CN115372613A - Method for qualitatively or quantitatively detecting neutralizing antibody in human or animal body based on NanoSPR biochip - Google Patents

Abstract

The invention discloses a method for quantitatively detecting neutralizing antibodies in human or animal bodies based on a NanoSPR biochip, and relates to the technical field of new coronavirus detection; adopting a two-step method, firstly, incubating and modifying virus fragment protein or receptor protein on a chip micro-porous plate integrated with a NanoSPR biochip; adding the sample diluents to be detected with different concentrations into each micropore of the chip micropore plate; recording the detection value of the initial state of the data by using a microplate reader; finally, correspondingly adding colloidal gold labeled by receptor protein or virus fragment protein; and after the reaction is finished, recording data by using an enzyme-labeling instrument to finish detection. Compared with an ELISA method, the method has the advantages that the time consumed in the whole detection stage is not more than 30min, repeated sample adding and plate washing are not needed, and high-throughput, rapid and ultra-sensitive detection of the neutralizing antibody can be realized.

Description

Method for qualitatively or quantitatively detecting neutralizing antibody in human or animal body based on NanoSPR biochip

Technical Field

The invention relates to the technical field of detection of neutralizing antibodies, in particular to a method for qualitatively or quantitatively detecting neutralizing antibodies in human or animal bodies based on a NanoSPR biochip.

Background

The ultra-fast mutation speed of the new coronavirus and the ultra-strong transmission and pathogenicity bring about a plurality of challenges to epidemic prevention and control and epidemic prevention scientific research. In order to rapidly adapt to the normalized prevention and control of the new coronary pneumonia epidemic variant strain, the development of variant strain vaccines and neutralizing antibody medicines is still two important directions. Neutralizing antibodies (Nautralizing antibodies) are naturally occurring antibodies that are part of the immune system response of the human body; can be triggered by infection or vaccination, and is used for inhibiting or destroying the influence of external factors invading human body. The neutralizing antibody can be directly combined with viruses to prevent the viruses from infecting cells, and is also one of important indexes of development, clinical evaluation and effectiveness evaluation after vaccination of new vaccine of variant strains.

In order to determine the effectiveness of vaccination (any vaccine against covi-19), measure social immunity, measure the immune status of previously infected individuals, search for antiserum donors (donated to ICU critically ill patients), aid in the development and clinical evaluation of variant vaccines, the current gold standard for detection of neutralizing antibodies is the infection inhibition assay, which mainly consists of a Plaque Reduction Neutralization Test (PRNT) with live virus, and a microcytotic neutralization assay (CPE) analyzed by detecting cytopathic effects. Both of these test methods are infectious and need to be performed in a high-safety P3 laboratory (P3 laboratory, which is a three-level biosafety protection laboratory, P stands for Physical contact. The whole laboratory is completely sealed, and the inside of the laboratory is in a negative pressure state, so that the gas inside the laboratory does not leak outside to cause pollution).

The nano plasma resonance (NanoSPR) detection technology is that a nano-pore array with the diameter not more than 500nm is manufactured on a nano-plasma resonance biochip, and detection on biochemical reaction is realized by utilizing the sensitivity of the wavelength of surface plasma resonance to the dielectric environment around a nano structure through the resonance coupling of incident light and a metal nano-pore structure. When the refractive index of the adsorbed molecules is different from that of the surrounding environment, the reaction between the biomolecules adsorbed on the surface of the substrate and the target molecules changes the refractive index of the surface of the substrate, so that the change of a resonance peak is caused, and the detection of the target substance to be detected is realized. Therefore, the resonance analysis process of the nano surface plasmon sensor does not need to use a complicated optical system like the conventional SPR technology.

At present, no method for carrying out qualitative/quantitative detection on a neutralizing antibody by utilizing a nano plasma resonance detection technology is found in the market.

Disclosure of Invention

In view of the defects of the prior art, the invention provides a method for qualitatively or quantitatively detecting neutralizing antibodies in human or animals based on a NanoSPR biochip, which can realize one-step operation without washing plates and any preventive measures. The test can be used for general automatic detection, the detection result can be obtained only in30 min, the detection has higher sensitivity and accuracy, and the detection process is simple and quick. Specifically, the following technique is used.

The method for quantitatively detecting the neutralizing antibody in the human or animal body based on the NanoSPR biochip comprises the following steps:

s1, taking a chip micro-porous plate (such as a chip micro-porous plate with 48, 96 or 192 holes) integrated with a nano plasma resonance biochip, and incubating and modifying virus fragment protein or receptor protein on the nano SPR biochip of each micro hole; diluting a sample to be detected in a gradient manner for later use;

s2, adding the gradient diluent of the sample to be detected prepared in the step S1 into the chip microporous plate obtained in the step S1 for reaction; recording the detection value of the initial state of the data by using a microplate reader; after reacting for a period of time, adding a colloidal gold solution marked with receptor protein or virus segments into a microporous plate of the chip for reaction, and recording a detection value of the terminal state by using an enzyme-labeling instrument after the reaction is finished;

the neutralizing antibody is an antibody which is generated when pathogenic microorganisms invade the human or animal body and has corresponding neutralizing capacity, 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.

According to the detection method, the neutralizing antibody and the receptor protein labeled colloidal gold particles in a sample to be detected are respectively combined with the virus fragment protein on the nano plasma resonance biochip in a competition manner, so that a substitute virus neutralizing test is established, and the total immunodominant neutralizing antibody of the targeted virus spike (S) protein receptor binding domain is detected in a species-independent manner. Taking the new coronavirus as an example, we simply and rapidly tested the blockade of the antibody-mediated interaction between angiotensin converting enzyme 2 (ACE 2) receptor protein and Receptor Binding Domain (RBD). By adopting the one-step detection method, the spectrometer can be directly used for detection, and operations such as washing, spin-drying and the like are not needed, so that the detection time is greatly saved, the detection efficiency is improved, and the sensitivity is higher.

Preferably, step S1 is specifically: taking a chip microporous plate integrated with a NanoSPR biochip, and firstly, carrying out primary incubation on the NanoSPR biochip of each micropore by using virus fragment protein or receptor protein, wherein the incubation temperature is 4-37 ℃, and the incubation time is 2-24h; cleaning with buffer solution, drying with nitrogen, adding casein blocking solution, and incubating at 4-37 deg.C for 0.5-2 hr; cleaning with buffer solution and drying with nitrogen for later use; and diluting the sample to be detected with a diluent in a gradient manner for later use.

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

Preferably, in step S2, the preparation method of the colloidal gold solution labeled with the receptor protein or the virus fragment protein specifically comprises: adding 6-20 μ L of 0.1M potassium carbonate solution into 1ml of colloidal gold solution, and mixing; then adding 1-10 mug receptor protein or virus fragment protein, mixing evenly and standing; adding 5-15 μ L10% PEG20000, mixing, and standing; freezing and centrifuging, removing supernatant, adding precipitate into 100-500 μ L redissolution, and mixing.

More preferably, the colloidal gold solution labeled with the receptor protein or the virus fragment protein is prepared in step S2 by performing refrigerated centrifugation at 7000-10000rpm at 2-8 ℃ for 20-40min.

Further preferably, in the step S2, when the colloidal gold solution labeled with the receptor protein or the virus fragment protein is prepared, the double solution is a PBST buffer containing sucrose in an amount of 1 to 5% by mass, glucose in an amount of 1 to 5% by mass, mannitol in an amount of 0.5 to 4% by mass, and BSA in an amount of 0.1 to 5% by mass.

Preferably, the infectious pathogenic microorganism is 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 fever, epidemic cerebrospinal meningitis, pertussis, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, human infection with H7N9 avian influenza, novel coronavirus pneumonia; the virus vector is adenovirus, adeno-associated virus and lentivirus.

Based on the principle of quantitative detection of NanoSPR biochip, the infectious pathogenic microorganisms or viral vectors can be detected based on corresponding neutralizing antibodies.

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

p1, taking a chip microporous plate integrated with a NanoSPR biochip, cleaning each micropore with ultrapure water and absolute ethyl alcohol in sequence, and drying by using nitrogen; adding 30-100 mu L of 2-100 mu g/ml new coronavirus fragment RBD protein solution or ACE2 protein solution into each micropore, performing primary incubation, washing with PBST buffer solution, and drying with nitrogen; adding casein sealing liquid with the mass fraction of 0.5-8% into each micropore for secondary incubation, washing with PBST buffer solution, and drying with nitrogen for later use; diluting a sample to be detected in a gradient manner for later use;

p2, adding the gradient diluent of the sample to be detected prepared in the step P1 into the chip microporous plate obtained in the step S1 for reaction; recording the detection value of the initial state of the data by using a microplate reader; adding a potassium carbonate solution into the colloidal gold solution, and uniformly mixing; then adding ACE2 protein or new coronavirus fragment RBD protein, mixing, and standing; adding PEG20000, mixing, and standing; freezing and centrifuging, removing supernatant, adding precipitate into redissolution, and mixing; and finally, adding a colloidal gold solution marked with new coronavirus segment RBD protein or ACE2 protein into a chip microporous plate for reaction for 10-40min, and recording the detection value of the end point state by using an enzyme-labeling instrument after the reaction is finished.

The method is suitable for qualitatively detecting the level of SARS-CoV-2 neutralizing antibody in plasma prepared from human serum or anticoagulant (heparin/EDTA/sodium citrate). Intended to help identify persons who have an adaptive immune response to SARS-CoV-2, indicating recent or previous infection. The detection result is the total neutralizing antibody level of SARS CoV-2.

A detection kit for qualitatively or quantitatively detecting neutralizing antibodies in human or animal bodies comprises all reagents and chip microplates used in any one of the detection methods.

Compared with the prior art, the invention has the advantages that: the invention provides a method for detecting neutralizing antibodies in human or animal bodies by using a NanoSPR biochip detection technology, and provides a detection kit for the neutralizing antibodies based on the method. The method can realize high-throughput, rapid and super-sensitive detection of the serum neutralizing antibody, compared with an ELISA method, the detection time is as long as 2h, the time consumption of the whole detection stage can be shortened to be not more than 30min, and the problem of missed detection caused by false negative of nucleic acid detection is solved to a certain extent; can be used for detecting the content of neutralizing antibodies in the body of a rehabilitation patient to judge whether the rehabilitation is possible or not and whether the re-infection risk exists or not.

Drawings

FIG. 1 is a flowchart of detection of neutralizing antibodies in human serum of test example 1;

FIG. 2 is a full spectrum of the detection of neutralizing antibodies in human serum of test example 1;

FIG. 3 is a detected whole spectrum of diluent 1 of test example 2 and OD values of 580 to 600 nm;

FIG. 4 is a detected whole spectrum of diluent 2 of test example 2 and OD values of 580 to 600 nm;

FIG. 5 is a detected whole spectrum of diluent 3 of test example 2 and OD values of 580 to 600 nm;

FIG. 6 is a detected whole spectrum of diluent 4 of test example 2 and OD values of 580 to 600 nm;

FIG. 7 is a detected whole spectrum of the gold particle complex solution 1 of test example 3 and OD values of 580 to 600 nm;

FIG. 8 is a detected whole spectrum of the gold particle complex solution 2 of test example 3 and OD values of 580 to 600 nm;

FIG. 9 is a detected whole spectrum of the gold particle complex solution 3 of test example 3 and OD values of 580 to 600 nm;

FIG. 10 is a detected whole spectrum of the gold particle complex solution 4 of test example 3 and OD values of 580 to 600 nm;

FIG. 11 shows the results of measurement of neutralizing antibody titer in different gradient dilutions of real human serum samples of test example 4;

FIG. 12 is a standard curve for detection of neutralizing antibodies in different gradient dilutions of real human serum samples of test example 4;

FIG. 13 shows the results of measurement of neutralizing antibody titer in different gradient diluted cat serum samples of test example 5;

FIG. 14 is a standard curve for detection of neutralizing antibodies in different gradient dilutions of cat serum samples of test example 5;

FIG. 15 shows the results of measurement of neutralizing antibody titer in dog serum samples diluted in different gradients in test example 5;

FIG. 16 is a standard curve for detection of neutralizing antibodies in dog serum samples at different gradient dilutions of Experimental example 5;

FIG. 17 shows the results of measurement of the titer of neutralizing antibodies in mink serum samples diluted in different gradients in test example 5;

FIG. 18 is a standard curve for detecting neutralizing antibodies in different gradient dilutions of mink serum samples of test example 5;

FIG. 19 is the result of the titer detection of the human neutralizing antibody against Neocrown Wild Type (WT) SARS-COV-2 in test example 6;

FIG. 20 is the result of potency assay of human neutralizing antibody against New crown variant (Omicron) SARS-COV-2 in test example 6;

FIG. 21 is a full spectrum of the detection of neutralizing antibodies in human serum of Experimental example 7.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The chip microplates integrated with nanoplasmon resonance biochips used in the following examples were prepared by the following methods:

(1) Firstly, a nano plasma substrate sheet is manufactured. A nano cup array with the period of 240nm, the height of 600nm, the upper diameter of 240nm and the lower diameter of 200nm is manufactured on the surface of a quartz plate by utilizing a laser interference photoetching technology. NOA-61 was applied uniformly over the mold and a sheet of Polyethylene Terephthalate (PET) was placed on top of the mold. Passing through 105mw/cm ² After 5min of ultraviolet irradiation, carefully stripping off the PET sheet to obtain a substrate sheet with periodic nanopore arrangement;

(2) Then, a nano plasma-based chip is manufactured. And sequentially depositing Ti with the thickness of 10nm, ag with the thickness of 50nm and Au with the thickness of 30nm on the nanocrystal array through electron beam evaporation deposition to obtain the nano plasma resonance sensing chip.

(3) Finally, the nano plasma resonance sensing chip is attached to a self-made bottomless 96-well plate manufactured by a 3D printer (Object 30 printer Stratasys Ltd.), and the assembly of the detection plate is completed. Bottomless 96-well plates may also be produced by other companies in a conventional manner.

Test example 1: detection of neutralizing antibodies in human serum

In this example, a method of detecting a neutralizing antibody against a human serum-containing sample is shown in FIG. 1, and the sample is provided by Tongji medical college, university of science and technology, huazhong. The method comprises the following specific steps:

1. taking a chip microporous plate integrated with a nano plasma resonance biochip, and incubating and modifying a cell Receptor Binding Domain (RBD) of a new coronavirus S protein structural domain on the nano plasma resonance biochip of each micropore.

The method specifically comprises the following steps: washing the prepared chip microporous plate of the nano plasma resonance biochip with absolute ethyl alcohol and ultrapure water for 1 time respectively at room temperature, and drying at 37 ℃; adding 3 μ L of RBD protein (purchased from Cassia Proteus) with concentration of 18 μ g/mL into the center of each well, and standing at 4 deg.C for 12h; adding 100 μ L of blocking solution (1% casein water solution) into each well, and incubating at 37 deg.C for 60min; drying the liquid in the plate, adding 100 μ L of protective solution (PBST buffer solution containing 3% sucrose, wherein the formula of the PBST buffer solution comprises 20-30g of disodium hydrogen phosphate, 2-5g of sodium dihydrogen phosphate, 1-3g of potassium chloride, 60-80g of sodium chloride, 5-10ml of Tween, 300-10 ml of preservative Proclin and 10L of double distilled water) into each hole, and incubating at 37 deg.C for 30min; drying the liquid in the plate, drying at 37 ℃, restoring the room temperature, then attaching a cover plate film, and storing at 4 ℃ for later use;

2. preparation of angiotensin converting enzyme 2 (ACE 2) -labeled colloidal gold particle solution

The specific method comprises the following steps: purchasing gold particles with the concentration of 0.1g/L and the particle size of 30nm from the quantitative (Wuhan) Life technologies Limited company, adding 5ul of 0.1Mol potassium carbonate solution, adding 5 mu L of 1mg/mL ACE2 protein solution (purchased from Yi Qiao Shen company), mixing uniformly, and incubating at room temperature for 15min; adding 3 μ L of Tris-HCl buffer solution (1M, pH = 8.0) to adjust pH, mixing, and incubating at room temperature for 15min; adding 1% PEG-20000 solution, mixing, and incubating at room temperature for 15min;7000rpm, centrifugation for 20min, removing the supernatant, 200 u L Tris-HCl buffer solution, 4 ℃ storage for use.

3. And detecting the sample.

The method comprises the following specific steps: diluting the sample to be detected by 100 times (the diluent adopts a Tris buffer solution containing NaCl with the mass fraction of 0.5% and PEG-20000 with the mass fraction of 0.5%), adding the diluted sample into the micropores of a microporous plate, and reading the absorbance of a specific starting point of a detection hole by using an enzyme-labeling instrument; then 10 mu L of ACE2 protein labeled colloidal gold solution is added, oscillation reaction is carried out for 15min, a specific end point light absorption value of a detection hole is read by a microplate reader, a light density difference value of specific wavelengths of an end point and a starting point is obtained, and quantitative detection of a neutralizing antibody is realized. The full spectrum is shown in FIG. 2. As can be seen from FIG. 2, the full spectrum curve of the neutralizing antibody curve from 580 to 640 is relatively flat, which represents that the human serum sample contains the neutralizing antibody capable of blocking the combination of human ACE2 and RBD, and the gold-labeled protein of human ACE2 is not combined with the RBD protein on the microplate of the chip; the non-specific antibody represents that no neutralizing antibody capable of blocking the binding of human ACE2 and RBD exists in the solution, but the non-specific antibody is an interfering antibody in serum; sample dilutions represent no neutralizing antibodies in human serum.

Test example 2: sensitivity evaluation of rapid detection of neutralizing antibody by using different dilutions of sample to be detected

In the detection of the NanoSPR technology, the sample diluent plays an important role in the final detection result and sensitivity. The test example adopts the same detection method as the test example 1, the detection conditions are consistent with those of the test example 1, and the difference is that the neutralizing antibody standard substance of the test example is purchased from Beijing Yinqiao Shenzhou company; four sets of comparative experiments were performed by replacing the dilutions of the samples to be tested in test example 1. The samples to be tested were selected 4: negative samples (no neutralizing antibody), 32ng/ml antibody, 512ng/ml antibody, 8192ng/ml antibody. The detection results are all subjected to zero treatment by using 8192ng/ml samples. The used diluents of the samples to be tested were respectively: diluent 1 (PBST buffer solution: 20-30g of disodium hydrogen phosphate dodecahydrate, 2-5g of sodium dihydrogen phosphate, 1-3g of potassium chloride, 60-80g of sodium chloride, 5-10ml of Tween-20, 300-10 ml of preservative Proclin and 10L of double distilled water); diluent 2 (TBST buffer TrisHCL (1M, pH 7.5): 50mL NaCl; diluent 3 (PBST buffer solution containing 2% by mass of nacl); diluent 4 (TBST buffer solution containing nacl at mass fraction 2%). The total spectra and OD values at 580-600nm of the 4 dilutions are shown in FIGS. 3-6. 3-6, the full spectrum gradient of the diluent 1 in FIG. 3 is shown in negativity, 32ng, 512ng and 8192ng, and 560nm-640nm has better gradient division and is most obviously distinguished at 580nm and 600 nm; 8192ng of neutralizing antibody has a flat full-spectrum curve, which represents that the human serum sample contains the neutralizing antibody capable of blocking the combination of human ACE2 and RBD, and the gold-labeled protein of human ACE2 is not combined with the RBD protein on the microporous plate of the chip; negative represents that no neutralizing antibody is added, and full spectrum curve represents that the added human ACE2 gold-labeled protein is combined with RBD protein on the micro-porous plate of the chip. The OD values of 600nm and 580nm of the histogram of the diluent 1 are used for making a difference, the gradients of negative samples, 32ng, 512ng and 8192ng are obvious, the relative OD values after the difference are made, and the OD value of the negative sample is 0.042. Diluent 2-4 was observed in accordance with the method used for the observation of Diluent 1. Therefore, in summary, the optimal sample diluent to be detected is the sample diluent 3, which can achieve the detection sensitivity of 32ng and has a large reaction value.

Test example 3: rapid detection of neutralizing antibody by using different gold particle complex solutions

The test example used the same test method as test example 1, the test conditions were the same as those of test example 1, except that the neutralizing antibody standard was purchased from Beijing Yinqiao Shenzhou, and four sets of comparative experiments were performed by replacing the gold particle reconstituted solution in test example 1. The gold particle complex solution is respectively as follows: the compound solution 1 is Tris buffer solution containing 0.5 percent of PEG-20000 by mass fraction; the reconstituted solution 2 is a Tris buffer containing 2.5% by mass of PEG-20000; the reconstituted solution 3 is a Tris buffer containing 0.5% by mass of NaCl and 0.5% by mass of PEG-20000; the reconstituted solution 4 was a Tris buffer containing 0.5% by mass of NaCl and 2.5% by mass of PEG-20000. The full spectrum and OD values at 580-600nm of the 4 kinds of gold particle complex solutions are shown in FIGS. 7-10. 7-10, the full spectrum gradient of the diluent 1 in the figure 7 is negative, 32ng, 512ng and 8192ng, 560nm-640nm has better gradient division, and the division is most obvious at 575nm and 600 nm; 8192ng of neutralizing antibody has a flat full-spectrum curve, which represents that the human serum sample contains the neutralizing antibody capable of blocking the combination of human ACE2 and RBD, and the gold-labeled protein of human ACE2 is not combined with the RBD protein on the microporous plate of the chip; negative represents that no neutralizing antibody is added, and full spectrum curve represents that the added human ACE2 gold-labeled protein is combined with RBD protein on the micro-porous plate of the chip. The OD values of 600nm and 575nm positions of the dilution 1 histogram are used for making a difference, the gradients of 32ng, 512ng and 8192ng are obvious, the negative OD value is lower than the 32ng value, the relative OD value after the difference is made is 0.34. Diluent 2-4 was observed in accordance with the method used for the observation of Diluent 1. Therefore, in summary, the best gold particle complex solution is obtained as the diluent 3, and a larger reaction can be obtained even with a higher detection sensitivity.

Test example 4: quantitative detection of neutralizing antibody titer in real human serum samples

The same test method as that of test example 1 was employed in this test example, the test conditions were kept the same as those of example 1, and the quantitative detection of neutralizing antibodies was carried out using a real human serum sample (provided by the college of Tongji medical college of Huazhong university of science) in a dilution range of 100-fold to 6400-fold gradient. As shown in fig. 11 and 12, the reaction still remained large when a high detection sensitivity was observed from the full spectrum. The standard curve is a four parameter fit, and the formula is: y = (a-D)/[ 1+ (x/C) ^ B ], where a =0.07802, B = -9.47145, C =2.59293, D = -0.02610, r2=0.98111.

Test example 5: rapid detection of neutralizing antibody in quantitative detection real animal serum sample

The test example used the same test method as test example 1, the test conditions were all the same as in example 1, and the neutralization antibodies in the animal serum were quantitatively detected using real animal serum samples (cat, dog, mink, provided by university of agriculture in Huazhong), with a dilution range of 100-6400-fold gradient. The ACE2 gold-labeled protein is alternating gold particles marked by ACE2 proteins of cats, dogs and minks respectively, and the ACE2 of the cats, the dogs and the minks is from university of agriculture in Huazhong.

The detection results of the neutralizing antibodies of the serum sample of the cat are shown in fig. 13 and 14, the reaction is still very large under the condition of higher detection sensitivity observed from the full spectrum, the standard curve of the cat is fit by a Hill curve, and the formula is as follows: y = ymax x ^ n/(k ^ n + x ^ n). The results of the detection of neutralizing antibodies in the dog serum samples are shown in fig. 15 and 16, the standard curve is a four-parameter fit, and the formula is: y = (A-D)/[ 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 detection of neutralizing antibodies in the mink serum samples are shown in FIGS. 17 and 18, and the standard curve isFour parameter fitting, formula is y = (A-D)/[ 1+ (x/C) ^ B]Wherein A =0.10162, B = -10.02895, C = -3.31771, D = -0.03415, r ² ＝0.99528。

Test example 6: quantitative determination of the potency of human neutralizing antibodies against New crown Wild Type (WT) SARS-COV-2 and mutant (Omicron) SARS-COV-2

The test example adopts the same detection method as the test example 1, the detection conditions are consistent with the test example 1, the neutralization antibody Nano SPR rapid detection kit for detecting the COVID-19 wild type and the omicron variant strains is replaced by wild type RBD and the omicron on the basis of the test example 5, the ACE2 gold-labeled protein is human ACE2 protein, and 2 sera inoculated with the new crown vaccine SARS-CoV-2 and 1 negative sera without the vaccine are detected at the same time. Wild RBD, omitron RBD, human ACE2 were all purchased from Beijing Yiqiao Shenzhou, and serum inoculated with new crown vaccine SARS-CoV-2 and 1 negative serum not inoculated with vaccine were from Tongji medical college of Huazhong university of science and technology.

As shown in Table 1, FIGS. 19-21, the results show that the neutralizing titers of COVID-19serum No.1 and No.2 against the wild-type strains using the NanoSPR platform were 38.021nM and 213.802nM, respectively, which are very close to the IC50 values obtained by the PRNT method (procedure completed by the college of Hospital medical university of Huazhong technology) of 42.638nM and 221.072nM, respectively. The neutralizing titers of the serum samples COVID-19serum No.1 and No.2 to the Omicron variant strain are 5.168nM and 9.450nM, respectively, which are very close to the IC50 values obtained by the PRNT method of 6.318nM and 10.655nM, respectively, and the resistance of the serum with the wild-type vaccine to the Omicron variant strain is also shown to be weakened.

TABLE 1 comparison of NanoSPR detection method to PRNT neutralization detection method

Test example 7: detection of neutralizing antibodies in human serum

In this example, a method for detecting a neutralizing antibody containing human serum was performed, and a sample to be tested was provided by Tongji medical college, university of science and technology, huazhong, unlike test example 1. The method comprises the following specific steps:

1. taking a chip microporous plate integrated with a nano plasma resonance biochip, and incubating and modifying the human ACE2 protein on the nano plasma resonance biochip of each micropore.

The method specifically comprises the following steps: washing the prepared chip microporous plate of the nano plasma resonance biochip with absolute ethyl alcohol and ultrapure water for 1 time respectively at room temperature, and drying at 37 ℃; 3 mu L of ACE2 protein with the concentration of 18 mu g/mL is added to the center of each hole, and the mixture is placed for 12 hours at 4 ℃; adding 100 μ L of blocking solution (1% casein water solution) into each well, and incubating at 37 deg.C for 60min; drying the liquid in the plate, adding 100 μ L of protective solution (PBST buffer solution containing 3% sucrose, wherein the formula of the PBST buffer solution is 20-30g of disodium hydrogen phosphate, 2-5g of sodium dihydrogen phosphate, 1-3g of potassium chloride, 60-80g of sodium chloride, 5-10ml of Tween-20, 300-10 ml of preservative Proclin and 10L of double distilled water) into each hole, and incubating at 37 ℃ for 30min; drying the liquid in the plate, drying at 37 ℃, restoring the room temperature, then attaching a cover plate film, and storing at 4 ℃ for later use;

2. preparing colloidal gold solution labeled with virus fragment protein

Purchasing gold particles with concentration of 0.1g/L and particle size of 30nm from Mass Spectrometry (Wuhan) Life technologies, inc., taking 1mL of colloidal gold solution, adding 12 mul of 0.1Mol potassium carbonate solution, adding 8 mul of 1mg/mL neocoronavirus RBD protein (purchased from Yiqiao Shenzhou company), uniformly mixing, and incubating at room temperature for 15min; adding 3 μ L of Tris-HCl buffer solution (1M, pH = 8.0) to adjust pH, mixing, and incubating at room temperature for 15min; adding 1% PEG-20000 solution, mixing, and incubating at room temperature for 15min;7000rpm, centrifugation for 20min, supernatant removal, 200 u L Tris-HCl buffer solution, 4 ℃ storage for use.

3. Detecting a sample to be detected

The specific method comprises the following steps: diluting the sample to be tested by 100 times (the diluent adopts 0.5% NaCl by mass and 0.5% PEG-20000 Tris buffer solution), adding into the micropores of the detection plate, reacting for 20min, and spin-drying the microporous plate; adding PBST, washing twice, and reading the absorbance of a specific starting point of the detection hole by using an enzyme-labeling instrument; then 10 mu L of ACE2 protein labeled colloidal gold solution is added, oscillation reaction is carried out for 15min, a specific end point light absorption value of a detection hole is read by a microplate reader, a light density difference value of specific wavelengths of an end point and a starting point is obtained, and quantitative detection of a neutralizing antibody is realized. The full spectrum is shown in FIG. 21.

As can be seen from fig. 21, the full spectrum curve of the neutralizing antibody curve from 580 to 640 is relatively flat, which represents that the human serum sample contains the neutralizing antibody capable of blocking the combination of human ACE2 and RBD, and the human RBD gold-labeled protein is not combined with ACE2 protein on the microplate of the chip; the non-specific antibody represents that no neutralizing antibody capable of blocking the binding of human ACE2 and RBD exists in the solution, but the non-specific antibody is an interfering antibody in serum; sample dilutions represent no neutralizing antibodies in human serum.

The practice of the present invention has been described in detail with reference to the foregoing detailed description, but the invention is not limited to the specific details of the foregoing embodiment. Within the scope of the claims and the technical idea of the invention, a number of simple modifications and changes can be made to the technical solution of the invention, and these simple modifications are within the scope of protection of the invention.

Claims (9)

1. The method for detecting the neutralizing antibody in the human or animal body based on the NanoSPR biochip is characterized by comprising the following steps:

s1, taking a chip microporous plate integrated with a nano plasma resonance biochip, and incubating and modifying virus fragment protein or receptor protein on the NanoSPR biochip of each micropore; diluting a sample to be detected in a gradient manner for later use;

s2, adding the gradient diluent of the sample to be detected prepared in the step S1 into the microporous plate of the chip obtained in the step S1 for reaction; recording the detection value of the initial state of the data by using a microplate reader; after reacting for a period of time, adding colloidal gold solution marked with receptor protein or virus segment into the microporous plate of the chip for reaction, and recording the detection value of the end point state by using an enzyme-labeling instrument after the reaction is finished;

the neutralizing antibody is an antibody with corresponding neutralizing capacity generated 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.

2. The method for detecting neutralizing antibody in human or animal body based on NanoSPR biochip according to claim 1, wherein step S1 is specifically: taking a chip microporous plate integrated with a NanoSPR biochip, and firstly, carrying out primary incubation on the NanoSPR biochip of each micropore by using virus fragment protein or receptor protein, wherein the incubation temperature is 4-37 ℃, and the incubation time is 2-24h; cleaning with buffer solution, drying with nitrogen, adding casein blocking solution, and incubating at 4-37 deg.C for 0.5-2 hr; cleaning with buffer solution and drying with nitrogen for later use; and diluting the sample to be detected with a diluent in a gradient manner for later use.

3. The NanoSPR biochip-based method for detecting neutralizing antibodies in human or animal bodies according to claim 1, wherein in step S1, the dilution used for diluting the sample to be tested is PBST buffer containing 2% by mass of NaCl.

4. The method for detecting neutralizing antibody in human or animal body based on NanoSPR biochip of claim 1, wherein the preparation method of the colloidal gold solution labeled with receptor protein or virus fragment protein in step S2 comprises: adding 6-20 μ L of 0.1M potassium carbonate solution into 1ml of colloidal gold solution, and mixing; then adding 1-10 mug receptor protein or virus fragment protein, mixing evenly and standing; adding 5-15 μ L10% PEG20000, mixing, and standing; freezing and centrifuging, removing supernatant, adding precipitate into 100-500 μ L redissolution, and mixing.

5. The method for detecting neutralizing antibodies in human or animal bodies based on NanoSPR biochip according to claim 4, wherein when the colloidal gold solution labeled with the receptor protein or the virus fragment protein is prepared in step S2, the frozen centrifugation is performed at 7000-10000rpm for 20-40min at 2-8 ℃.

6. The method for detecting neutralizing antibodies in human or animals according to claim 4 or 5, wherein the complex solution is PBST buffer containing sucrose in 1-5% by mass, glucose in 1-5% by mass, mannitol in 0.5-4% by mass, BSA in 0.1-5% by mass when preparing the colloidal gold solution labeled with the receptor protein or the virus fragment protein in step S2.

7. The NanoSPR biochip-based method for detecting neutralizing antibodies in human or animals according to claim 1, wherein 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 fever, epidemic cerebrospinal meningitis, pertussis, diphtheria, neonatal tetanus, scarlet fever, brucellosis, gonorrhea, syphilis, leptospirosis, schistosomiasis, malaria, human infection with H7N9 avian influenza, novel coronavirus pneumonia; the virus vector is adenovirus, adeno-associated virus and lentivirus.

8. The method for detecting neutralizing antibody in human or animal body based on NanoSPR biochip of claim 1, wherein the neutralizing antibody is anti-SARS-COV-2 neutralizing antibody, and the specific detection method is as follows:

p1, taking a chip microporous plate integrated with a NanoSPR biochip, cleaning each micropore with ultrapure water and absolute ethyl alcohol in sequence, and drying by using nitrogen; adding 30-100 mu L of 2-100 mu g/ml new coronavirus fragment RBD protein solution or ACE2 protein solution into each micropore, performing primary incubation, washing with PBST buffer solution, and drying with nitrogen; adding casein sealing liquid with the mass fraction of 0.5-8% into each micropore for secondary incubation, washing with PBST buffer solution, and drying with nitrogen for later use; diluting a sample to be detected in a gradient manner for later use;

p2, adding the gradient diluent of the sample to be detected prepared in the step P1 into the microporous plate of the chip obtained in the step S1 for reaction; recording the detection value of the initial state of the data by using a microplate reader; adding a potassium carbonate solution into the colloidal gold solution, and uniformly mixing; then adding ACE2 protein or new coronavirus fragment RBD protein, mixing, and standing; adding PEG20000, mixing, and standing; freezing and centrifuging, removing supernatant, adding precipitate into redissolution, and mixing; and finally, adding a colloidal gold solution marked with new coronavirus segment RBD protein or ACE2 protein into a microporous plate of the chip for reaction for 10-40min, and recording the detection value of the terminal state by using an enzyme-labeling instrument after the reaction is finished.

9. A test kit for the detection of neutralizing antibodies in humans or animals, characterized in that it contains all the reagents and microtitre plates used in the method according to any one of claims 1 to 5.

Images