CN116148475A - ELISA detection method of rabies virus neutralizing antibody - Google Patents

ELISA detection method of rabies virus neutralizing antibody Download PDF

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CN116148475A
CN116148475A CN202111373926.1A CN202111373926A CN116148475A CN 116148475 A CN116148475 A CN 116148475A CN 202111373926 A CN202111373926 A CN 202111373926A CN 116148475 A CN116148475 A CN 116148475A
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丁小静
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Jiangsu Uninovo Biological Technology Co ltd
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Abstract

The invention discloses an ELISA detection method of rabies virus neutralizing antibodies, which belongs to the technical field of biology. The optimized rabies virus G protein nucleotide sequence is shown as SEQ No.1, the VSV framework pseudovirus carrying the rabies virus G protein is obtained through expression and purification, and the pseudovirus is used as an antigen to prepare an ELISA plate for rabies virus antibody detection. The invention has the beneficial effects that: during detection, the real virus and cultured cells are not required to be operated, so that the risk of biosafety and the requirements on production conditions are reduced; the VSV skeleton pseudovirus carrying rabies virus G protein has large expression quantity, is easy to purify, and is convenient for large-scale industrialized production; the antigen VSV-CVSG which mainly induces and generates the neutralizing antibody is used as a coating antigen, and the detection result can better reflect the neutralizing antibody level in the body; in addition, the stability of the pseudovirus is higher than that of the true virus, so that the method is easier to popularize.

Description

ELISA detection method of rabies virus neutralizing antibody
Technical Field
The invention relates to the technical field of biology, in particular to an ELISA detection method of rabies virus neutralizing antibodies.
Background
Rabies is an acute human and animal zoonotic infectious disease caused by rabies virus, and once the host is ill, the mortality rate is almost 100%. Over 5 tens of thousands of people die from the disease worldwide each year, mainly focusing on developing countries such as asia and africa, where india dies as much as 3 tens of thousands of people each year. The main infectious sources of China are sick dogs and dogs with toxins, and secondly warm-blooded animals such as cats, pigs, cattle and the like, and the main infectious sources of China in European and American countries with better rabies control are wild animals such as wolves, foxes, skunks, raccoons and the like. Rabies virus is mainly infected through wounds or mucous membranes, and the bite of rabies animals is the most important way for transmitting rabies virus.
Rabies virus is a single-strand negative strand RNA virus, the total length of the RNA is about 11kb, the rabies virus belongs to the rabies genus of Rhabdoviridae, and the virus shape accords with the typical characteristics of Rhabdoviridae: the front end is semicircular, and the rear end is flush. The viruses of Rhabdoviridae are almost bullet-shaped or rod-shaped, and the major structural components are proteins and RNAs. The RNA contains 5 segments of different genes, and N, P, M, G, L is arranged from the 3 'end to the 5' end. Each gene segment encodes a corresponding protein, namely NP (nucleoprotein), PP (phosphoprotein), MP (matrix protein), GP (glycoprotein), LP (RNA polymerase large protein).
At present, human rabies vaccine effective antigens mainly comprise nucleoprotein and glycoprotein (G protein). Wherein the G protein is composed of 524 amino acids, is the only glycosylated protein in the rabies virus, and can form the thorn-shaped protrusion of the trimer, thereby forming the important component of the surface of the rabies virus envelope. It is the primary protein affecting viral virulence and the most predominant antigen that induces the production of neutralizing antibodies. Accounting for as much as 42% of the total protein content of rabies virus and being the only protein structure outside the viral envelope. Whether the antigen can make organism produce neutralizing antibody is critical to whether the three-dimensional conformation of rabies virus glycoprotein trimer is maintained, and the complete natural structure plays a decisive role in the effectiveness and immune effect of rabies vaccine. Glycoprotein G is also reported to be involved in inducing and binding virus neutralizing antibodies, stimulating proliferation of specific T lymphocytes, determining molecular localization of virulence, and the like.
At present, the number of people infected by animal media such as dogs, cats and the like in China exceeds thousands of people, and the number of dead people is the third in various reports of infectious diseases. Therefore, effective prevention and control of rabies has become urgent. Immunization remains an important means of controlling rabies transmission. According to WHO and OIE reports, the vaccine can effectively control the rabies epidemic only when the immune coverage rate reaches more than 70%. The research on the method for detecting the serum antibody of the rabies virus after immunization actively monitors the rabies immunity of animals, especially dogs, and provides necessary technical support for effective prevention and control of rabies.
In order to ensure the resistance of the organism to rabies virus attack, the quantitative detection of anti-rabies virus antibodies or neutralizing antibodies to rabies virus is a good monitoring means. The most commonly used quantitative detection method of the anti-rabies virus neutralizing antibody comprises the following steps: mouse virus neutralization assay (MNT), fluorescent antibody virus neutralization assay (FAVN), rapid foci inhibition assay (RFFIT), enzyme-linked immunosorbent assay (ELISA), and the like.
The methods recommended by WHO and OIE for detecting RV antibodies are RFFIT and FAVN, and both methods can quantitatively detect antibody levels, but involve living cells and viruses to be operated, and have high requirements on instrument equipment, operation technology of workers and laboratory biosafety, and are limited to detection of a small number of serum samples. The ELISA is a classical test method for detecting serum antibodies, has short test period, simple operation and easy standardization, does not need to operate live viruses and cultured cells, has low dependence on the quality of serum to be detected, and does not need to consider the problem of cytotoxicity caused by hemolysis. Therefore, the method is a feasible method for clinically detecting the serum antibody level of the rabies virus, and is widely applied in the related fields.
In the rabies-associated enzyme-linked immunosorbent assay, 5 proteins encoded by the rabies virus gene and corresponding antibodies thereof are reported and studied. Since the antibodies detected by ELISA test are coated antigen antibodies, and deviations are unavoidable, the neutralizing antibody titer unit detected by ELISA is IU/mL of the EU/mL of the substitution fluorescent antibody virus neutralization test and the rapid fluorescence focus inhibition test, and certain limitations still exist.
In China, a commercial rabies virus serum antibody ELISA detection kit is available, and can be used for rapidly detecting serum antibody levels of humans and other animals (mainly dogs). However, most of the kits adopt inactivated RV whole virus as a diagnosis antigen, the use of the whole virus as the diagnosis antigen has undisputed advantages in the aspects of detection accuracy, reliability and the like, but RV is used as infectious disease pathogen seriously harming human and animal health, the preparation and use processes are relatively complicated, and potential biosafety problems exist.
The invention uses the VSV skeleton pseudovirus (VSV-CVSG) carrying rabies virus G protein as a diagnostic antigen to establish an ELISA detection method, has a plurality of advantages which are incomparable with the use of whole virus as a coating antigen, firstly, the virus and the cultured cells are not required to be operated during detection, and the biological safety risk and the production condition requirement are reduced; secondly, the VSV skeleton pseudovirus carrying rabies virus G protein has large expression quantity, is easy to purify, and is convenient for large-scale industrialized production; third, the antigen VSV-CVSG which mainly induces the generation of neutralizing antibodies is used as a coating antigen, and the detection result can better reflect the neutralizing antibody level in the body. Finally, the pseudovirus has better stability than the whole virus, so that the method is easy to store and popularize.
Disclosure of Invention
The invention aims to solve the technical problems of complex operation, higher biosafety risk, high production condition, inconvenience for large-scale industrialized production and the like in the prior art.
The technical scheme for solving the problems is as follows:
the ELISA detection method of the rabies virus neutralizing antibody is characterized by comprising the steps of antigen preparation, antigen purification, ELISA plate preparation, sample detection, observation result and the like.
Preferably, the antigen preparation method comprises the following steps: and (3) downloading a rabies virus G protein sequence (AF 085333.1) from NCBI, performing codon optimization, constructing a recombinant plasmid vector, converting the constructed recombinant plasmid vector into competent cells, extracting plasmids by using Qiagen EndoFree Plasmid Maxi Kit, transfecting the cells for 24 hours, and infecting the cells by using commercial VSV-G to enable the G protein of the cells to be replaced by the rabies virus G protein, so that the VSV pseudovirus carrying the rabies virus G protein, namely VSV-CVSG, can be finally obtained.
Preferably, the pseudoviral antigen is purified, comprising the following purification steps:
(1) The collected pseudovirus supernatant was centrifuged at 7500 r/min for 15 minutes to remove dead cells and cell debris from the supernatant.
(2) The centrifuged supernatant was filtered through a 0.45 μm filter to further remove impurities from the supernatant.
(3) Adding 7.5 ml of 40% PEG8000 mother liquor and 7.5 ml of 20% NaCl mother liquor into 30ml of pseudovirus initial liquor, and uniformly mixing; 4. standing overnight at a temperature; 12000rpm, centrifuging for 1h; the supernatant is sucked and removed, and the tube is kept stand for 1 to 2 minutes to suck residual liquid; adding 1mL of complete culture medium to dissolve the pseudovirus precipitate;
(4) The resuspension was collected into a 1.5 mL EP tube, a 15 mL EP tube of the new EPPENDORF was prepared, 11mL of pre-chilled filtered sterilized 20% sucrose solution was added, 1mL of virus resuspension was gently added to the top of the sucrose solution, and the added 15 mL EP tube was placed into an adapted rotor for centrifugation at 4 ℃,5591g, 18 hours.
(5) After centrifugation, the supernatant was discarded, the pellet was resuspended in DMEM and split into PCR tubes, 5. Mu.L per tube, split-filled, labeled, and frozen at-80℃while the virus was titered after concentration.
Preferably, the preparation method of the ELISA plate comprises the following steps: at 10 5 TCID50/mL purified VSV-CVSG coated ELISA plate, 100 μl per well, overnight at 4deg.C, 1% BSA blocked at 37deg.C for 90min, washing the plate 3 times with washing solution, and air drying.
Preferably, the sequence of the G protein after codon optimization is shown as SEQ No. 1.
Preferably, the sample detection step comprises the steps of:
(1) Adding serum to be detected: the serum to be tested is diluted by a dilution liquid in a multiple ratio, 100 mu l of sample is taken in each dilution gradient and added into the enzyme-labeled plate hole coated with the antigen, blank control and negative control are simultaneously carried out, the mixture is placed at 37 ℃ for 0.5-2h, and the washing liquid repeatedly washes the plate for 3 times (250 mu l each time), and the washing is the same.
(2) Adding enzyme-labeled secondary antibodies: the corresponding horseradish peroxidase-labeled antibody was added and placed at 37℃for 0.5-2h per well and the plate was washed 3 times (250. Mu.l per well) repeatedly with wash solution (PBST).
(3) Adding a substrate solution for color development: 100 μl of the color development solution was left in the dark at room temperature for 10-15 minutes.
(4) Adding a stop solution: 50 μl per well.
The invention has the beneficial effects that: the method uses the VSV framework pseudovirus (VSV-CVSG) carrying rabies virus G protein as a diagnostic antigen to establish an ELISA detection method, has a plurality of advantages which are incomparable with the use of whole virus as a coating antigen, firstly, the virus and the cultured cells are not required to be operated during detection, and the biological safety risk and the production condition requirement are reduced; secondly, the VSV skeleton pseudovirus carrying rabies virus G protein has large expression quantity, is easy to purify, and is convenient for large-scale industrialized production; third, the antigen VSV-CVSG which mainly induces the generation of neutralizing antibodies is used as a coating antigen, and the detection result can better reflect the neutralizing antibody level in the body. Finally, the pseudovirus has better stability than the whole virus, so that the method is easy to popularize.
Drawings
FIG. 1 shows the OD of a rabies virus antibody standard after gradient dilution using the present method 450 Values.
Detailed Description
In order to more clearly describe the technical contents of the present invention, a further description will be made below in connection with specific embodiments.
The experimental methods in which specific conditions are not specified in the following examples are generally performed according to conventional methods known to those skilled in the art or according to conditions recommended by manufacturers of actual equipment and the like. The various chemicals commonly used in the examples are commercially available.
Example 1 ELISA detection method of rabies virus neutralizing antibody
1. CVSG vector construction
The gene sequence was codon optimized with reference to rabies virus G protein sequence (AF 085333.1) in NCBI to improve protein stability and increase expression. The gene sequence is shown as SEQ No.1 (the amino acid sequence corresponding to the sequence is EFTRATMVPQVLLFVLLLGFSLCFGKFPIYTIPDELGPWSPIDIHHLSCPNNLVVEDEGCTNLSEFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGDPRYEESLHNPYPDYHWLRTVRTTKESLIIISPSVTDLDPYDKSLHSRVFPGGKCSGITVSSTYCSTNHDYTIWMPENPRPRTPCDIFTNSRGKRASNGNKTCGFVDERGLYKSLKGACRLKLCGVLGLRLMDGTWVAMQTSDETKWCPPDQLVNLHDFRSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLKVGGRCHPHVNGVFFNGIILGPDDHVLIPEMQSSLLQQHMELLKSSVIPLMHPLADPSTVFKEGDEAEDFVEVHLPDVYKQISGVDLGLPNWGKYVLMTAGAMIGLVLIFSLMTWCRRANRPESKQRSFGGTGGNVSVTSQSGKVIPSWESYRSGGEIRLPGKL), the optimized sequence is connected to pUC57 to obtain a recombinant expression vector, and the synthetic connection process is completed by general biological systems (Anhui) limited company.
2. Antigen preparation
Transforming the constructed plasmid vector into competent cells, extracting plasmids by using Qiagen EndoFree Plasmid Maxi Kit, transfecting the cells for 24 hours, infecting the cells by using commercial VSV-G to enable G protein of the cells to be replaced by rabies virus G protein, and finally obtaining the VSV pseudovirus carrying the rabies virus G protein, namely VSV-CVSG.
3. Antigen purification
The collected pseudovirus supernatant was centrifuged at 7500 r/min for 15 minutes to remove dead cells and cell debris from the supernatant. The centrifuged supernatant was filtered through a 0.45 μm filter to further remove impurities from the supernatant. Adding 7.5 ml of 40% PEG8000 mother liquor and 7.5 ml of 20% NaCl mother liquor into 30ml of pseudovirus initial liquor, and uniformly mixing; 4. standing overnight at a temperature; 12000rpm, centrifuging for 1h; the supernatant is sucked and removed, and the tube is kept stand for 1 to 2 minutes to suck residual liquid; 1mL of complete medium was added to solubilize the pseudoviral pellet. Collecting the heavy suspension into 1.5 mL EP tube, preparing new 15 mL EP tube of EPPENDORF, adding 11mL of precooled filtered sterilized 20% sucrose solution, gently adding 1mL of virus heavy suspension to the top of sucrose solution, centrifuging the added 15 mL EP tube in an adaptive rotor, centrifuging at 4deg.C, 5591xg, centrifuging for 18 hrWhen (1). After centrifugation, the supernatant was discarded, the pellet was resuspended in DMEM and split into PCR tubes, 5uL per tube, split-packed, labeled, and frozen at-80℃while the concentrated purified pseudovirus was subjected to titer determination. 4. Preparation of ELISA plate at 10 5 TCID 50 Each well of the purified VSV-CVSG coated ELISA plate was 100. Mu.l/well, blocked at 4℃overnight with 1% BSA at 37℃for 90min, washed 3 times with washing solution (250. Mu.l/well), and dried.
5. Sample detection
And (3) adding rabies virus antibody standard substances of 4.5IU, 2.25IU, 1.125IU and 0.45IU into the coated ELISA plate, setting a duplicate well, a blank control and a negative control, incubating for 1h at 37 ℃, and washing the plate for 3 times by using a washing solution. Mu.l of HRP-labeled secondary antibody was added, incubated for 1h at 37℃and the plates were washed 3 times. Mu.l of a color-developing solution was added to each well, and incubated at room temperature in the dark for 15 minutes. Mu.l of stop solution was added to each well.
6. Observation result
The 450nm reading was recorded with an enzyme-linked immunosorbent assay. This step was performed within 15 minutes of adding the stop solution.
Although certain embodiments of the present invention have been described above by way of example in a preferred manner, it will be understood by those skilled in the art that the present invention is not limited to the embodiments disclosed above, but may be modified in light of the knowledge of those skilled in the art to which the present invention pertains without exceeding the scope of the claimed invention.
Sequence listing
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<120> ELISA detection method of rabies virus neutralizing antibody
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actatcttca acaaaacctt gatggaggca gatgctcact ataagtccgt gaggacttgg 1080
aacgagataa ttccctcaaa aggctgcctg aaggtgggag gccgctgtca tcctcacgtg 1140
aacggggtgt tctttaatgg gattatactg ggtccagatg atcacgtcct gattccggaa 1200
atgcagtcta gtcttctcca gcaacatatg gagctgttga aatctagcgt catacccctg 1260
atgcacccac tggcagatcc ttccaccgtc tttaaggagg gcgacgaagc agaagacttc 1320
gtcgaggtgc acctgcctga cgtctataag cagatttcag gtgtggatct gggccttcca 1380
aattggggaa aatatgttct gatgactgca ggggccatga ttggccttgt cctgatcttc 1440
tcccttatga cgtggtgcag gagggctaat cgccccgaga gcaagcagag gtcattcgga 1500
ggaactggag gcaacgtgag tgtgacctcc cagagtggta aggtgattcc atcttgggaa 1560
tcttatcgat caggagggga gatccggttg tgacccggga agctt 1605

Claims (6)

1. The ELISA detection method of the rabies virus neutralizing antibody is characterized by comprising the steps of antigen preparation, antigen purification, ELISA plate preparation, sample detection, observation result and the like.
2. The method for detecting the rabies virus neutralizing antibody according to claim 1, wherein the antigen preparing method comprises the following steps: NCBI downloads rabies virus G protein sequence (AF 085333.1) to carry out codon optimization, and constructs a recombinant expression plasmid vector, the constructed recombinant plasmid vector is transformed into competent cells, qiagen EndoFree Plasmid Maxi Kit is used for extracting plasmids, and after transfection into cell culture for 24 hours, commercial VSV-G infected cells are used for replacing G protein with rabies virus G protein, and finally VSV pseudovirus carrying rabies virus G protein, namely VSV-CVSG, can be obtained.
3. The method for ELISA detection of rabies virus neutralizing antibody according to claim 1, characterized in that the antigen purification comprises the steps of: (1) centrifuging the collected pseudovirus supernatant for 15 minutes at 7500 r/min to remove dead cells and cell fragments in the supernatant (2) filtering the centrifuged supernatant with a 0.45 mu m filter membrane to further remove impurities in the supernatant (3) per 30ml of pseudovirus initial solution, adding 7.5 ml of 40% PEG8000 mother solution and 7.5 ml% of 20% NaCl mother solution, and mixing; 4. standing overnight at a temperature; 12000rpm, centrifuging for 1h; the supernatant is sucked and removed, and the tube is kept stand for 1 to 2 minutes to suck residual liquid; adding 1mL of complete medium to dissolve pseudovirus precipitate (4), collecting the resuspension into a 1.5 m L EP tube, preparing a new 15 m L EP tube of EPPENDORF, adding 11mL of precooled filtered sterilized 20% sucrose solution, lightly adding 1mL of virus resuspension to the top end of the sucrose solution, placing the added 15 m L EP tube into an adaptive rotor for centrifugation, centrifuging at 4 ℃ and 5591g, centrifuging for 18 hours (5), discarding the supernatant, resuspension precipitation by DMEM, subpackaging into PCR tubes, 5 mu L each tube, marking after subpackaging, freezing at-80 ℃, and simultaneously performing toxicity determination on the concentrated virus.
4. The ELISA detection method of rabies virus neutralizing antibody according to claim 1, wherein the ELISA plate preparation method comprises the following steps: at 10 5 TCID 50 Each well of the purified VSV-CVSG was coated with an ELISA plate, 100. Mu.l each well was blocked at 4℃overnight with 1% BSA at 37℃for 90min, washed 3 times with washing solution, and dried.
5. The method for preparing antigen according to claim 2, wherein the codon-optimized G protein sequence is shown in SEQ No. 1.
6. The method for ELISA detection of rabies virus neutralizing antibody according to claim 1, characterized in that the sample detection comprises the steps of: (1) adding serum to be detected: diluting the serum to be tested by a dilution solution in a multiple ratio, taking 100ul of samples at each dilution gradient, adding the samples into the enzyme-labeled plate holes coated with the antigen, simultaneously taking blank control and negative control, standing at 37 ℃ for 0.5-2h, repeatedly washing the plate 3 times (250 mu l each time) by using a washing solution, and washing the plate with the enzyme-labeled secondary antibody (2): adding corresponding horseradish peroxidase-labeled antibody, placing 100ul of the antibody in each well at 37 ℃ for 0.5-2h, and repeatedly washing the plate 3 times (250 mu l of the antibody in each well) with a washing solution (PBST) (3) adding a primer solution for color development: 100ul of color development liquid, 10-15 minutes (4) of stop solution in dark at room temperature: 50 microliters per well.
CN202111373926.1A 2021-11-19 2021-11-19 ELISA detection method of rabies virus neutralizing antibody Pending CN116148475A (en)

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