CN113391067B

CN113391067B - Indirect ELISA detection method for anti-Nipah virus G protein antibody

Info

Publication number: CN113391067B
Application number: CN202110667511.9A
Authority: CN
Inventors: 金宁一; 李昌; 高子函; 李乐天; 许汪; 郝鹏飞; 鲁会军; 李霄; 田明尧
Original assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Current assignee: Military Veterinary Research Institute Academy Of Military Medical Sciences
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2023-07-25
Anticipated expiration: 2041-06-16
Also published as: CN113391067A

Abstract

The invention relates to the technical field of biology, in particular to an indirect ELISA detection method for an anti-Nipag virus G protein antibody. The invention uses Nipah virus G protein to coat the ELISA plate, uses square matrix titration method to determine the optimum working condition of indirect ELISA, and analyzes the specificity, sensitivity, stability and the like of the method. The invention successfully establishes an indirect ELISA detection method for the mouse serum nipah virus G protein antibody, and can be applied to the analysis of the anti-nipah virus G protein antibody in the mouse serum and the evaluation and research of the specific antibody titer excited by the vaccine.

Description

Indirect ELISA detection method for anti-Nipah virus G protein antibody

Technical Field

The invention relates to the technical field of biology, in particular to an indirect ELISA detection method for an anti-Nipag virus G protein antibody.

Background

Nipah virus disease, which is a newly emerging virulent zoonotic infectious disease, can cause severe and high mortality (40% -70%) in humans, is a Nipah virus (NiV),is a single-stranded negative strand RNA virus belonging to Paramyxoviridae (Paramyxoviridae) Huntiepaviral (Henipavirus) ^[1] NiV can be transmitted by direct contact or in the form of an aerosol, resulting in infection and even death of humans or animals. The virus was first discovered after the outbreak of encephalitis in swine farmers in malaysia and singapore, and then almost every year in bangladesh or india. The virus was named because it was isolated in the Nipath region. NiV currently has mainly 2 genetic lineages: niV Malaysia strain (NiV Malaysia, niV-MY) and NiV Bengal strain (NiV Bangladesh, niV-BD). Wherein the genome of the NiV-MY strain is 18246 nucleotides, and the genome of the NiV-BD strain is 18252 nucleotides, the RNA genome of the NiV-MY strain is formed by continuously arranging 6 genes of nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (G) and long polymerase (L), and the genes are N, P and L attached to viral RNA to form viral ribonucleoprotein (vRNP). M, F, G constitutes the envelope of the virion, where M is involved in maintaining the virus morphology and mediating viral budding, while F, G is involved in mediating viral attachment and invasion of host cells.

NiV outbreaks with mortality rates exceeding 70% -100% have occurred in Bengala and India almost annually since 2001, whereas the last outbreak was in Kerikende (Kozhikode) in India, resulting in 17 deaths in 19 people infected, with mortality rates as high as 89%, and our country bordered by India and adjacent to Bengala, which, although no case has been reported, has a risk of afferent China. Because of the high pathogenicity, potential pandemic, and the currently unapproved vaccine and mature therapeutic system of this virus, there is an urgent need to study and develop antiviral drugs and vaccines and effective detection methods to help prevent and control future epidemic situations that may be outbreak.

Disclosure of Invention

In view of this, the present invention provides an indirect ELISA detection method for mouse anti-Nipah virus (NiV) G protein antibodies. The invention establishes an indirect ELISA detection method for the mouse serum Nipag virus G protein antibody, and lays a solid foundation for the subsequent development of vaccines and the detection and evaluation of specific antibodies.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an indirect ELISA detection method for anti-Nipag virus G protein antibody, which comprises the steps of taking Nipag virus G protein as antigen to coat an ELISA plate, beating liquid, washing, sealing, washing, adding diluted primary antibody for incubation, beating primary antibody for washing, adding ELISA secondary antibody for incubation, beating secondary antibody for incubation, washing, developing color for incubation, and stopping reaction to obtain OD ₄₅₀ A value;

the primary antibody comprises positive sample serum or negative sample serum;

the coating concentration of the antigen is 2-10 mug/mL; the dilution of the primary antibody was 1: 200-1: 800; the incubation time of the primary antibody is 30-90 min.

In some embodiments of the invention, the antigen is coated at a concentration of 10 μg/mL; the dilution of the primary antibody was 1:400, the incubation time of the primary antibody was 60min.

In some specific embodiments of the invention, the working concentration of the enzyme-labeled secondary antibody is 1:5000-1:20000; the incubation time of the enzyme-labeled secondary antibody is 30-120 min.

In some embodiments of the invention, the dilution of the second enzyme-labeled antibody is 1:20000, and the incubation time of the second enzyme-labeled antibody is 30min.

In some embodiments of the invention, the closure is specifically: 5% skim milk-TBST was used as a blocking solution, and the mixture was blocked at 37℃for 1.5 hours.

In some embodiments of the invention, the time for the color development is from 5 minutes to 20 minutes.

In some embodiments of the invention, the time for development is 15 minutes.

In some embodiments of the invention, the Cut-off value is the OD of a negative control serum ₄₅₀ Mean x 2.1.

Based on the research, the invention also provides a method for determining the Nipagin virus candidate vaccine, which takes the candidate vaccine as a primary antibody and detects according to the indirect ELISA detection method.

The invention also provides a method for determining the anti-Nipag virus G protein antibody in the serum to be detected, wherein the serum to be detected is used as a primary antibody, and the detection is carried out according to the indirect ELISA detection method.

The invention uses Nipah virus G protein to coat the ELISA plate, uses square matrix titration method to determine the optimum working condition of indirect ELISA, and analyzes the specificity, sensitivity, stability and the like of the method. Results the optimal dilution of the ELISA method samples was 1:400, the optimal incubation time is 60min; HRP-labeled goat anti-mouse IgG optimal dilution is 1:20000, and optimal incubation time is 30min; the optimal coating concentration of the antigen is 10 mug/mL; the optimal color development time is 15min, and the negative control serum OD is used ₄₅₀ Mean X2.1 (positive control serum greater than 4.00 calculated as 4.00) is the Cut-off value. The Coefficient of Variation (CV) of 3 replicates in the sample experiment is less than 10%, and the CV of 3 replicates in the sample experiment is less than 10%. Conclusion the indirect ELISA detection method of the mouse serum Nipag virus G protein antibody is successfully established, and the method can be applied to evaluation and research of specific antibody titers excited by vaccines and used for analysis of anti-Nipag virus G protein antibodies in mouse serum.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the results of negative serum indirect ELISA assays.

Detailed Description

The invention discloses an indirect ELISA detection method for anti-Nipagin G protein antibodies, and a person skilled in the art can properly improve the process parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

Nipah virus as a pathogen with a broad host spectrum ^[7] Its host comprises common animals such as fruit bat, pig, cow, horse, goat, cat, dog, mouse, rabbit, fox, glancing bird, and mexico ^[8] The range of movement of the bats is wider, and NiV is likely to spread among the bats of fruit eating in a certain area, so that countries and regions with the same type of bats are exposed to the risks of virus mutation and epidemic outbreak. As a big pig raising country, once epidemic situation outbreaks occur, the method can cause great threat to the life and property safety of people in China. Although NiV is not detected in China at present, the southeast coastal area and the Yunnan area of China have ecological environment and host animals suitable for NiV epidemic, and a natural host of NiV is found, nipah virus antibodies exist in bat bodies, which suggests that China possibly faces epidemic situation outbreak epidemic threat, and therefore related researches on NiV detection means and vaccines are necessary to be carried out. Indirect ELISA developed using viral antigens for serological detection (IgM and IgG) has been used for serological antibody detection in humans and field studies in bats and other animals. However, since the virus is highly dangerous, it is difficult to detect the virus using whole virus as an antigen because the virus cannot be cultured under ordinary laboratory conditions. Eshaghi et al ^[10] The expression of nipah virus N protein using baculovirus expression system was established and found to be useful in place of whole virus as antigen for IgG antibody capture ELISA. Kaku et al established sandwich ELISA for antigen capture by using polyclonal antibody, constructed two vector plasmids (F protein encoding plasmid and G protein encoding plasmid) immune rabbit to obtain polyclonal antibody IgG as antigen capture antibody, respectively adding horseradish peroxidase as enzyme-labeled conjugate antibody on the two IgG, respectively comparing reactivity by ELISA, finally obtaining the polyclonal antibody of G protein with optimal reactivity, respectively testing pseudotyped VSV, niV of two different strains and HeV live virus to obtain lower detection limit, and using common VSV as negative control, and the result shows that the detection sensitivity of pseudotyped VSV and NiV is acceptable, and the detection lower limit of HeV is higher and not sensitive, the method can only be used for detecting henpa virus, and can not distinguish NiV and HeV, but the preparation of polyclonal antibody is simplerThe ELISA can be used as a standby detection method in view of the problems of genetic variation of henipa virus and reliability of various existing PCR detection.

At present, no commercial nipah virus G protein antibody detection kit is used for evaluating nipah virus candidate vaccines, so a worker tries to establish an indirect ELISA method for detecting nipah virus G protein IgG, and ELISA is very suitable for being used as an epidemiological investigation and serological screening tool due to the characteristics of sensitivity, rapidness, safety and high flux. The invention takes eukaryotic expression of Nipah virus G protein as a coating antigen, and finally successfully establishes an indirect ELISA method with high specificity, sensitivity and stability through fumbling on various conditions, thereby being applicable to epidemiological investigation and evaluation of candidate vaccines.

In the indirect ELISA detection method of the anti-Nipagin G protein antibody provided by the invention, raw materials and reagents used can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 basic procedure of an indirect ELISA method

The preparation method of the Nipag G protein comprises the following steps:

nipah virus G protein sequence analysis, design and synthesis

The gene sequences of the G proteins were compared according to the 28 whole genome sequences of nipah virus on NCBI, the evolutionary tree was drawn, and the amino acid homology of the G proteins was analyzed. Analysis was performed by on-line hydrophobicity analysis means, epitope prediction means (http:// www.detaibio.com/tools /) to obtain the G protein sequence of the nipah virus representative strain (NCBI accession number: AF 212302.2), deleting cytoplasmic tail, transmembrane hydrophobic and part of the stem region, leaving only the extracellular globular head region (designated sG). The N end is added with a human tissue plasminogen activator signal peptide (tPA) sequence and Strep tag, and the two ends are respectively added with enzyme cutting sites HindIII and BamHI, after the sequence design is completed, the sequence is subjected to codon optimization and synthesis by Shanghai Biotechnology, thereby obtaining the glycerinum containing target gene plasmid (pUC 57-NiV-sG).

Construction of recombinant plasmids

The synthesized 10 mu L glycerol bacteria containing the target gene plasmid (pUC 57-NiV-sG) are inoculated in 5mL liquid LB culture medium with ampicillin resistance, shake culture is carried out for 12h at 37 ℃ to carry out small extraction preparation of plasmids, hindIII and Bam HI are connected to eukaryotic expression vector pcDNA3.1 (+) after double enzyme digestion to obtain eukaryotic expression plasmid pcDNA-sG, the recombinant plasmid is transformed into competent cells Trans1-T1 by a heat shock method, positive colonies are selected through ampicillin resistance screening, plasmids are extracted, double enzyme digestion identification is carried out, the plasmids with correct identification are sent to Shanghai bioengineering limited company for sequencing, and after sequence comparison confirmation, a large amount of plasmids are prepared and purified to determine plasmid concentration for cell transfection.

Cell transfection

The day before transfection, the Expi293F cells were cultured in suspension at 8% CO2 at 37℃under 125r/min, 60mL of a system of 293F cell cultures were assayed for viability and viable cell density, viable cells were grown to 4.2X106/mL with a viability of 97%, the cell cultures were diluted to 2.1X106/mL, the day of transfection with a cell density of 3X 106/mL, 60. Mu.g eukaryotic expression plasmid pcDNA-sG was isolated from 3mL Opti-MEM ^TM I Reduced Serum Medium and 160. Mu.L of Expiectamine ^TM 293 Reagent and 2.8mL Opti-MEM ^TM I Reduced Serum Medium, incubating at room temperature for 5min, mixing the two, and incubating at room temperature for 20min. The solution was then slowly transferred to a cell culture shake flask, which was gently rotated during addition.

Collecting the protein of interest

After 12h of transfection, 300. Mu.L of transfection enhancer 1 and 3mL of transfection enhancer 2 were added, culture was continued for 96h, the culture was centrifuged at 800g for 10min, the culture supernatant and cell pellet were collected, respectively, the culture supernatant was designated sG-MS, the cell pellet was resuspended in IP lysate and lysed on ice for 10min, disrupted by an ultrasonic cell disruptor for 10min, and the supernatant was collected by centrifugation and designated sG-DS.

The antigen (Nipag G protein) prepared and stored in molecular virology and immunology laboratory of military veterinary institute of medical science was prepared by ELISCoating an ELISA plate after diluting the protein A coating liquid, and coating the ELISA plate at 4 ℃ overnight (16-24 h); the liquid is beaten off, and TBST is added for washing twice for 2min each time; adding 5% skimmed milk-TBST, sealing at 37deg.C for 1.5 hr; cleaning the sealing liquid, adding TBST for washing for 3 times, and 2min each time; after diluting sample serum (primary antibody) with PBS, incubating the ELISA plate at 37 ℃ for 60min; the primary antibody was then removed by patting, washing with TBST three times for 5min each, adding TBST diluted goat anti-mouse IgG-HRP (secondary antibody), and incubating at 37deg.C for 60min; the secondary antibody is beaten, washed three times by TBST for 10min each time, 100 mu LTMB single-component color development liquid is added into each hole, and incubated for 15min at 37 ℃; the reaction was stopped by adding 50. Mu. LELISA stop solution to each well. Determination of OD Using an enzyme-labeled Instrument ₄₅₀ Values. The above is the initial basic flow of the method, and then the optimal conditions are determined through fumbling of various conditions.

Example 2 determination of optimal coating concentration of antigen and optimal dilution of serum

The optimal coating concentration of antigen and the optimal dilution of serum are determined by a square titration method, nipag G protein is respectively diluted into 2 mug/mL, 4 mug/mL, 8 mug/mL and 10 mug/mL, 50 mug/well is used for coating an ELISA plate, and the ELISA plate is incubated overnight at 4 ℃. Positive sample serum and negative sample serum were diluted to 1: 200. matrix titration was performed with 1:400, 1:800 and the protein coating concentrations described above, and three parallel experiments were performed. The optimal dilution of serum and the optimal coating concentration are obtained by using the serum dilution times and the antigen coating concentration when the P/N value (positive control serum A450 mean/negative control serum A450 mean) is more than 2.1 and the maximum.

When the antigen coating concentration was 10. Mu.g/mL, the serum dilution was 1: positive control serum OD at 400 ₄₅₀ And the combination of the P/N values satisfies the set condition, and the P/N value is highest, see Table 1. Thus, the optimal coating concentration for antigen was determined to be 10. Mu.g/ml and the serum dilution was 1:400.

TABLE 1 results of square titration of optimal coating concentration of antigen and optimal dilution of serum (OD ₄₅₀ )

Tab 1.Block titration results at various antigen concentrations for coating and serum dilutions(OD ₄₅₀ )

Example 3 determination of optimal working concentration and reaction time for enzyme-labeled secondary antibody

The method comprises the steps of performing three groups of parallel experiments by respectively diluting goat anti-mouse IgG-HRP to 1:5000, 1:10000 and 1:20000 with the optimal antigen coating concentration and the optimal serum dilution determined in the previous step, incubating an ELISA plate at 37 ℃ for 30min, 60min, 90min and 120min, and displaying the reading of the ELISA plate as OVER and OD ₄₅₀ The value is calculated to be 4.000, and the optimal working concentration and the optimal reaction time of the enzyme-labeled secondary antibody are taken when the P/N value is larger than 2.1 and the enzyme-labeled secondary antibody is maximum.

Goat anti-mouse IgG-HRP (enzyme-labeled secondary antibody) dilution is 1:20000, reaction time is 30min, and the result is the most suitable for the set condition, as shown in Table 2. Therefore, the optimal working concentration of the enzyme-labeled secondary antibody is 1:20000, and the optimal reaction time is 30min.

TABLE 2 results of square titration of different enzyme-labeled antibody concentrations and reaction times (OD ₄₅₀ )

Tab 2.Block titration results of various HRP-labeled antibody concentrations and reaction time(OD ₄₅₀ )

Example 4 determination of closed conditions

Three parallel experiments were performed using 5% skim milk-TBST and 3% BSA-TBST, respectively, at 37℃for 1h, 1.5h, and 2h, respectively, with P/N values greater than 2.1 and at maximum as optimal blocking conditions.

The two different sealing liquids were used for sealing, and 3 times were set, and as can be seen from Table 3, when 5% skim milk-TBST was used as the sealing liquid for sealing at 37℃for 1.5 hours, the P/N value was the largest, indicating that the set conditions were most satisfied. Therefore, the optimal blocking condition is 5% skim milk-TBST, and blocking is carried out at 37 ℃ for 1.5 hours.

TABLE 3 results of square matrix titration of blocking fluid and blocking time (OD ₄₅₀ )

Tab 1.Block titration results at various blocking solution and blocking time(OD ₄₅₀ )

Example 5 determination of the optimal response time of serum to be examined

The positive serum sample and the negative serum sample are respectively incubated for 30min, 60min and 90min at 37 ℃ under the optimal conditions determined in the previous step, and three parallel experiments are carried out on each group, wherein the optimal reaction time is the maximum P/N value which is larger than 2.1.

The best reaction time was determined by incubating the serum to be tested at 37℃for 60min, which best meets the set conditions as set forth in Table 4.

TABLE 4 results (OD ₄₅₀ )

Tab 4.Values of different incubation time of serum to be tested (OD ₄₅₀ )

Example 6 determination of optimal color development time

According to the determined optimal conditions, after adding the substrate, respectively reacting for 5min, 10min, 15min and 20min at 37 ℃, carrying out three parallel experiments on each group, and determining the optimal color development time when the P/N value is larger than 2.1 and the maximum value is the maximum value.

The color of the substrate deepened with the extension of the color development time, and the time node of P/N maximum at the time of development for 10min was shown in Table 5, so that the optimum development time was determined to be 15min.

TABLE 5 results (OD) ₄₅₀ )

Tab 5.Values of samples after coloration for various minutes (OD ₄₅₀ )

Example 7 determination of cut-off value

32 negative control sera were assayed by established indirect ELISA to calculate OD ₄₅₀ The mean (x) and Standard Deviation (SD) of the method, the Cut-off value of the method is determined.

Experiments were performed on 32 negative control sera according to the established indirect ELISA method, with an OD450 average of 0.071 and a standard deviation of 0.012, according to the formulaThe theoretical threshold (Cut-off) of the indirect ELISA method was 0.095, but the main objective of the method was to evaluate the ability of the vaccine to excite specific antibodies, so that the improvement criterion was calculated as 0.149 according to the OD450 mean of the negative control serum X2.1 as the Cut-off of the method, as shown in FIG. 1.

Effect example 1 specificity verification

The established method is used for detecting positive serum and negative control serum of novel coronaviruses, chikungunya viruses, katavirus, porcine circovirus type 2, porcine circovirus type 3 and transmissible gastroenteritis viruses, and judging whether the positive serum and the negative control serum are positive so as to verify the specificity of the method.

In order to verify the specificity of the established method, the indirect ELISA method is used for detecting the positive serum of the novel coronavirus, chikungunya virus, katavirus, porcine circovirus type 2, porcine circovirus type 3 and transmissible gastroenteritis virus mice, and the results are negative, and are shown in Table 6, so that the indirect ELISA method has good specificity.

TABLE 6 method specificity test results [ (]OD ₄₅₀ )

Tab 6.Result of specificity test

Effect example 2 stability verification

Three laboratory-prepared Nipag G protein mouse positive sera were respectively provided with 3 duplicate wells in the group and 3 repeated experiments between the groups, CV was calculated, and the stability of the method was judged.

To verify the repeatability of the study setup method, 3 positive serum samples prepared in the laboratory were selected, and 3 replicates in the group and three replicates between groups were tested, with an intra-group CV of 1.1% -8.5% and an inter-group CV of 6.2% -7.0%, as shown in table 7, to demonstrate that the indirect ELISA method has good reproducibility.

TABLE 7 stability test results

Tab 7.Results of stability test

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. An indirect ELISA detection method of anti-Nipah virus G protein antibody, which is characterized in that,

coating an ELISA plate with Nipah virus G protein as an antigen, cleaning liquid, washing, sealing, washing, adding diluted primary antibody, incubating, cleaning primary antibody, washing, adding ELISA secondary antibody, incubating, cleaning secondary antibody, washing, developing, incubating, and terminating reaction to obtain OD ₄₅₀ A value;

the primary antibody comprises positive sample serum or negative sample serum;

the coating concentration of the antigen is 2-10 mug/mL; the dilution of the primary antibody was 1: 200-1: 800; the incubation time of the primary antibody is 30-90 min;

the working concentration of the enzyme-labeled secondary antibody is 1:5000-1:20000; the incubation time of the enzyme-labeled secondary antibody is 30-120 min;

the sealing is specifically as follows: taking 5% skim milk-TBST as a sealing liquid, and sealing for 1.5h at 37 ℃;

the color development time is 5-20 min;

the coating concentration of the antigen is 10 mug/mL; the dilution of the primary antibody was 1:400, wherein the incubation time of the primary antibody is 60min;

the preparation method of the Nipagin virus G protein comprises the following steps:

nipah virus G protein sequence analysis, design and synthesis

Comparing the gene sequences of the G proteins according to 28 whole genome sequences of the Nipagin viruses on NCBI, drawing a evolutionary tree, and analyzing the amino acid homology of the G proteins; analysis was performed by an on-line hydrophobicity analysis tool and an epitope prediction tool to obtain the nipah virus representative strain NCBI accession number: the sequence of the protein G of AF212302.2, which is named sG by design, is deleted from the tail region, the transmembrane hydrophobic region and part of the stem region of the cytoplasm, and only the extracellular spherical head region is reserved; adding a human tissue plasminogen activator signal peptide sequence and a Strep tag at the N end, respectively adding enzyme cutting sites HindIII and BamHI at the two ends, and performing codon optimization and synthesis by Shanghai Biotechnology limited company after sequence design is completed to obtain glycerol bacteria containing a target gene plasmid pUC 57-NiV-sG;

construction of recombinant plasmids

Inoculating 10 mu L of glycerol bacteria containing a target gene plasmid pUC57-NiV-sG into 5mL of liquid LB culture medium with ampicillin resistance, performing shake culture at 37 ℃ for 12h, performing small-scale extraction preparation of plasmids, connecting HindIII and Bam HI to eukaryotic expression vectors pcDNA3.1 (+) after double enzyme digestion to obtain eukaryotic expression plasmids pcDNA-sG, transforming competent cells Trans1-T1 of the recombinant plasmids by a heat shock method, screening by ampicillin resistance, picking positive colonies, extracting plasmids, performing double enzyme digestion identification, sending the plasmids with correct identification to Shanghai bioengineering limited company for sequencing, performing mass preparation and purification after sequence comparison confirmation, and determining plasmid concentration for cell transfection;

cell transfection

The day before transfection, the Expi293F cells were incubated at 8% CO ₂ 、37℃And suspension culturing at 125r/min, and measuring the survival rate and living cell density of 293F cell culture of 60mL system, and growing living cells to 4.2X10 ⁶ Cell cultures were diluted to 2.1X10 at a viability of 97% per mL ⁶ Cell density was 3X 10 cells per mL, day of transfection ⁶ Mu.g eukaryotic expression plasmid pcDNA-sG was combined with 3mL Opti-MEM at a concentration of 60. Mu.g/mL ^TM I Reduced Serum Medium mixing, 160 mu LExPiFectamine ^TM 293 Reagent and 2.8mL Opti-MEM ^TM I Reduced Serum Medium, mixing, incubating at room temperature for 5min, mixing the two, and incubating at room temperature for 20min; slowly transferring the solution into a cell culture shake flask, and slightly rotating the shake flask during the adding process;

collecting the protein of interest

After 12h of transfection, 300. Mu.L of transfection enhancer 1 and 3mL of transfection enhancer 2 were added, culture was continued for 96h, the culture was centrifuged at 800g for 10min, the culture supernatant and cell pellet were collected separately, the cell pellet was resuspended in IP lysate and lysed on ice for 10min, and disrupted with an ultrasonic cell disruptor for 10min.

2. The indirect ELISA detection method of claim 1, wherein the dilution of the second enzyme-labeled antibody is 1:20000 and the incubation time of the second enzyme-labeled antibody is 30min.

3. The indirect ELISA detection method according to any of claims 1 to 2, characterized in that the development time is 15min.

4. The indirect ELISA detection method as claimed in any one of claims 1 to 2 wherein the Cut-off value is the OD of the negative control serum ₄₅₀ Mean x 2.1.

5. A method for determining a candidate vaccine against nipah virus, wherein antibodies induced after immunization of mice with the candidate vaccine are used as primary antibodies, and are detected according to the indirect ELISA detection method according to any of claims 1 to 4.

6. A method for determining anti-nipah virus G protein antibodies in a test serum, characterized in that the test serum is used as a primary antibody, and the detection is performed according to the indirect ELISA detection method according to any of claims 1 to 4.