required, multiple tests can be performed according to step 4 of the detection method. The corresponding relationship between the test results and the test in the virus is shown in Table 1.

[0073] Table 1:

[0074]

[0075] Example 3 Detection of blood samples of vaccine recipients

[0076] 1. Experimental Methods

[0077] The latex microsphere test strips of Examples 1 and 2 were used to detect clinical samples. 411 serum samples with clear neutralizing antibody titers in virus neutralization experiments were tested. To evaluate the detection effect of the test strips of Examples 1 and 2 on the neutralizing antibodies of the new coronavirus, after the preliminary judgment of the positive and negative, the positive samples can be further diluted to estimate the range of serum neutralizing antibody titers to achieve quantitative detection effects.

[0078] Among them, the method of virus neutralization test is: use a quantitative amount of new coronavirus and a serial dilution of the test serum for neutralization, add the neutralization solution to Vero-E6 cells for culture, and the unneutralized virus causes cell morphological changes and cytopathic effect (CPE). After the specific neutralizing antibody binds to the virus, the virus particles lose their infectivity and inhibit the occurrence of CPE. The highest serum dilution that protects more than 50% of the cells from cytopathic effect (CPE) is used as the neutralization titer.

[0079] 2. Experimental Results

[0080] The test results are shown in Table 2. The overall compliance rate of the latex microsphere test strips of Examples 1 and 2 with the neutralization test is 97.32%, and the performance is good.

[0081] Table 2:

[0082]

[0083] The 60 positive samples were further tested using the latex microsphere test strips of Examples 1 and 2. The results showed that the sample dilution ratio corresponded to the titer of the virus neutralization test, which solved the limitation of conventional latex test strips that can only interpret positive and negative results. The results are shown in Table 3 and Figure 2 As shown, after further dilution of the positive sample, the result can correspond to the neutralization test titer.

[0084] Table 3:

[0085]

[0086]

[0087] Example 4 Neutralizing Antibody Latex Test Strip Performance Evaluation

[0088] 1. Experimental Methods

[0089] The latex microsphere test strips of Examples 1 and 2 were used to perform performance evaluation, including:

[0090] ① Cross-reaction experiment, using samples from people who are not clinically infected with SARS-CoV-2, have not been vaccinated with SARS-CoV-2, and are positive for other respiratory virus antibodies, to detect whether the latex microsphere test strip is interfered by other virus antibodies.

[0091] ② Interference experiment, using samples from people with high expression of clinical serum cholesterol, triglycerides, and bilirubin, who were not infected with SARS-CoV-2 and had not been injected with SARS-CoV-2 vaccine, to detect whether the results of the latex microsphere test strip were interfered by substances such as cholesterol, triglycerides, and bilirubin.

[0092] ③Sensitivity experiment, to detect whether the latex microsphere test strip has detection sensitivity for SARS-CoV-2 recovered samples.

[0093] ④ Matrix equivalence experiment, to detect whether the test results of the latex microsphere test strip are affected under different matrix conditions.

[0094] 2. Experimental Results

[0095] ① Cross-reaction experiment: The test results are shown in Table 4. Other virus antibodies have no effect on the neutralizing antibody latex test strip test results, and there is no cross-reaction.

[0096] Table 4:

[0097]

[0098] ②Interference experiment: The test results are shown in Table 5. Serum cholesterol, triglycerides, bilirubin and other substances have no effect on the test results of the neutralizing antibody latex test strips, and there is no interference reaction.

[0099] Table 5:

[0100]

[0101] ③ Sensitivity experiment: The test results are shown in Table 6. The neutralizing antibody latex test strip can detect neutralizing antibodies in SARS-CoV-2 infected samples and recovered samples.

[0102] Table 6:

[0103]

[0104] ④ Matrix equivalence experiment: The test results are shown in Table 7. The neutralizing antibody latex test strip is not affected by matrices such as venous whole blood, capillary blood, plasma, and serum.

[0105] Table 7:

[0106]

[0107] Comparative Example 1 Effect of different RBDs on detection results

[0108] 1. Experimental Methods

[0109] According to the method of Example 1, latex microsphere test strips were prepared using the following different RBDs:

[0110] RBD1: B.X.Bio COV-RBD2,

[0111] RBD2: Feipeng Bio NCOV-PS-AG27,

[0112] RBD3: GenScript Bio SARS-CoV-2 Spike Protein (RBD) Z03491,

[0113] RBD4: Huamei Biotech CSB-MP3324GMY1.

[0114] The four RBDs were coupled to red latex microspheres and coated with detection lines using the chessboard method. Latex microsphere test strips were prepared by pairing them two by two. 250 ng of positive quality control IgG (Feipeng Biotechnology, catalog number: ncov-PS-ConAbG3) was used for spotting. The one with the strongest red band was the best pairing combination.

[0115] 2. Experimental Results

[0116] The results are shown in Table 8, which show that the latex microsphere test strip prepared by the preparation method of Example 1 (RBD1 coupled with red latex microspheres, RBD2 coated with the detection line) has the most obvious red band when detecting equal amounts of positive quality control.

[0117] Table 8:

[0118]

[0119] Comparative Example 2 Effect of the amount of RBD1 coupled with red latex microspheres and the concentration of RBD2 coated on the detection effect

[0120] 1. Experimental Methods

[0121] According to the method of Example 1, the amount of RBD1 (Baixi Bio COV-RBD2) coupled to the red latex microspheres and the concentration of the coated detection line RBD2 (Feipeng Bio NCOV-PS-AG27) were changed to prepare the latex microsphere test strips.

[0122] The dosage of RBD coupled with red latex microspheres was 20, 25, 30, 35, and 40 μg, respectively.

[0123] The concentrations of RBD coated test lines were 0.25, 0.5, 1, 1.5, and 2 mg / mL, respectively.

[0124] The latex microsphere test strips were prepared by pairing two by two using the chessboard method, and 250 ng of positive quality control IgG (Feipeng Bio, catalog number: ncov-PS-ConAbG3) was used for spotting. The best pairing combination was the one with the strongest red band.

[0125] 2. Experimental Results

[0126] The results are shown in Table 9, which show that the latex microsphere test strip prepared by the preparation method of Example 1 (the amount of RBD coupled to the red latex microspheres is 30 μg, and the concentration of RBD coated on the detection line is 1 mg / mL) has the most obvious red band when detecting the same amount of positive quality control. Table 9:

[0127]

[0128] Comparative Example 3 Effect of the coating amount of the test line on the test effect

[0129] 1. Experimental Methods

[0130] According to the method of Example 1, the spray volume of the marker mixture was changed to 2, 3, 4, 5, 6, 7, and 8 μL / cm, respectively, to prepare latex microsphere test strips, and 250 ng of positive control IgG (250 ng IgG) diluted 40 times with the sample diluent or 1% (mass fraction) phosphate Tween buffer (1% PBST) was used for spotting. The strongest red band was the best.

[0131] 2. Experimental Results

[0132] The results are shown in Table 10, which show that the red band of the positive quality control with the same amount of the latex microsphere test strip prepared by the preparation method of Example 1 (6 μL / cm) is the most obvious.

[0133] Table 10:

[0134]

[0135] Comparative Example Effect of the Volume Ratio of 4-color Carboxyl Latex Microspheres Latex-labeled RBD1 and Red Carboxyl Latex Microspheres Sheep Anti-chicken IgY on the Detection Effect

[0136] 1. Experimental Methods

[0137] According to the method of Example 1, the volume ratio of red carboxyl latex microsphere latex-labeled RBD1 and red carboxyl latex microsphere sheep anti-chicken IgY was changed to prepare latex microsphere test strips, and 250ng positive quality control IgG (250ng IgG) diluted 40 times with sample diluent or 1% (mass fraction) phosphate Tween buffer (1% PBST) was used for spotting. The strongest red band was the best.

[0138] 2. Experimental Results

[0139] The results are shown in Table 11, which show that the latex microsphere test strip prepared by the preparation method of Example 1 (red carboxyl latex microspheres labeled RBD1 and red carboxyl latex microspheres sheep anti-chicken IgY are mixed in a volume ratio of 4:1) has the most obvious red band when detecting equal amounts of positive quality control.

[0140] Table 11:

[0141]

[0142] Comparative Example Effect of the Volume Ratio of Five-color Carboxyl Latex Microspheres Latex-labeled RBD1 and Red Carboxyl Latex Microspheres Sheep Anti-chicken IgY on the Detection Effect

[0143] 1. Experimental Methods

[0144] According to the method of Example 1, the sample pad buffer was changed to prepare a latex microsphere test strip, and 250ng positive control IgG (250ng IgG) diluted 40 times with the sample diluent or 1% (mass fraction) phosphate Tween buffer (1% PBST) was used for spotting. The strongest red band was the best.

[0145] Sample pad buffer 1: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in phosphate buffer;

[0146] Sample pad buffer 2: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in 0.1M pH 9.0 borax buffer;

[0147] Sample pad buffer 3: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in 0.1M pH 9.0 borax-casein buffer;

[0148] Sample pad buffer 4: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in 0.1M pH 7.4 Tris-HCl buffer;

[0149] Sample pad buffer 5: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, 0.5% (mass fraction) surfactant S9 dissolved in 0.1M pH8.0 Tris-HCl buffer;

[0150] Sample pad buffer 6: 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, and 0.5% (mass fraction) surfactant S9 dissolved in 0.1M pH8.5 Tris-HCl buffer.

[0151] 2. Experimental Results

[0152] The results are shown in Table 12, which show that the latex microsphere test strips prepared by the preparation method of Example 1 (containing 0.1% (mass fraction) NaN3, 0.50% (mass fraction) NaCl, 0.80% (mass fraction) BSA, 0.20% (mass fraction) EDTA·2Na, 1.00% (mass fraction) PVP40, 1.00% (mass fraction) CaseinNa, 15.00% (mass fraction) sucrose, 7.00% (mass fraction) trehalose, and 0.5% (mass fraction) surfactant S9 dissolved in 10mM pH8.0 Tris-HCl dissolved in 10mM pH8.0 Tris-HCl buffer) detected the same amount of positive quality control red bands most obviously.

[0153] Table 12:

[0154]

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A latex test strip for detecting neutralizing antibodies of the novel coronavirus that can be traced back to the titer of the neutralization test of the novel coronavirus, characterized in that: The latex microsphere test strip contains a sample pad, a conjugate pad, a reaction membrane and absorbent paper which are sequentially connected to the bottom plate. The sample pad is obtained by soaking in a sample pad buffer and then drying. The sample pad buffer has the following formula: 0.1% NaN3 by mass, 0.50% NaCl by mass, 0.80% BSA by mass, 0.20% EDTA·2Na by mass, 1.00% PVP40 by mass, 1.00% CaseinNa by mass, 15.00% sucrose by mass, 7.00% trehalose by mass, and 0.5% surfactant S9 by mass dissolved in 10mM pH7.4 Tris-HCl buffer; The conjugate pad is coated with latex microspheres coupled with RBD protein and goat anti-chicken IgY. The coupled RBD protein is from Baixi Biotechnology, with the product number COV-RBD2. The amount used is 30 μg based on the mass of the RBD protein. The latex microspheres are red carboxyl latex microspheres. The RBD coupled with the red carboxyl latex microspheres and the goat anti-chicken IgY coupled with the red carboxyl latex microspheres are in a dosage ratio of 120:25 based on the mass of the RBD protein and the goat anti-chicken IgY. The reaction membrane is provided with a detection line and a quality control line, the detection line is coated with 1 mg / mL RBD protein, the RBD protein of the detection line is: Feipeng Biology, item number: NCOV-PS-AG27, and the quality control line is coated with chicken IgY antibody.

2. The novel coronavirus neutralizing antibody latex test strip according to claim 1, characterized in that: The latex microspheres are latex microspheres coupled with RBD protein and latex microspheres coupled with sheep anti-chicken IgY.

3. The novel coronavirus neutralizing antibody latex test strip according to claim 2, characterized in that: The preparation method of the latex microspheres coupled with RBD protein is as follows: using EDC and suflo-NHS to activate latex microspheres, mixing with RBD protein, covalently coupling, and adding blocking buffer for blocking after sufficient reaction; the preparation method of the latex microspheres coupled with goat anti-chicken IgY is as follows: using EDC and suflo-NHS to activate latex microspheres, mixing with goat anti-chicken IgY, covalently coupling, and adding blocking buffer for blocking after sufficient reaction.

4. The novel coronavirus neutralizing antibody latex test strip according to claim 1, characterized in that: The bonding pad is made of polyester film material.

5. The novel coronavirus neutralizing antibody latex test strip according to claim 1, characterized in that: The reaction membrane is made of nitrocellulose membrane material.

6. The novel coronavirus neutralizing antibody latex test strip according to claim 1, characterized in that: The latex microspheres have a diameter of 400 nm.

7. Use of the coronavirus neutralizing antibody latex test strip described in any one of claims 1 to 6 in the preparation of a coronavirus neutralization test kit.

8. The use according to claim 7, characterized in that: If the test result of the kit is positive, the sample to be tested is further diluted to obtain the titer range of the neutralizing antibody in the sample to be tested.

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