Method for qualitative or quantitative detection of neutralizing antibodies in humans or animals based on NanoSPR biochip

Abstract

The present invention discloses a method for quantitatively detecting neutralizing antibodies in humans or animals based on NanoSPR biochips, and relates to the technical field of novel coronavirus detection; a two-step method is adopted, firstly incubating and modifying virus fragment proteins or receptor proteins on a chip microplate integrated with NanoSPR biochips; then adding different concentrations of the sample dilution to be tested to each microwell of the chip microplate; using an ELISA instrument to record the data initial state detection value; finally, colloidal gold labeled with receptor protein or virus fragment protein is added accordingly; after the reaction is completed, the ELISA instrument is used to record the data to complete the detection. This method can detect neutralizing antibodies of most infectious pathogenic microorganisms or viral vectors of humans or animals. Compared with the ELISA method, the entire detection phase of this method takes no more than 30 minutes, and there is no need for repeated sample addition and plate washing, which can achieve high-throughput, rapid, and ultra-sensitive neutralizing antibody detection.

Description

Technical Field

[0001] The present invention relates to the technical field of neutralizing antibody detection, and in particular to a method for qualitatively or quantitatively detecting neutralizing antibodies in human or animal bodies based on a NanoSPR biochip. Background Art

[0002] Neutralizing antibodies are naturally produced antibodies that are part of the human immune system response; they can be triggered by infection or vaccination and are used to inhibit or destroy the effects of foreign factors that invade the human body. In addition to being able to directly bind to viruses and prevent them from infecting cells, neutralizing antibodies are also one of the important indicators for the development of new vaccines for variants, clinical evaluation, and evaluation of effectiveness after vaccination.

[0003] In order to judge the effectiveness of vaccination (any vaccine for COVID-19), measure the immunity of society, detect the immune status of previously infected people, find antiserum donors (donated to critically ill patients in ICU), and assist in the development and clinical evaluation of variant vaccines, the current gold standard for detecting neutralizing antibodies is the infection inhibition test, which mainly includes the plaque reduction neutralization test (PRNT) using live viruses, and the micro-cell neutralization test analyzed by detecting the cytopathic effect (CPE). Both test methods are infectious and need to be carried out in a high-security P3 laboratory (biosafety level 3 laboratory, referred to as P3 laboratory, P stands for Physical containment. The entire laboratory is completely sealed and the room is under negative pressure, so that the gas inside the laboratory will not leak to the outside and cause contamination).

[0004] Nanoplasmon resonance (NanoSPR) detection technology is to make a nanopore array with a diameter not exceeding 500nm on a nanoplasmon resonance biochip, and detect biochemical reactions by resonant coupling between incident light and the metal nanopore structure, and by using the sensitivity of the wavelength of surface plasmon resonance to the dielectric environment around the nanostructure. When there is a difference between the refractive index of the adsorbed molecule and the refractive index of the surrounding environment, the reaction between the biomolecules adsorbed on the substrate surface and the target molecules will change the refractive index of the substrate surface, thereby causing a change in the resonance peak and realizing the detection of the target substance to be tested. Therefore, the resonance analysis process of the nano surface plasmon sensor does not require the use of a complex optical system like the traditional SPR technology.

[0005] Currently, there is no method on the market for qualitative / quantitative detection of neutralizing antibodies using nanoplasmon resonance detection technology. Summary of the invention

[0006] In view of the above shortcomings of the prior art, the present invention provides a method for qualitatively or quantitatively detecting neutralizing antibodies in humans or animals based on NanoSPR biochips, which can achieve a one-step operation without washing the plate and without any precautions. The test can be used for general automated detection, and the test results can be obtained in just 30 minutes. The detection has high sensitivity and accuracy, and the detection process is simple and fast. It is specifically achieved through the following technologies.

[0007] A method for quantitatively detecting neutralizing antibodies in humans or animals based on NanoSPR biochips comprises the following steps:

[0008] S1. Take a chip microplate with integrated nanoplasmonic resonance biochip (e.g., a chip microplate with 48, 96 or 192 wells), incubate and modify the virus fragment protein or receptor protein on each micropore of the NanoSPR biochip; dilute the sample to be tested in a gradient manner for later use;

[0009] S2, adding the gradient dilution of the sample to be tested prepared in step S1 to the chip microplate obtained in step S1 for reaction; using an ELISA reader to record the data initial state detection value; after a period of reaction, adding a colloidal gold solution labeled with a receptor protein or a virus fragment to the chip microplate for reaction, and after the reaction is completed, using an ELISA reader to record the end state detection value;

[0010] The neutralizing antibodies are antibodies with corresponding neutralizing ability produced when pathogenic microorganisms invade the human or animal body, and the pathogenic microorganisms are infectious pathogenic microorganisms or viral vectors; the receptor protein is a protein that specifically binds to the viral fragment protein.

[0011] The above detection method uses neutralizing antibodies and receptor protein-labeled colloidal gold particles in the sample to be tested to compete with the virus fragment protein on the nanoplasmon resonance biochip, respectively, to establish an alternative virus neutralization test, and detect total immunodominant neutralizing antibodies targeting the receptor binding domain of the virus spike (S) protein in a species-independent manner. Taking the new coronavirus as an example, our simple and rapid test is based on the antibody-mediated blocking of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor binding domain (RBD). Using the above-mentioned one-step detection method, it can be directly detected by a spectrometer without the need for washing, drying and other operations, which greatly saves detection time, improves detection efficiency, and has high sensitivity.

[0012] Preferably, step S1 is specifically as follows: take a chip microplate integrated with a NanoSPR biochip, and first incubate the NanoSPR biochip in each microwell with a virus fragment protein or a receptor protein for the first time, at an incubation temperature of 4-37°C and an incubation time of 2-24h; after washing with a buffer solution and blowing dry with nitrogen, add a casein blocking solution for a second incubation, at an incubation temperature of 4-37°C and an incubation time of 0.5-2h; after washing with a buffer solution and blowing dry with nitrogen, set aside; dilute the sample to be tested with a diluent for a gradient and set aside.

[0013] Preferably, in step S3, the sample diluent is a PBST buffer containing 2% NaCl by mass.

[0014] Preferably, in step S2, the preparation method of the colloidal gold solution labeled with the receptor protein or the viral fragment protein is specifically as follows: take 1 ml of the colloidal gold solution, add 6-20 μL of 0.1M potassium carbonate solution, and mix; then add 1-10 μg of the receptor protein or the viral fragment protein, mix, and let stand; then add 5-15 μL of 10% PEG 20000, mix, and let stand; perform refrigerated centrifugation, remove the supernatant, take the precipitate, add 100-500 μL of the reconstitution solution and mix.

[0015] More preferably, when preparing the colloidal gold solution labeled with receptor protein or virus fragment protein in step S2, the conditions of refrigerated centrifugation are 2-8° C., 7000-10000 rpm, and 20-40 min.

[0016] Further preferably, when preparing the colloidal gold solution labeled with receptor protein or viral fragment protein in step S2, the reconstituted solution is a PBST buffer containing 1-5% by mass of sucrose, 1-5% by mass of glucose, 0.5-4% by mass of mannitol, and 0.1-5% by mass of BSA.

[0017] Preferably, the infectious pathogenic microorganisms are infectious atypical pneumonia, AIDS, viral hepatitis, poliomyelitis, human infection with highly pathogenic avian influenza, measles, epidemic hemorrhagic fever, rabies, epidemic encephalitis B, dengue fever, anthrax, bacterial and amebic dysentery, tuberculosis, typhoid and paratyphoid, epidemic cerebrospinal meningitis, whooping cough, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, human infection with H7N9 avian influenza, and new coronavirus infection; the viral vector is adenovirus, adeno-associated virus, or lentivirus.

[0018] Based on the principle of quantitative detection of NanoSPR biochip, the above-mentioned infectious disease pathogens or viral vectors can be detected based on the corresponding neutralizing antibodies.

[0019] Preferably, when the neutralizing antibody is an anti-SARS-COV-2 neutralizing antibody (e.g., an anti-SARS-COV-2Omicron variant neutralizing antibody, an anti-SARS-COV-2Delta variant neutralizing antibody), the detection method is specifically as follows:

[0020] P1. Take the chip microplate with the NanoSPR biochip integrated, wash each microwell with ultrapure water and anhydrous ethanol, and blow dry with nitrogen; add 30-100μL of 2-100µg / ml SARS-CoV-2 fragment RBD protein solution or ACE2 protein solution to each microwell for the first incubation, wash with PBST buffer, and blow dry with nitrogen; then add 0.5-8% casein blocking solution to each microwell for the second incubation, wash with PBST buffer, blow dry with nitrogen and set aside; dilute the sample to be tested in a gradient manner and set aside;

[0021] P2. Add the gradient dilution of the sample to be tested prepared in step P1 to the chip microplate obtained in step S1 for reaction; record the data initial state detection value with an enzyme reader; take the colloidal gold solution, add the potassium carbonate solution, and mix; then add the ACE2 protein or the new coronavirus fragment RBD protein, mix, and let stand; then add PEG 20000, mix, and let stand; freeze centrifuge, remove the supernatant, take the precipitate, add the reconstitution solution and mix; finally, add the colloidal gold solution labeled with the new coronavirus fragment RBD protein or ACE2 protein to the chip microplate for reaction for 10-40 minutes. After the reaction is completed, record the endpoint state detection value with an enzyme reader.

[0022] The above method is suitable for in vitro qualitative detection of SARS-CoV-2 neutralizing antibody levels in human serum or plasma prepared with anticoagulants (heparin / EDTA / sodium citrate). It is intended to help identify people who have an adaptive immune response to SARS-CoV-2, indicating recent or past infection. The test result is the level of total neutralizing antibodies to SARS CoV-2.

[0023] A detection kit for qualitatively or quantitatively detecting neutralizing antibodies in human or animal bodies, comprising all reagents and chip microplates used in any of the above detection methods.

[0024] Compared with the prior art, the present invention is beneficial in that: the present invention provides a method for detecting neutralizing antibodies in humans or animals using NanoSPR biochip detection technology, and based on this method, provides a neutralizing antibody detection kit. The method can achieve high-throughput, rapid, and ultra-sensitive serum neutralizing antibody detection. Compared with the ELISA method, which takes up to 2 hours to detect, the time consumption of the entire detection phase of the present invention can be shortened to no more than 30 minutes, which to a certain extent makes up for the missed detection problem caused by false negative nucleic acid detection; it can be used to detect the neutralizing antibody content in recovered patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The detection process of neutralizing antibodies in human serum of Test Example 1;

[0026] Figure 2 This is a full spectrum graph of the detection of neutralizing antibodies in human serum in Experimental Example 1;

[0027] Figure 3 The full spectrum of the dilution 1 of Test Example 2 and the OD value at 580-600nm;

[0028] Figure 4 The full spectrum of the dilution 2 of Test Example 2 and the OD value at 580-600nm;

[0029] Figure 5 The full spectrum of the dilution 3 of Test Example 2 and the OD value at 580-600nm;

[0030] Figure 6 The full spectrum of the dilution 4 of Test Example 2 and the OD value at 580-600nm;

[0031] Figure 7 The full spectrum of the gold particle complex solution 1 of Experimental Example 3 and the OD value at 580-600nm;

[0032] Figure 8 The full spectrum of the gold particle complex solution 2 of Experimental Example 3 and the OD value at 580-600nm;

[0033] Fig. 9 The full spectrum of the gold particle complex solution 3 of Experimental Example 3 and the OD value at 580-600nm;

[0034] Fig.10 The full spectrum of the gold particle complex solution 4 of Experimental Example 3 and the OD value at 580-600nm;

[0035] Fig.11 The neutralizing antibody titer test results in real human serum samples with different gradient dilutions in Test Example 4;

[0036] Fig.12 The neutralizing antibody detection standard curve in real human serum samples with different gradient dilutions of Experimental Example 4;

[0037] Fig.13 The neutralizing antibody titer test results in cat serum samples of different gradient dilutions in Test Example 5;

[0038] Fig.14 The neutralizing antibody detection standard curve in cat serum samples of different gradient dilutions in Experimental Example 5;

[0039] Fig.15 The neutralizing antibody titer test results in dog serum samples with different gradient dilutions in Experimental Example 5;

[0040] Fig.16 The neutralizing antibody detection standard curve in dog serum samples with different gradient dilutions in Experimental Example 5;

[0041] Fig.17 The neutralizing antibody titer test results in mink serum samples with different gradient dilutions in Experimental Example 5;

[0042] Fig.18 The standard curve for neutralizing antibody detection in mink serum samples with different gradient dilutions in Experimental Example 5;

[0043] Fig.19 The quantitative detection results of the human neutralizing antibody titer against the wild type (WT) SARS-COV-2 in Test Example 6;

[0044] Fig. 20 The test results of quantitative detection of the potency of human neutralizing antibodies against the new coronavirus mutant (Omicron) SARS-COV-2 in Test Example 6;

[0045] Fig.21 This is the full spectrum diagram of the detection of neutralizing antibodies in human serum of Experimental Example 7. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The chip microplates integrated with nanoplasmon resonance biochips used in the following examples are all prepared by the following method:

[0048] (1) First, make a nanoplasmonic substrate. Use laser interference lithography to make a nanocup array with a period of 240nm, a height of 600nm, an upper diameter of 240nm, and a lower diameter of 200nm on the surface of a quartz plate. Apply NOA-61 evenly on the mold and place a polyethylene terephthalate (PET) sheet on top of the mold. After 105mw / cm 2 After irradiation with ultraviolet light for 5 min, the PET sheet was carefully peeled off to obtain a substrate sheet with periodic nanopore arrangement;

[0049] (2) Then, a nanoplasmonic chip is made. Ti with a thickness of 10 nm, Ag with a thickness of 50 nm, and Au with a thickness of 30 nm are deposited on the nanocrystal array in sequence by electron beam evaporation deposition, thus obtaining a nanoplasmonic resonance sensor chip.

[0050] (3) Finally, the nanoplasmonic resonance sensor chip is attached to a self-made bottomless 96-well plate made by a 3D printer (Object 30 primerTMStratasys Ltd.), thus completing the assembly of the test plate. The bottomless 96-well plate can also be made by other companies using conventional methods.

[0051] Test Example 1: Detection of neutralizing antibodies in human serum

[0052] The method for detecting neutralizing antibodies in human serum in this embodiment is as follows: Figure 1 As shown, the sample is provided by Tongji Medical College of Huazhong University of Science and Technology. The specific steps are as follows:

[0053] 1. Take a chip microplate with integrated nanoplasmonic resonance biochips, and incubate and modify the cell receptor binding domain (RBD) of the SARS-CoV-2 S protein domain on each microwell of the nanoplasmonic resonance biochip.

[0054] Specifically, at room temperature, the prepared nanoplasmon resonance biochip microplate was washed with anhydrous ethanol and ultrapure water once each, and dried at 37°C; 3 μL of 18 μg / mL RBD protein (purchased from Sino Biological Shenzhou Company) was added to the center of each well and placed at 4°C for 12 hours; 100 μL of blocking solution (1% casein aqueous solution by mass fraction) was added to each well and incubated at 37°C for 60 minutes; the liquid in the plate was dried, and 100 μL of protective solution (PBST buffer containing 3% sucrose, wherein the formula of PBST buffer is: 20-30g disodium hydrogen phosphate, 2-5g sodium dihydrogen phosphate, 1-3g potassium chloride, 60-80g sodium chloride, 5-10ml Tween-20, 5-10ml preservative Proclin300, 10L double distilled water) was added to each well and incubated at 37°C for 30 minutes; the liquid in the plate was dried, dried at 37°C, and after returning to room temperature, the cover film was attached and stored at 4°C for use;

[0055] 2. Preparation of angiotensin-converting enzyme 2 (ACE2) labeled colloidal gold particle solution

[0056] The specific method is as follows: purchase gold particles with a concentration of 0.1g / L and a particle size of 30nm from Liangzhun (Wuhan) Life Science Technology Co., Ltd., add 5ul 0.1Mol potassium carbonate solution, add 5μL 1mg / mL ACE2 protein solution (purchased from Yiqiao Shenzhou Company), mix well and incubate at room temperature for 15min; then add 3μL Tris-HCl buffer solution (1M, pH=8.0) to adjust the pH value, mix well and incubate at room temperature for 15min; then add PEG-20000 solution with a final concentration of 1%, mix well and incubate at room temperature for 15min; centrifuge at 7000rpm for 20min, remove the supernatant, reconstitute with 200μL Tris-HCl buffer solution, and store at 4℃ for later use.

[0057] 3. Test the samples.

[0058] Specifically, the sample to be tested is diluted 100 times (the diluent uses a Tris buffer containing 0.5% NaCl and 0.5% PEG-20000 by mass fraction), added to the microwells of the microplate, and the specific starting point absorbance of the detection well is read with an enzyme reader; then 10μL of ACE2 protein-labeled colloidal gold solution is added, and the reaction is oscillated for 15 minutes. The specific end point absorbance value of the detection well is read with an enzyme reader, and the optical density difference at the specific wavelength between the end point and the starting point is obtained to achieve quantitative detection of neutralizing antibodies. The full spectrum is shown in the figure below: Figure 2 As shown. Figure 2 It can be seen that the neutralizing antibody curve is relatively flat in the full spectrum curve of 580-640, which means that there are neutralizing antibodies in the human serum sample that can block the binding of human ACE2 and RBD, and the human ACE2 gold-labeled protein does not bind to the RBD protein on the chip microplate; non-specific antibodies mean that there are no neutralizing antibodies in the solution that can block the binding of human ACE2 and RBD, but it is a type of interfering antibody in the serum; the sample diluent means that there are no neutralizing antibodies in the human serum.

[0059] Experimental Example 2: Sensitivity evaluation of rapid neutralizing antibody detection using different dilutions of the test sample

[0060] In the NanoSPR technology detection, the sample diluent plays an important role in the final detection results and sensitivity. This test example uses the same detection method as Test Example 1, and the detection conditions are consistent with Example 1. The difference is that the neutralizing antibody standard in this test example is purchased from Beijing Yiqiao Shenzhou Company; the diluent of the sample to be tested in Test Example 1 is replaced, and four groups of comparative experiments are carried out. Four samples to be tested were selected: negative samples (without neutralizing antibodies), 32ng / ml antibodies, 512ng / ml antibodies, and 8192ng / ml antibodies. The test results were all reset to zero using the 8192ng / ml sample. The diluents of the samples to be tested were: diluent 1 (PBST buffer: 20-30g disodium hydrogen phosphate dodecahydrate, 2-5g sodium dihydrogen phosphate, 1-3g potassium chloride, 60-80g sodium chloride, 5-10ml Tween-20, 5-10ml preservative Proclin300, 10L double distilled water); diluent 2 (TBST buffer: TrisHCL (1M, pH7.5): 50mL Nacl: 8g KCL: 0.2g Tween: 0.5ml distilled water to 1L); diluent 3 (PBST buffer with 2% NaCl); diluent 4 (TBST buffer with 2% NaCl). The full spectra and OD values ​​at 580-600nm of the four diluents are shown in the figure. Figure 3-6 As shown. Figure 3-6 As you can see, Figure 3 The full spectrum gradient of diluent 1, from negative, 32ng, 512ng, and 8192ng, has a good gradient of 560nm-640nm, and the distinction is most obvious at 580nm and 600nm; the full spectrum curve of 8192ng neutralizing antibody is relatively flat, indicating that there are neutralizing antibodies in human serum samples that can block the binding of human ACE2 to RBD, and the human ACE2 gold-labeled protein does not bind to the RBD protein on the chip microplate; negative means that no neutralizing antibody is added, and the full spectrum curve represents the binding of the human ACE2 gold-labeled protein added later to the RBD protein on the chip microplate. The histogram of diluent 1 uses the OD values ​​at 600nm and 580nm as the difference, and the gradients of negative, 32ng, 512ng, and 8192ng are more obvious. The relative OD value after the difference is 0.042 for the negative sample. The observation method of diluents 2-4 is consistent with that of diluent 1. Therefore, in summary, the best dilution of the sample to be tested is sample dilution 3, which can achieve a detection sensitivity of 32 ng and a larger reaction value.

[0061] Experimental Example 3: Rapid detection of neutralizing antibodies using different gold particle complex solutions

[0062] This test example uses the same detection method as in Test Example 1, and the detection conditions are consistent with those in Test Example 1, with the difference that the neutralizing antibody standard was purchased from Beijing Yiqiao Shenzhou Company, and the gold particle reconstitution solution in Test Example 1 was replaced to conduct four groups of comparative experiments. The gold particle reconstitution solutions are: reconstitution solution 1 is a Tris buffer containing a mass fraction of 0.5% PEG-20000; reconstitution solution 2 is a Tris buffer containing a mass fraction of 2.5% PEG-20000; reconstitution solution 3 is a Tris buffer containing a mass fraction of 0.5% NaCl and a mass fraction of 0.5% PEG-20000; reconstitution solution 4 is a Tris buffer containing a mass fraction of 0.5% NaCl and a mass fraction of 2.5% PEG-20000. The full spectra and OD values ​​of 580-600nm of the four gold particle reconstitution solutions are shown in the figure. Figure 7-10 As shown. Figure 7-10 As you can see, Figure 7 The full spectrum gradient of diluent 1, from negative, 32ng, 512ng, and 8192ng, has a good gradient of 560nm-640nm, and the distinction is most obvious at 575nm and 600nm; the full spectrum curve of 8192ng neutralizing antibody is relatively flat, indicating that there are neutralizing antibodies in human serum samples that can block the binding of human ACE2 to RBD, and the human ACE2 gold-labeled protein does not bind to the RBD protein on the chip microplate; negative means that no neutralizing antibody is added, and the full spectrum curve represents the binding of the human ACE2 gold-labeled protein added later to the RBD protein on the chip microplate. The histogram of diluent 1 uses the OD values ​​at 600nm and 575nm as the difference. The gradients of 32ng, 512ng, and 8192ng are more obvious, and the negative OD value is lower than the 32ng value. The relative OD value after the difference is 0.34 for the negative sample. The observation method of diluents 2-4 is consistent with that of diluent 1. Therefore, in summary, the best gold particle reconstitution solution is dilution 3, which can achieve a higher detection sensitivity and a greater response.

[0063] Experimental Example 4: Quantitative detection of neutralizing antibody titer in real human serum samples

[0064] This test example adopts the same detection method as in Test Example 1, and the detection conditions are consistent with those in Example 1. Real human serum samples (provided by Tongji Medical College of Huazhong University of Science and Technology) are used for quantitative detection of neutralizing antibodies, and the dilution range is from 100 times to 6400 times. The test results are as follows Fig.11 , 12As shown, it can be observed from the full spectrum that the reaction is still large even with high detection sensitivity. The standard curve is a four-parameter fit, and the formula is: y=(AD) / [1+(x / C)^B], where A=0.07802, B=-9.47145, C=2.59293, D=-0.02610, r 2 =0.98111.

[0065] Experimental Example 5: Rapid detection of neutralizing antibodies in real animal serum samples

[0066] This test example adopts the same detection method as in Test Example 1, and the detection conditions are consistent with those in Example 1. Real animal serum samples (cats, dogs, minks, provided by Huazhong Agricultural University) are used for quantitative detection of neutralizing antibodies in animal serum, and the dilution range is from 100 times to 6400 times. The ACE2 gold-labeled proteins are alternating gold particles labeled with ACE2 proteins of cats, dogs, and minks, and cat, dog, and mink ACE2 are from Huazhong Agricultural University.

[0067] The results of neutralizing antibody test of cat serum samples are as follows Fig.13 , 14 As shown in the figure, it can be observed from the full spectrum that the reaction is still large even with high detection sensitivity. The standard curve for cats is a Hill curve fit, and the formula is: y=ymax*x^n / (k^n+x^n). The neutralizing antibody test results of dog serum samples are shown in the figure. Fig.15 , 16 As shown, the standard curve is a four-parameter fit, and the formula is: y=(AD) / [1+(x / C)^B], where A=0.09372, B=-6.74396, C=2.5132, D=-0.01974, r 2 =0.98734. The results of the neutralizing antibody test of the mink serum samples are as follows Fig.17 , 18 As shown, the standard curve is a four-parameter fit, and the formula is y=(AD) / [1+(x / C)^B], where A=0.10162, B=-10.02895, C=3.31771, D=-0.03415, r 2 =0.99528.

[0068] Test Example 6: Quantitative detection of the titer of human neutralizing antibodies against wild-type (WT) SARS-COV-2 and mutant (Omicron) SARS-COV-2

[0069] This test example adopts the same detection method as in Test Example 1, and the detection conditions are consistent with those in Example 1. The Nano SPR rapid detection kit for neutralizing antibodies against COVID-19 wild-type and omicron variants is replaced with wild-type RBD and omicron on the basis of Test Example 5. The ACE2 gold-labeled protein is human ACE2 protein, and 2 sera vaccinated with the new crown vaccine SARS-CoV-2 and 1 negative serum that was not vaccinated are tested at the same time. Wild-type RBD, omicron RBD, and human ACE2 were purchased from Beijing Yiqiao Shenzhou, and the serum vaccinated with the new crown vaccine SARS-CoV-2 and 1 negative serum that was not vaccinated were from Tongji Medical College of Huazhong University of Science and Technology.

[0070] As shown in Table 1, Figure 19-21 As shown, the results showed that the neutralization titers of serum samples COVID-19 serum no.1 and no.2 against wild-type virus strains detected by NanoSPR platform were 38.021nM and 213.802nM, respectively, which were very close to the IC50 of 42.638nM and 221.072nM obtained by PRNT method (the test process was completed by Tongji Medical College of Huazhong University of Science and Technology). The neutralization titers of serum samples COVID-19 serum no.1 and no.2 against Omicron variants were 5.168nM and 9.450nM, respectively, which were also very close to the IC50 of 6.318nM and 10.655nM obtained by PRNT method, respectively. It also shows that the resistance of serum vaccinated with wild-type vaccine to Omicron variants is weakened.

[0071] Table 1 Comparison of NanoSPR detection method and PRNT neutralization detection method

[0072]

[0073] Test Example 7: Detection of neutralizing antibodies in human serum

[0074] The method for detecting neutralizing antibodies in human serum in this embodiment is different from that in Experimental Example 1. The samples to be tested are provided by Tongji Medical College of Huazhong University of Science and Technology. The specific steps are as follows:

[0075] 1. Take a chip microplate with integrated nanoplasmonic resonance biochips, and incubate and modify human ACE2 protein on the nanoplasmonic resonance biochip in each microwell.

[0076] Specifically, at room temperature, the prepared nanoplasmon resonance biochip chip microplate was washed once with anhydrous ethanol and ultrapure water, and dried at 37°C; 3 μL of ACE2 protein at a concentration of 18 μg / mL was added to the center of each well, and placed at 4°C for 12 hours; 100 μL of blocking solution (casein aqueous solution with a mass fraction of 1%) was added to each well, and incubated at 37°C for 60 minutes; the liquid in the plate was dried, and 100 μL of protective solution (PBST buffer containing 3% sucrose, wherein the formula of PBST buffer is: disodium hydrogen phosphate 20-30g, sodium dihydrogen phosphate 2-5g, potassium chloride 1-3g, sodium chloride 60-80g, Tween-20 5-10ml, preservative Proclin300 5-10ml, double distilled water 10L) was added to each well, and incubated at 37°C for 30 minutes; the liquid in the plate was dried, dried at 37°C, and the cover film was attached after returning to room temperature, and stored at 4°C for use;

[0077] 2. Preparation of colloidal gold solution labeled with viral fragment protein

[0078] Gold particles with a concentration of 0.1 g / L and a particle size of 30 nm were purchased from Liangzhun (Wuhan) Life Science Co., Ltd., 1 ml of colloidal gold solution was taken, 12 μl of 0.1 Mol potassium carbonate solution was added, and 8 μL of 1 mg / mL SARS-CoV-2 RBD protein (purchased from Sino Biological Shenzhou Co., Ltd.) was added, mixed and incubated at room temperature for 15 min; then 3 μL of Tris-HCl buffer solution (1 M, pH = 8.0) was added to adjust the pH value, mixed and incubated at room temperature for 15 min; then PEG-20000 solution with a final concentration of 1% was added, mixed and incubated at room temperature for 15 min; centrifuged at 7000 rpm for 20 min, removed the supernatant, re-dissolved with 200 μL of Tris-HCl buffer solution, and stored at 4 °C for later use.

[0079] 3. Test the samples to be tested

[0080] The specific method is: dilute the sample to be tested at a 100-fold concentration (the diluent uses a Tris buffer containing 0.5% NaCl and 0.5% PEG-20000 by mass fraction), add it to the microwells of the detection plate, and after the sample reacts for 20 minutes, spin dry the chip microwell plate; add PBST to wash twice, and use an enzyme reader to read the specific starting point absorbance of the detection well; then add 10μL of ACE2 protein-labeled colloidal gold solution, oscillate and react for 15 minutes, and use an enzyme reader to read the specific end point absorbance value of the detection well, and obtain the optical density difference between the end point and the specific wavelength of the starting point to achieve quantitative detection of neutralizing antibodies. The full spectrum is shown in the figure below: Fig.21 shown.

[0081] from Fig.21It can be seen that the neutralizing antibody curve is relatively flat in the full spectrum curve of 580-640, which means that there are neutralizing antibodies in the human serum sample that can block the binding of human ACE2 and RBD, and the human RBD gold-labeled protein does not bind to the ACE2 protein on the chip microplate; non-specific antibodies mean that there are no neutralizing antibodies in the solution that can block the binding of human ACE2 and RBD, but it is a type of interfering antibody in the serum; the sample diluent means that there are no neutralizing antibodies in the human serum.

[0082] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A detection kit for detecting neutralizing antibodies in humans or animals, characterized in that: The operation method includes the following steps: S1. Take a chip microplate with integrated NanoSPR biochip, first incubate with virus fragment protein or receptor protein on each micropore of NanoSPR biochip, incubate at 4-37°C for 2-24h; wash with buffer and blow dry with nitrogen, then add casein blocking solution for second incubation, incubate at 4-37°C for 0.5-2h; wash with buffer and blow dry with nitrogen, and set aside; dilute the sample to be tested with diluent for gradient dilution; the diluent for diluting the sample to be tested is PBST buffer with 2% NaCl by mass; S2, adding the gradient dilution of the sample to be tested prepared in step S1 to the chip microplate obtained in step S1 for reaction; using an ELISA reader to record the data initial state detection value; after a period of reaction, adding a colloidal gold solution labeled with a receptor protein or a virus fragment to the chip microplate for reaction, and after the reaction is completed, using an ELISA reader to record the end state detection value; The neutralizing antibodies are antibodies with corresponding neutralizing ability produced when pathogenic microorganisms invade the human or animal body, and the pathogenic microorganisms are infectious pathogenic microorganisms or viral vectors; the receptor protein is a protein that specifically binds to a viral fragment protein; The NanoSPR biochip is prepared by the following method: (1) First, prepare the nanoplasmonic substrate: use laser interference lithography to produce a nanocup array with a period of 240nm, a height of 600nm, an upper diameter of 240nm, and a lower diameter of 200nm on the surface of a quartz plate; evenly apply NOA-61 on the mold and place a polyethylene terephthalate sheet on the top of the mold; and then apply 105mw / cm 2 After irradiation with ultraviolet light for 5 min, the PET sheet was carefully peeled off to obtain a substrate sheet with periodic nanopore arrangement; (2) Then, a nanoplasmonic chip is fabricated: Ti with a thickness of 10 nm, Ag with a thickness of 50 nm, and Au with a thickness of 30 nm are sequentially deposited on the nanocrystal array by electron beam evaporation, thereby obtaining a NanoSPR biochip; The detection kit contains all reagents and chip microplates used in the operation method.

2. A detection kit for detecting neutralizing antibodies in humans or animals according to claim 1, characterized in that: In step S2, the preparation method of the colloidal gold solution labeled with the receptor protein or the viral fragment protein is specifically as follows: take 1 ml of the colloidal gold solution, add 6-20 μL of 0.1M potassium carbonate solution, and mix; then add 1-10 μg of the receptor protein or the viral fragment protein, mix, and let stand; then add 5-15 μL of 10% PEG 20000, mix, and let stand; perform refrigerated centrifugation, remove the supernatant, take the precipitate, add 100-500 μL of the reconstitution solution and mix.

3. A detection kit for detecting neutralizing antibodies in humans or animals according to claim 2, characterized in that: When preparing the colloidal gold solution labeled with receptor protein or virus fragment protein in step S2, the conditions of refrigerated centrifugation are 2-8° C., 7000-10000 rpm, and 20-40 min.

4. A detection kit for detecting neutralizing antibodies in humans or animals according to claim 2 or 3, characterized in that: When preparing the colloidal gold solution labeled with receptor protein or virus fragment protein in step S2, the reconstituted solution is a PBST buffer containing 1-5% by mass of sucrose, 1-5% by mass of glucose, 0.5-4% by mass of mannitol, and 0.1-5% by mass of BSA.

5. A detection kit for detecting neutralizing antibodies in humans or animals according to claim 1, characterized in that: The infectious disease pathogenic microorganisms are pathogenic microorganisms that cause infectious atypical pneumonia, AIDS, viral hepatitis, poliomyelitis, human infection with highly pathogenic avian influenza, measles, epidemic hemorrhagic fever, rabies, epidemic encephalitis B, dengue fever, anthrax, bacterial and amoebic dysentery, tuberculosis, typhoid and paratyphoid, epidemic cerebrospinal meningitis, whooping cough, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, human infection with H7N9 avian influenza or new coronavirus infection; the viral vector is adenovirus, adeno-associated virus or lentivirus.

6. A detection kit for detecting neutralizing antibodies in humans or animals according to claim 1, characterized in that: The neutralizing antibody is an anti-SARS-COV-2 neutralizing antibody, and the specific operation method is as follows: P1. Take the chip microplate with the NanoSPR biochip integrated, wash each microwell with ultrapure water and anhydrous ethanol, and blow dry with nitrogen gas; add 30-100μL of 2-100µg / ml SARS-CoV-2 fragment RBD protein solution or ACE2 protein solution to each microwell, incubate for the first time, wash with PBST buffer, and blow dry with nitrogen gas; Then, add 0.5-8% casein blocking solution to each microwell for a second incubation, wash with PBST buffer, blow dry with nitrogen gas and set aside; dilute the sample to be tested in a gradient manner and set aside; P2. Add the gradient dilution of the sample to be tested prepared in step P1 to the chip microplate obtained in step S1 for reaction; record the data initial state detection value with an ELISA instrument; take the colloidal gold solution, add the potassium carbonate solution, and mix well; then add the ACE2 protein or the new coronavirus fragment RBD protein, mix well, and let stand; then add PEG 20000, mix well, and let stand; After freezing centrifugation, remove the supernatant, take the precipitate and add the reconstitution solution to mix well; finally, add the colloidal gold solution labeled with the RBD protein or ACE2 protein of the new coronavirus fragment to the chip microplate and react for 10-40 minutes. After the reaction is completed, use an enzyme reader to record the end state detection value.

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