A method for fluorescent labeling of specific antibodies and its application in antigen detection

Abstract

The application discloses a fluorescence labeling method of specific antibodies and application thereof in antigen detection, and comprises the following steps: preparing complete sfGFP fluorescent protein carrying a cleavage site by using a method of co-expression of sfGFP1-10 (with a 6his purification tag) and sfGFP11 (without a 6his purification tag); the obtained fluorescent protein is subjected to a nickel column for protein purification, then the nickel column is flushed with a guanidine hydrochloride solution to denature the sfGFP, the sfGFP11 is washed away, finally the nickel column is washed with an imidazole solution to obtain a large fragment of sfGFP1-10-ch; the sfGFP11 part is fused with specific antibodies for expression, the obtained antibodies with the sfGFP11 and the sfGFP1-10-ch are mixed to recombine into complete sfGFP, i.e. fluorescence labeled antibodies. The method is simple, and can realize directional fluorescence labeling of the antibodies. The fluorescence labeled antibodies can be applied in antigen detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of antibodies labeled in antigen detection, and relates to a method for labeling antibodies with fluorescent proteins based on fluorescent protein splitting technology. BACKGROUND

[0002] Since the 21st century, coronaviruses have begun to spread on a large scale, especially SARS-CoV-2 (new coronavirus). SARS-CoV-2 invades cells by using a highly glycosylated S protein. The S protein is a trimeric protein belonging to type I fusion proteins, which exists in a metastable prefusion state and will undergo structural rearrangement. When the S1 subunit binds to the host receptor, it will trigger the membrane fusion between the virus and the host cell, so that the RBD (receptor binding domain) region of the S1 subunit binds to the host receptor [i] . The host receptor to which the S protein binds is the PD (peptidase domain) domain of ACE2 (angiotensin-converting enzyme 2). The dissociation constant Kd of the extracellular region segment of the S protein and the PD domain of ACE2 is about 14.7 nM, which proves that SARS-CoV-2 invades the host by binding to ACE2 through the S protein. Researchers have also obtained the protein crystal structure analysis of ACE2 and the RBD region [ii] . Further, people have analyzed the human full-length ACE2 receptor by cryo-EM, revealing the pathway of SARS-CoV-2 invading cells [iii] . Therefore, the S protein is confirmed to be an important target for SARS-CoV-2 neutralizing antibodies.

[0003] The current S protein antigen detection method based on fluorescently labeled antibodies for coronaviruses mainly uses chemical reactions to couple small fluorescent chemical molecules to antibodies. However, the chemical modification method requires strict requirements, needs high purity of antibodies, and a large amount of antibodies is used. After the coupling is completed, the unreacted components need to be removed by dialysis and other means. In addition, the chemical reaction coupling site cannot be located at a specific amino acid site, but may exist in all active sites of the entire antibody, so this modification may change the original properties of the antibody.

[0004] And the operation process of the conventional ELISA enzyme label detection method for new coronavirus antigen is relatively complex, and many steps of rinsing are needed in the middle, which greatly increases the complexity of operation. The antibody of the new coronavirus is labeled by using split protein technology in the application. The split protein technology is to split a complete protein at a certain specific site of amino acid sequence into two parts. The two parts are not active when they exist alone, and when the two parts of protein are close, they will recombine into an active protein.

[0005] Green fluorescent protein GFP (Green Fluorescent Protein) is also suitable for split protein technology. When the two halves of the fluorescent protein are separated, they have no fluorescence characteristics. Only when the two halves recombine into a complete protein, an environment conducive to the formation of a fluorescent group will be formed, and the fluorescent protein will produce fluorescence. sfGFP is composed of 230 amino acids, and is a barrel-shaped structure composed of 11 beta-folded peptide chains. Studies have shown that the sfGFP fluorescent protein is split at the 214th amino acid site to obtain a large amino acid fragment of 1-214 and a small amino acid fragment of 215-230, and the two parts after splitting can recombine to form a complete fluorescent protein. Since the amino acid of 1-214 contains the first to tenth beta-folded polypeptide, it is named sfGFP1-10; the 215-230 amino acid fragment contains the eleventh beta-folded polypeptide, and is named sfGFP11.

[0006] sfGFP1-10 large fragment amino acid sequence:

[0007] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK

[0008] sfGFP11 small fragment polypeptide sequence:

[0009] RDHMVLHEYVNAAGIT

[0010] Split sfGFP has been reported to be used to detect protein expression solubility, protein interaction, and cell contact. Figure 11 If the protein is soluble, sfGFP11 fragment will be exposed, and sfGFP1-10 will produce fluorescence after complexing with the exposed sfGFP11 fragment [iv] .

[0011] Meanwhile, sfGFP1-10 and sfGFP11 complexed by separate expression can be used for cell contact research [v] , as shown in Figure 12 .

[0012] For the sfGFP protein splitting method, in addition to the method of separately expressing sfGFP1-10, researchers have tried to first express the complete sfGFP protein with a trypsin cleavage site inserted between the 10th and 11th beta folds, then cleave the site by trypsin, and then remove the sfGFP11 small fragment by molecular sieve after guanidine hydrochloride denaturation, leaving the large fragment sfGFP1-10-ch [vi] . The difference between this method and the method of separately expressing large fragments is that it contains a fluorescent group, but the preparation process requires inserting a trypsin cleavage sequence between the large and small fragments in advance and using protease cleavage to split sfGFP, which may have non-specific sites. Long-time cleavage will increase the occurrence of non-specific site cleavage, resulting in cleavage of other parts of the fluorescent protein. The preparation process also needs to use molecular exclusion chromatography to separate large and small fragments, which increases the complexity of the preparation process with a fluorescent group.

[0013] Currently, there is no report on using split protein technology to label antibodies with fluorescent groups. SUMMARY

[0014] The purpose of the present application is to provide a method for labeling antibodies with fluorescent groups using split protein technology to solve the problem of the overly complex process of preparing split fluorescent proteins with fluorescent groups.

[0015] The purpose of the present application is achieved by the following technical solutions:

[0016] The present application improves the method of preparing split fluorescent proteins with fluorescent groups by splitting sfGFP fluorescent proteins, obtains sfGFP1-10-ch containing fluorescent groups, and uses the split fluorescent protein method to quickly and specifically label new coronavirus antibodies (P2B-2F6), and uses microspheres to realize new coronavirus S protein antigen detection. The present application uses sfGFP1-10-ch (ch: chromophore fluorescent group) to represent the case with a fluorescent group, and sfGFP1-10 to represent the case of separate expression without a fluorescent group.

[0017] In order to avoid the shortcomings of the current reporting method, get rid of the cumbersome enzyme cutting, and express complete sfGFP and conveniently and quickly and efficiently split sfGFP1-10-ch, the present application proposes a method of using co-expression of sfGFP1-10 and sfGFP11 to prepare complete sfGFP fluorescent protein carrying a split site. The expression plasmid containing the sfGFP1-10 (with 6his purification tag) and sfGFP11 (without 6his purification tag) gene coding is introduced into eukaryotic cells (HEK293) to express complete complex sfGFP (sfGFP1-10:sfGFP11). Such sfGFP1-10 and sfGFP11 are not covalently linked together by a peptide chain, but are combined together by the interaction of sfGFP1-10 and sfGFP11. Therefore, the sfGFP at this time is complete and complex, so that the three amino acids in sfGFP1-10 are in the correct spatial structure environment, and the fluorescent group can mature and have fluorescence ability, becoming sfGFP1-10-ch; at the same time, sfGFP1-10 and sfGFP11 are connected by non-covalent bonds, and the separation of sfGFP1-10-ch and sfGFP11 is easy to realize.

[0018] After expression, the sfGFP is subjected to protein purification nickel column (Ni column), and since sfGFP1-10 has a 6his purification tag, sfGFP can be combined to the Ni column. Then, the Ni column is washed with 6M guanidine hydrochloride, and sfGFP will be denatured under the action of guanidine hydrochloride, and since sfGFP11 does not have a 6his purification tag, it will be washed away with guanidine hydrochloride, and sfGFP1-10-ch is still combined to the Ni column. Finally, the sfGFP1-10-ch combined to the Ni column is washed down by washing the Ni column with imidazole solution, and the large fragment sfGFP1-10-ch is obtained. The sfGFP11 part is expressed by protein fusion expression method to fuse with new coronavirus antibody (P2B-2F6), so that the antibody carries the sfGFP11 small fragment.

[0019] The antibody with sfGFP11 is mixed with sfGFP1-10-ch, and since sfGFP11 and sfGFP1-10-ch can recombine into complete sfGFP, the antibody is connected with the sfGFP fluorescent protein, so that the fluorescence labeling of the antibody is realized.

[0020] A fluorescence labeling method of specific antibody, comprising the following steps:

[0021] (1) The method for preparing complete sfGFP fluorescent protein carrying a break site by co-expression of sfGFP1-10 and sfGFP11; After co-expression, the sfGFP fluorescent protein is passed through a nickel column for protein purification, then the nickel column is washed with guanidine hydrochloride solution to denature sfGFP, sfGFP11 is washed away, and sfGFP1-10-ch is still bound to the Ni column, finally the nickel column is washed with imidazole solution to wash down sfGFP1-10-ch bound to the nickel column, and sfGFP1-10-ch with large fragments is obtained; The sfGFP1-10 has a 6his purification tag; The sfGFP11 does not have a 6his purification tag;

[0022] (2) The method for fusing sfGFP11 to a specific antibody by protein fusion expression, so that the antibody carries sfGFP11 small fragments;

[0023] (3) The antibody with sfGFP11 is mixed with sfGFP1-10-ch to recombine into complete sfGFP, that is, the fluorescence-labeled antibody is prepared.

[0024] Preferably, the co-expression method of step (1) is: respectively introducing the expression plasmids containing the genes encoding sfGFP1-10 and sfGFP11 into eukaryotic cells for expression to express complete and combined sfGFP.

[0025] Preferably, the eukaryotic cells are HEK293; the concentration of the guanidine hydrochloride solution is 6±3M.

[0026] Preferably, the antibody with sfGFP11 and sfGFP1-10-ch in step (3) are mixed according to a molar ratio of 1:2.

[0027] The fluorescence-labeled antibody prepared by the above method is applied in antigen detection.

[0028] The method for detecting viral proteins by microspheres, comprising the following steps:

[0029] (1) Precombining an antibody recognizing a target protein to a microsphere;

[0030] (2) Adding the fluorescence-labeled antibody and the microsphere connected with the antibody recognizing the target protein in step (1) to the detection sample;

[0031] If the target protein exists in the detection sample, the protein will be captured to the microsphere, and then the fluorescently labeled antibody will also be adsorbed to the microsphere by combining with the target protein, and whether the target protein exists in the detection sample can be judged by observing whether the microsphere carries fluorescence through the fluorescence microscope.

[0032] Preferably, the virus is a new coronavirus, the target protein is S protein, the antibody recognizing S protein is 4A8, and the specific antibody in the fluorescently labeled antibody is a new coronavirus antibody P2B-2F6.

[0033] A kit for detecting viral proteins by using microspheres comprises the fluorescently labeled antibody prepared by the above method and the microspheres connected with the antibody recognizing the target protein.

[0034] A kit for detecting new coronavirus proteins by using microspheres comprises the fluorescently labeled antibody and the microspheres connected with the antibody 4A8 recognizing S protein, and the specific antibody in the fluorescently labeled antibody is a new coronavirus antibody P2B-2F6.

[0035] The antibody 4A8 recognizing S protein is pre-bound to the microspheres for detecting new coronavirus proteins (taking S protein as an example), if the S protein exists in the sample to be detected, the S protein will be captured to the microspheres, and then the antibody P2B-2F6-2sfGFP carrying the sfGFP fluorescent protein label will also be adsorbed to the microspheres by combining with the S protein, so that the microspheres will carry fluorescence. Whether the S protein exists in the solution can be judged by observing whether the microspheres carry fluorescence through the fluorescence microscope.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] (1) Antibody fluorescent labeling can be realized quickly and simply. The labeled antibody does not need to remove the fluorescent group as in chemical coupling, because sfGFP1-10-ch is specifically combined with sfGFP11, and the antibody carrying sfGFP11 will not be labeled with fluorescence.

[0038] (2) Antibody directional fluorescent labeling can be realized. The fluorescent sfGFP1-10-ch is only labeled on the part carrying sfGFP11.

[0039] (3) The process for preparing the large fragment sfGFP1-10-ch is simpler than the currently disclosed method. The currently disclosed method for obtaining the large fragment sfGFP1-10-ch is to first use a protease to cut the cleavage site, then perform size fragment splitting by guanidine hydrochloride denaturation, and then separate the small fragments using molecular exclusion chromatography (molecular sieve). The present application does not require protease cleavage and does not require exclusion chromatography separation. Only a Ni column is used to separate small fragments during protein purification, greatly improving the preparation efficiency of large fragments containing fluorescent groups.

[0040] (4) Compared with the existing large fragment sfGFP1-10 expressed alone, since sfGFP1-10-ch is derived from complete sfGFP, contains fluorescent groups, and the fluorescence modification of the antibody after complexing with sfGFP11 is greatly shortened compared with the fluorescence modification of the antibody after complexing with sfGFP1-10 and sfGFP11.

[0041] (5) Compared with the existing ELISA method, the most obvious advantage of the present application for detecting viral proteins using microspheres is that after adding the sample to be detected, no further experimental operation is required. Only the fluorescence of the microspheres needs to be observed, greatly simplifying the detection process. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Preparation of sfGFP1-10-ch containing fluorescent groups and method for realizing antibody fluorescence labeling after complexing with antibody with sfGFP11 tag (compared with sfGFP1-10 expressed alone and complexed with antibody with sfGFP11 tag).

[0043] Figure 2 Principle diagram for detecting S protein on microspheres using antibody labeled with sfGFP1-10-ch.

[0044] Figure 3 Co-expression of sfGFP1-10-ch and sfGFP11 (left) and co-expression of sfGFP1-10-ch and sfGFP11-RBD (right).

[0045] Figure 4 Binding of sfGFP1-10-ch to microspheres through strep tag.

[0046] Figure 5 Fluorescence excitation spectrum of the prepared large fragment sfGFP1-10-ch.

[0047] Figure 6 Change in fluorescence intensity of sfGFP1-10-ch after complexing with sfGFP11 over time.

[0048] Figure 7The P2B-2F6 antibody was fluorescently labeled using sfGFP1-10-ch (to verify the fluorescent labeling effects of three different numbers of sfGFP11 tags: sfGFP11, 2sfGFP11, and 3sfGFP11).

[0049] Figure 8 Sensitivity of detecting SARS-CoV-2 S protein on microspheres using sfGFP1-10-ch complexed with P2B-2F6-2sfGFP11.

[0050] Figure 9 After sfGFP1-10 was complexed with sfGFP11, the fluorescence recovery over time was observed on the microspheres.

[0051] Figure 10 The changes in fluorescence intensity over time after sfGFP1-10 complexed with sfGFP11.

[0052] Figure 11 Schematic diagram of monitoring protein solubility using sfGFP1-10 expressed alone without a fluorescent group.

[0053] Figure 12 This is a diagram showing the effects of expressing sfGFP1-10 and sfGFP11 separately for cell contact research. DETAILED DESCRIPTION

[0054] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0055] Eukaryotic cells HEK293 were purchased from Saiku Biotechnology Co., Ltd., guanidine hydrochloride was purchased from Shanghai Sangon Biotechnology Co., Ltd., and Strep-TactinXT microspheres were purchased from IBA Biotechnology Co., Ltd.

[0056] Example 1

[0057] Preparation of sfGFP1-10-ch with fluorescent groups: When we prepare sfGFP1-10-ch with fluorescent groups, we first need to express a large amount of complete complex sfGFP (sfGFP1-10:sfGFP11). Therefore, we need to co-introduce the gene encoding expression plasmids containing sfGFP1-10 (with 6his purification tag) and sfGFP11 (without 6his purification tag) into eukaryotic cells (HEK293) for expression. However, since sfGFP11 is a short peptide of only a dozen amino acids, it is not suitable for cell expression. Therefore, in the actual implementation process, we connected sfGFP11 to an RBD protein and then co-expressed it with sfGFP1-10. The experimental results found that ( Figure 3), sfGFP1-10 can express a large amount of complex sfGFP with sfGFP11-RBD, greatly improving the expression efficiency of sfGFP. To obtain sfGFP1-10-ch with a fluorescent group, we similarly added 6 histidines (6His tag) to the end of the sfGFP1-10-ch fragment, while sfGFP11-RBD did not have a 6His tag. In the purification process, the protein is hung on the nickel column, and then guanidine hydrochloride (6M) is used to denature the sfGFP1-10-ch and sfGFP11-RBD complex protein. Because the sfGFP1-10-ch fragment can be fixed to the solid protein purification medium Ni column, while sfGFP11-RBD cannot bind to the nickel column, guanidine hydrochloride can be used to denature sfGFP11-RBD in the purification process, leaving only the sfGFP1-10-ch fragment.

[0058] The specific experimental steps for mass expression of sfGFP1-10-ch and sfGFP11-RBD complex protein. A large amount of sfGFP1-10 and sfGFP11-RBD plasmids were extracted, endotoxin was removed, and the plasmids were transiently transfected in the HEK293 suspension culture expression system according to the mass ratio of 1:1 (50ug:50ug per 100ml culture medium) for co-expression. The culture volume was 400ml, and the culture was carried out at 37℃, 120rpm on a shaking table. After 6 days, the supernatant was collected by centrifugation. The culture solution was passed through a nickel column, and after the protein was hung on the nickel column, the nickel column was washed with a washing buffer containing 15mM imidazole, the washing buffer was drained, and the nickel column was washed with 6M guanidine hydrochloride, and the washing guanidine hydrochloride was completely drained. Turn off the outlet below the nickel column, add guanidine hydrochloride to the nickel column again, and let the nickel column soak in 6M guanidine hydrochloride for 6 hours to completely denature the protein hung on the nickel column. Finally, the guanidine hydrochloride was completely drained. The column was washed with 6M guanidine hydrochloride to remove the sfGFP11-RBD fragment, and the guanidine hydrochloride was completely washed with a washing buffer containing no imidazole. The last liquid was drained, and the elution buffer containing 500mM imidazole was used for elution. The sfGFP1-10-ch fragment with a fluorescent group was obtained after elution.

[0059] The eluted sfGFP1-10-ch is combined with Strep-Tactin XT microspheres through the strep tag, and the microspheres will immediately have green fluorescence Figure 4 ), indicating that the sfGFP1-10-ch prepared by this method has fluorescent properties. At the same time, under 460nm wavelength excitation, the sfGFP1-10-ch fluorescence emission spectrum was measured, and the strongest emission peak was found at about 510nm Figure 5 ), similar to the complete sfGFP, indicating that the prepared sfGFP1-10-ch also retains the original fluorescent properties.

[0060] Further observation of sfGFP1-10-ch fluorescence change over time in the presence of sfGFP11 fragment: sfGFP1-10-ch was added to a final concentration of 1 uM in a 96-well plate, followed by the addition of sfGFP11-RBD to a concentration of 1 uM, and fluorescence was measured over different time periods. The results, as shown in Figure 6 , indicate that the fluorescence value of sfGFP1-10-ch does not increase substantially over time, indicating that the fluorescent group sfGFP1-10-ch already has fluorescence at the beginning, and does not need to wait for the fluorescent group to mature. Therefore, sfGFP1-10-ch has the advantage of faster fluorescence generation after complexing with sfGFP11.

[0061] Fluorescent modification of antibodies using sfGFP1-10-ch: We selected the P2B-2F6 antibody. P2B-2F6 antibody is an antibody that can bind to the S protein of SARS-CoV-2 [vii] . We compared the fusion of antibody P2B-2F6 with different numbers of sfGFP11. In the case of antibody P2B-2F6, 1 sfGFP11 (P2B-2F6-sfGFP11), 2 sfGFP11 (P2B-2F6-2sfGFP11), and 3 sfGFP11 (P2B-2F6-3sfGFP11) were fused and expressed at the C-terminus of the light chain. In the microsphere system containing the 4A8 antibody [viii] (could bind to the S protein) was added an equal amount of P2B-2F6-sfGFP11, P2B-2F6-2sfGFP11, and P2B-2F6-3sfGFP11, and an equal amount of sfGFP1-10-ch and S protein was also added. After 4 h, the fluorescence of the microspheres was observed under a fluorescence microscope. The results, as shown in Figure 7 , indicate that the prepared sfGFP1-10-ch can successfully label the antibody P2B-2F6, and multiple sfGFP labeling of an antibody can be achieved, with the case of 2 sfGFP11 (P2B-2F6-2sfGFP11) having the brightest fluorescence.

[0062] Method for detecting SARS-CoV-2 using sfGFP1-10-ch fluorescently modified antibody: ELISA is an important method for detecting proteins at present, but the operation process of ELISA experiment is relatively cumbersome. In order to detect proteins more simply, we used a method for detecting fluorescence on microspheres. First, the 4A8 antibody with Twin-Strep-tag label was combined to Strep-Tactin XT microspheres, and the S protein was captured with these microspheres. If the S protein exists, the P2B-2F6-2sfGFP detection antibody with fluorescent signal can bind to the S protein and be adsorbed to the microspheres, so that the microspheres carry the fluorescent signal. The difference between this method and ELISA in the detection process is that the microspheres are not rinsed, and the presence or absence of S protein can be judged by directly observing the fluorescent signal of the microspheres.

[0063] Specific experimental process: P2B-2F6-2sfGFP11 and sfGFP1-10-ch were mixed according to a molar ratio of 1:2, and were left for 15 min for complexation. In a 100 ul system, 4 ul of 4A8 antibody connected microspheres were added, so that the final concentration of P2B-2F6-2sfGFP11 and 800 nM sfGFP1-10-ch was 400 nM. Then the S protein was added to the 100 ul system, so that the final concentration of the S protein was 0, 60 nM, 125 nM and 250 nM, respectively. After 4 h, imaging was performed under a fluorescence microscope. The results are shown in Figure 8 0 nM as a control, set the fluorescence threshold to show red signal after deducting the background from the control group, and the results show that the signal of the group containing S protein is very strong. And the lowest concentration of S protein detected can reach 60 nM, reaching the nM level.

[0064] Example 2

[0065] To prove that the sfGFP1-10-ch obtained by the method of the application has a fluorescent group, and the sfGFP1-10 expressed alone has no fluorescent group, we used the method of prokaryotic expression in E. coli to express sfGFP1-10 alone. The prokaryotic expression plasmid pET-sfGFP1-10 was introduced into BL21 (DE3) E. coli, and the inclusion body sfGFP1-10 produced by prokaryotic expression was dissolved with 6M guanidine hydrochloride. The dissolved solution was purified by nickel column, and finally sfGFP1-10 fragment was obtained by elution.

[0066] The sfGFP1-10 fragment was added to the microspheres with sfGFP11, and imaging was performed on the fluorescence microscope after 15 min, 2 h, 4 h, 6 h and 8 h. The results are shown in Figure 9As shown, it indicates that sfGFP1-10 is different from sfGFP1-10-ch, and the recovery of fluorescence after sfGFP1-10 is complexed with sfGFP11 needs a long process.

[0067] Further quantitative study of the change of sfGFP1-10 fluorescence over time in the presence of sfGFP11: in a 96-well plate, sfGFP1-10 is added to a final concentration of 1 uM, followed by the addition of sfGFP11-RBD to a concentration of 1 uM, and the fluorescence is measured at different time intervals. The results are shown in Figure 10 As shown, the fluorescence value of sfGFP1-10 slowly increases over time. It indicates that without a fluorescent group, there is a maturation process of the fluorescent group, and the fluorescence slowly increases over time. Therefore, compared with sfGFP1-10-ch obtained by our method, the recovery of fluorescence after sfGFP1-10 is complexed with sfGFP11 needs a slow process. The sfGFP large fragment prepared by the present application has a fluorescent group and does not need to go through the maturation process of the fluorescent group, and has the ability to produce fluorescence faster after being complexed with the sfGFP11 fragment.

[0068] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

[0069] __________________________

[0070] [i]Wrapp D, Wang N, Corbett K S, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation [J]. Science, 2020, 367(6483): 1260-1263.

[0071] [ii]Lan J, Ge J, Yu J, et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor [J]. nature, 2020, 581(7807): 215-220.

[0072] [iii]Yan R, Zhang Y, Li Y, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2 [J]. Science, 2020, 367(6485): 1444-1448.

[0073] [iv]Cabantous S, Terwilliger T C, Waldo G S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein [J]. Nature biotechnology, 2005, 23(1): 102-107.

[0074] [v]Tsetsenis T, Boucard A A, D, et al. Direct visualization of trans-synaptic neurexin–neuroligin interactions during synapse formation [J]. Journal of Neuroscience, 2014, 34(45): 15083-15096.

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[0076] [vii]Ju B, Zhang Q, Ge J, et al. Human neutralizing antibodies elicited by SARS-CoV-2 infection [J]. Nature, 2020, 584(7819): 115-119.

[0077] [viii] Chi X, Yan R, Zhang J, et al. A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2 [J]. Science, 2020, 369(6504): 650-655.

Claims

1. A fluorescent labeling method for specific antibodies, characterized in that: The steps include: (1) The complete sfGFP fluorescent protein with a breakage site was prepared by co-expressing sfGFP1-10 and sfGFP11; the co-expressed sfGFP fluorescent protein was passed through a nickel column for protein purification, and then the nickel column was rinsed with a guanidine hydrochloride solution to denature sfGFP and wash away sfGFP11, and finally the sfGFP1-10-ch bound to the nickel column was washed off with an imidazole solution to obtain a large fragment of sfGFP1-10-ch; the sfGFP1-10 had a 6his purification tag; the sfGFP11 did not have a 6his purification tag; (2) The sfGFP11 part was fused with a specific antibody through protein fusion expression, allowing the antibody to carry a small fragment of sfGFP11; (3) The antibody containing sfGFP11 is mixed with sfGFP1-10-ch to reconstitute the complete sfGFP, thereby obtaining a fluorescently labeled antibody.

2. The method according to claim 1, characterized in that The co-expression method in step (1) is: respectively introducing the expression plasmids containing the sfGFP1-10 and sfGFP11 genes into eukaryotic cells for expression, thereby expressing the complete composite sfGFP.

3. The method according to claim 2, characterized in that The eukaryotic cells are HEK293 cells; the concentration of the guanidine hydrochloride solution is 6M.

4. The method according to claim 3, characterized in that The antibody containing sfGFP11 and sfGFP1-10-ch described in step (3) were mixed at a molar ratio of 1:

2.

5. A fluorescently labeled antibody obtained by the method according to any one of claims 1 to 4.

6. Use of the fluorescently labeled antibody according to claim 5 in antigen detection.

7. The use according to claim 6, characterized in that Detection of viral proteins using microspheres includes the following steps: (1) Pre-bind the antibody that recognizes the target protein to the microspheres; (2) adding fluorescently labeled antibodies and microspheres linked to antibodies that recognize the target protein in step (1) to the test sample; If the target protein exists in the test sample, the protein will be captured by the microspheres, and then the fluorescently labeled antibody will bind to the target protein and be adsorbed to the microspheres. The presence of the target protein in the test sample can be determined by observing whether the microspheres are fluorescent under a fluorescence microscope.

8. The use according to claim 7, characterized in that The virus is the new coronavirus, the target protein is the S protein, the antibody that recognizes the S protein is 4A8, and the specific antibody among the fluorescently labeled antibodies is the new coronavirus antibody P2B-2F6.

9. A kit for detecting viral proteins using microspheres, characterized in that: The invention comprises a fluorescently labeled antibody prepared by the method according to any one of claims 1 to 4 and microspheres connected with an antibody that recognizes a target protein.

10. A kit for detecting novel coronavirus protein using microspheres, characterized in that: The invention comprises a microsphere comprising the fluorescently labeled antibody according to claim 8 and the antibody 4A8 that recognizes the S protein.

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