CN113391067A

CN113391067A - Indirect ELISA detection method of anti-nipah virus G protein antibody

Info

Publication number: CN113391067A
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: 2021-09-14
Anticipated expiration: 2041-06-16
Also published as: CN113391067B

Abstract

The invention relates to the technical field of biology, in particular to an indirect ELISA detection method of an anti-Nipavirus G protein antibody. The invention uses Nipah virus G protein to coat the ELISA plate, uses the 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 vaccine-stimulated specific antibody titer.

Description

Indirect ELISA detection method of 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 of an anti-Nipavirus G protein antibody.

Background

Nipah virus disease, a newly emerging virulent zoonotic infectious disease, can cause severe and highly fatal (40% -70% fatality) in humans, and is caused by Nipah virus (NiV), a mononegavirale RNA virus, a member of the Paramyxoviridae (Paramyxoviridae) Henipah virus (Henipavirus) genus^[1]NiV can be transmitted by direct contact or in aerosol form, causing infection or even death in humans or animals. The virus was first discovered after encephalitis in pig breeders in malaysia and singapore, and subsequently encephalitis in bangladesh or india almost every year. The virus was named because it was isolated in the nipalobixi (Sungai Nipah) area. NiV currently has mainly 2 genetic lineages: NiV Malaysia strain (NiV Malaysia, NiV-MY) and NiV Bengal strain (NiV Bangladesh, NiV-BD). The genome of NiV-MY strain 18246 nucleotides and the genome of NiV-BD strain 18252 nucleotides have RNA genomes 3 '-5' formed by 6 genes of nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion glycoprotein (F), attachment glycoprotein (G) and long polymerase (L) which are arranged in series, N, P and L are attached to the viral RNA to form viral ribonucleoprotein (vRNP). M, F, G constitute the envelope of virions, where M is involved in maintaining virus morphology and mediating virus budding, and F, G is involved in mediating virus attachment and invasion to host cells.

NiV outbreaks with fatality rates of over 70% -100% have occurred almost annually in bangladesh and india since 2001, and the last outbreak was in indian krinke de (Kozhikode), causing 19 deaths with 17 deaths with fatality rates as high as 89%, china borders india and is near bangladesh, with the risk of introducing this disease into our country although no cases have been reported. Due to the high pathogenicity, potential pandemic, and the lack of currently approved vaccines and mature therapeutic systems, there is an urgent need to research and develop antiviral drugs and vaccines and effective detection methods to help prevent and control future outbreaks.

Disclosure of Invention

In view of the above, the present invention provides an indirect ELISA method for detecting mouse anti-Nipah virus (NiV) G protein antibody. The invention establishes an indirect ELISA detection method for the mouse serum Nipah 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 of an anti-Nipah virus G protein antibody, which comprises the steps of coating an ELISA plate with the Nipah virus G protein as an antigen, washing, sealing, washing, adding diluted primary antibody for incubation, washing, adding an enzyme-labeled secondary antibody for incubation, washing, developing for incubation, and terminating the reaction to obtain OD₄₅₀A value;

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

the coating concentration of the antigen is 2 mug/mL-10 mug/mL; the dilution of the primary antibody is 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 μ g/mL; the dilution of the primary antibody is 1:400, the incubation time of the primary antibody is 60 min.

In some 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 enzyme-labeled secondary antibody is 1:20000, and the incubation time of the enzyme-labeled secondary antibody is 30 min.

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

In some embodiments of the invention, the time for the development is 5min to 20 min.

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

In some embodiments of the invention, the Cut-off value is the negative control serum OD₄₅₀Mean × 2.1.

Based on the research, the invention also provides a determination method of the nipah virus candidate vaccine, which takes the candidate vaccine as a primary antibody to carry out detection according to the indirect ELISA detection method.

The invention also provides a determination method of the anti-Nipah virus G protein antibody in the serum to be detected, which takes the serum to be detected as a primary antibody and carries out detection according to the indirect ELISA detection method.

The invention uses Nipah virus G protein to coat the ELISA plate, uses the 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 60 min; optimal dilution of HRP-labeled goat anti-mouse IgG is 1:20000, and optimal incubation time is 30 min; the optimal coating concentration of the antigen is 10 mug/mL; the optimal color development time is 15min, and negative control serum OD is used₄₅₀The mean value X2.1 (positive control serum > 4.00 calculated as 4.00) is the Cut-off value. The Coefficient of Variation (CV) was less than 10% for 3 replicates in a sample experiment and less than 10% for 3 replicates between sample experiments. The conclusion successfully establishes the indirect ELISA detection method of 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 research of the vaccine-stimulated specific antibody titer.

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.

Detailed Description

The invention discloses an indirect ELISA detection method of an anti-Nipah virus G protein antibody, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Nipah virus as pathogen with wide host spectrum^[7]The host includes common animals of Foliuzidae such as Hepialus, pig, cattle, horse, goat, cat, dog, mouse, rabbit, fox, bird, and myna^[8]The range of movement of the bat is wide, and the NiV can be spread among the fruit-eating bats in a certain area, so that countries and regions where the same kind of bats are distributed can be exposed to the risks of virus mutation and epidemic disease outbreak. As a big country for pig breeding in China, once epidemic outbreaks occur, the pig breeding method can bring great threat to the life and property safety of people in China. Although no NiV is detected in China at present, the coastal region and the Yunnan region of China have ecological environments and host animals suitable for the prevalence of the NiV, and the natural hosts of the NiV are found, and Nipah virus antibodies exist in bats, which indicates that China possibly faces the threat of epidemic outbreak, so that a related research aiming at an NiV detection means and a vaccine is necessary to be developed. Indirect ELISA developed using viral antigens for serological detection (IgM and IgG) has been used for serological antibody detection in humans and field studies of bats and other animals. However, since this virus is highly dangerous, it cannot be cultured under ordinary laboratory conditions, and thus it is difficult to detect the virus using the whole virus as an antigen. Eshaghi et al^[10]A baculovirus expression system is established for expressing the Nipavirus N protein, and the N protein is found to replace a whole virus to be used as an antigen for carrying out IgG antibody capture ELISA. Kaku et al established that the polyclonal antibody is utilized to carry out antigen capture sandwich ELISA, two carrier plasmids (F protein-encoding plasmid and G protein-encoding plasmid) are constructed to immunize rabbits to obtain polyclonal antibody IgG serving as an antigen capture antibody, and horseradish peroxide is added to the two IgG respectivelyThe method can only be used for detecting the Henipavirus virus, cannot distinguish NiV from HeV, but the preparation of the polyclonal antibody is simpler, more economical, does not need to prepare monoclonal antibodies, and has higher detection sensitivity to NiV.

At present, no commercial detection kit for the G protein antibody of the Nipah virus is used for evaluating a Nipah virus candidate vaccine in China, so a worker tries to establish an indirect ELISA method for detecting the IgG of the G protein of the Nipah virus, and the 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 eukaryon expressed Nipah virus G protein as coating antigen, finally successfully establishes an indirect ELISA method with high specificity, sensitivity and stability by groping various conditions, and is suitable for epidemiological investigation and evaluation of candidate vaccines.

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

The invention is further illustrated by the following examples:

example 1 basic procedure for Indirect ELISA method

The preparation method of the nepag protein comprises the following steps:

nipah virus G protein sequence analysis, design and synthesis

And comparing the gene sequences of the G protein according to 28 whole genome sequences of the Nipah virus on NCBI, drawing an evolutionary tree, and analyzing the amino acid homology of the G protein. The G protein sequence of a representative strain of Nipah virus (NCBI accession No.: AF212302.2) was obtained by on-line analysis of hydrophobicity and antigenic determinant prediction tools (http:// www.detaibio.com/tools /), and the cytoplasmic tail region, transmembrane hydrophobic region and partial stem region were deleted, leaving only the extracellular globular head region (designated as sG). Adding a human tissue plasminogen activator signal peptide (tPA) sequence and a Strep label at the N end, respectively adding enzyme cutting sites Hind III and Bam H I at the two ends, and after the sequence design is finished, carrying out codon optimization and synthesis by Shanghai Producer company Limited to obtain the glycerol strain containing the target gene plasmid (pUC 57-NiV-sG).

Construction of recombinant plasmid

Inoculating 10 mu L of glycerol strain containing a synthetic 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-amount extraction preparation of the plasmid, performing double enzyme digestion on Hind III and Bam HI, connecting the Hind III and Bam HI to a eukaryotic expression vector pcDNA3.1(+), obtaining a eukaryotic expression plasmid pcDNA-sG, transforming a competent cell Trans1-T1 by adopting a heat shock method on a recombinant plasmid, screening for ampicillin resistance, selecting a positive bacterial colony, extracting the plasmid, performing double enzyme digestion identification, sending the correctly identified plasmid to Shanghai biological engineering Limited company for sequencing, performing sequence comparison to confirm no errors, performing large-amount preparation and purification, determining the concentration of the plasmid, and using the plasmid for cell transfection.

Cell transfection

One day before transfection, Expi293F cells were suspension cultured at 8% CO2, 37 ℃ and 125r/min, the viability and viable cell density of 293F cell cultures in 60mL system were determined, viable cells were grown to 4.2X 106/mL and the viability was 97%, the cell cultures were diluted to 2.1X 106/mL, the cell density was 3X 106/mL on the day of transfection, 60. mu.g eukaryotic expression plasmid pcDNA-sG and 3mL Opti-MEM were added^TMMixing I Reduced Serum Medium, 160 μ L Expifeacylamine^TM293 Reagent and 2.8mL Opti-MEM^TMMixing I Reduced Serum Medium, incubating at room temperature for 5min, mixing, and incubating at room temperature for 20 min. The solution was then slowly transferred to a cell culture shake flask, and the flask was gently swirled during the addition.

Collecting the protein of interest

Adding 300 mu L of transfection enhancing agent 1 and 3mL of transfection enhancing agent 212 h after transfection, continuously culturing for 96h, centrifuging the culture for 10min at 800g, respectively collecting culture medium supernatant and cell sediment, wherein the culture medium supernatant is named as sG-MS, the cell sediment is cracked for 10min on ice after being resuspended by using IP lysate, and is crushed for 10min by using an ultrasonic cell crusher, and the supernatant is named as sG-DS after centrifugation.

Diluting an antigen (NipaG protein) prepared and stored by a molecular virology and immunology laboratory of military veterinary research institute of medical science by using an ELISA protein coating solution, coating an ELISA plate, and standing overnight at 4 ℃ (16h-24 h); patting out the liquid, adding TBST to wash twice, and each time for 2 min; adding 5% skimmed milk-TBST 37 deg.C, and sealing for 1.5 hr; cleaning the sealing solution, adding TBST, and washing for 3 times, each for 2 min; after diluting sample serum (primary antibody) with PBS, incubating the ELISA plate for 60min at 37 ℃; beating off primary antibody, washing with TBST for three times, each time for 5min, adding goat anti-mouse IgG-HRP (secondary antibody) diluted with TBST, and incubating at 37 deg.C for 60 min; cleaning the secondary antibody, washing with TBST for 10min for three times, adding 100 mu LTMB single-component color developing solution into each hole, and incubating for 15min at 37 ℃; the reaction was stopped by adding 50. mu.L LISA stop solution to each well. Determination of OD Using microplate reader₄₅₀The value is obtained. The above is the initial basic flow of the method, and then the optimal conditions are determined by groping various conditions.

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

The optimal coating concentration of the antigen and the optimal dilution of serum are determined by an array titration method, the nepag protein is respectively diluted into 2 mu G/mL, 4 mu G/mL, 8 mu G/mL and 10 mu G/mL, 50 mu L of the nepag protein is coated on an enzyme label plate, and the enzyme label plate is incubated overnight at 4 ℃. The positive sample serum and the negative sample serum were diluted to 1: 200. carrying out matrix titration on the protein coating concentration at the ratio of 1:400 and 1:800, and carrying out three groups of parallel experiments. The serum dilution and the antigen coating concentration when the P/N value (positive control serum A450 mean value/negative control serum A450 mean value) is greater than 2.1 and is maximum are the serum optimal dilution antigen and the optimal coating concentration.

When the antigen coating concentration was 10. mu.g/mL, the serum dilution was 1: positive control serum OD 400₄₅₀And the combination of the P/N values meets the set conditions, and the P/N value is the highest, see Table 1. Thus, it isThe optimal coating concentration of the antigen is 10 mug/ml, and the serum dilution is 1: 400.

TABLE 1 Square matrix titration results (OD) for optimal antigen coating concentration and optimal serum dilution₄₅₀)

Tab 1.Block titration results at various antigen concentrations for coating and serum dilutions(OD₄₅₀)

EXAMPLE 3 determination of optimal working concentration and reaction time of enzyme-labeled Secondary antibody

Performing operation by using the determined optimal antigen coating concentration and the determined optimal serum dilution, respectively diluting goat anti-mouse IgG-HRP to 1:5000, 1: 10000 and 1:20000, incubating the ELISA plate, incubating at 37 ℃ for 30min, 60min, 90min and 120min, performing three groups of parallel experiments, wherein the readings of the ELISA plate reader are shown as OVER in OD₄₅₀The value is 4.000, and the optimal working concentration and the optimal reaction time of the enzyme-labeled secondary antibody are determined when the P/N value is greater than 2.1 and the maximum value is obtained.

The dilution of goat anti-mouse IgG-HRP (enzyme-labeled secondary antibody) is 1:20000, the reaction time is 30min, and the result most conforms to the set conditions, and is shown in Table 2. Therefore, the optimal working concentration of the enzyme-labeled secondary antibody is 1:20000, and the optimal reaction time is 30 min.

TABLE 2 Square matrix titration results (OD) for different enzyme-labeled antibody concentrations and reaction times₄₅₀)

Tab 2.Block titration results of various HRP-labeled antibody concentrations and reaction time(OD₄₅₀)

EXAMPLE 4 determination of blocking conditions

The procedure was carried out under the optimum conditions determined above, using 5% skim milk-TBST and 3% BSA-TBST for blocking, respectively, and 1h, 1.5h and 2h at 37 deg.C, and three parallel experiments were carried out, with the P/N value greater than 2.1 and the maximum being the optimum blocking condition.

The two different sealing liquids were used for sealing, and 3 times were set, and it can be seen from table 3 that when 5% skim milk-TBST was used as the sealing liquid and sealed at 37 ℃ for 1.5 hours, the P/N value was the greatest, indicating that the set conditions were best met. Therefore, the optimal sealing condition is 5% skim milk-TBST and sealing for 1.5h at 37 ℃.

TABLE 3 Block fluid and Block time Square matrix titration results (OD)₄₅₀)

Tab 1.Block titration results at various blocking solution and blocking time(OD₄₅₀)

EXAMPLE 5 determination of optimal reaction time of serum to be tested

The operation is carried out under the optimal conditions determined in the foregoing, the positive serum sample and the negative serum sample are respectively incubated for 30min, 60min and 90min at 37 ℃, each group is subjected to three parallel experiments, and the optimal reaction time is determined when the P/N value is greater than 2.1 and the maximum value is obtained.

The serum to be detected is incubated for 60min at 37 ℃ and best meets the set conditions, which are shown in the table 4, so that the optimal reaction time is determined.

TABLE 4 results (OD) of incubation times of different sera to be tested₄₅₀)

Tab 4.Values of different incubation time of serum to be tested (OD₄₅₀)

EXAMPLE 6 determination of optimum color development time

According to the determined optimal conditions, after adding a 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 greater than 2.1 and the maximum value is obtained.

The color of the substrate deepens with the increase of the color development time, and the color is the time node with the maximum P/N when the color is developed for 10min, as shown in Table 5, so that the optimal color development time is determined to be 15 min.

TABLE 5 results (OD) for different development times₄₅₀)

Tab 5.Values of samples after coloration for various minutes (OD₄₅₀)

EXAMPLE 7 determination of cut-off value

The OD was calculated by measuring 32 negative control sera by the established indirect ELISA method₄₅₀Mean (x) and Standard Deviation (SD), determining the Cut-off value of the method.

32 negative control sera were tested by the established indirect ELISA method with an OD450 mean of 0.071 and a standard deviation of 0.012 according to the formula

Therefore, the theoretical critical value (Cut-off value) of the indirect ELISA method is 0.095, but the main objective of the method is to evaluate the ability of the vaccine to elicit specific antibodies, so the improvement standard is 0.149 determined by calculation using the negative control serum OD450 mean value x 2.1 as the Cut-off value of the method, as shown in FIG. 1.

Effect example 1 specificity test

The established method is used for detecting the mouse positive serum and the negative control serum of the novel coronavirus, the chikungunya virus, the Getta virus, the porcine circovirus type 2, the porcine circovirus type 3, the porcine transmissible gastroenteritis virus and judging whether the mouse 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 mouse positive serum of the novel coronavirus, the chikungunya virus, the Getavirus, the porcine circovirus type 2, the porcine circovirus type 3 and the porcine transmissible gastroenteritis virus, the results are negative, and the indirect ELISA method is proved to have good specificity as shown in Table 6.

TABLE 6 methodsResults of the anisotropy assay (

OD₄₅₀)

Tab 6.Result of specificity test

Effect example 2 stability verification

And (3) respectively arranging the positive serum of the three NipaG protein mice prepared in a laboratory in 3 multiple holes in the group and 3 times of repeated tests among the groups, calculating CV, and judging the stability of the method.

In order to verify the repeatability of the establishment method of the research, 3 positive serum samples prepared in the laboratory are selected, and through 3 times of repeated experiments in groups and three times of repeated experiments among the groups, the CV in the groups is 1.1% -8.5%, and the CV among the groups is 6.2% -7.0%, which is shown in Table 7, the indirect ELISA method is proved to have good repeatability.

Table 7 stability test results

Tab 7.Results of stability test

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

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

coating an ELISA plate with Nipah virus G protein as antigen, washing, sealing, washing, adding diluted primary antibody, incubating, washing, adding enzyme-labeled secondary antibody, incubating, washing, developing, terminating reaction, and collectingTo obtain OD₄₅₀A value;

2. The indirect ELISA detection method of claim 1 wherein the antigen is coated at a concentration of 10 μ g/mL; the dilution of the primary antibody is 1:400, the incubation time of the primary antibody is 60 min.

3. The indirect ELISA detection method of claim 1 or 2 wherein 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.

4. The indirect ELISA detection method of any one of claims 1 to 3 wherein the enzyme-labeled secondary antibody is diluted at 1:20000 and the incubation time of the enzyme-labeled secondary antibody is 30 min.

5. The indirect ELISA detection method of any one of claims 1 to 4 wherein the blocking is specifically: 5% skim milk-TBST was used as a blocking solution, and blocked at 37 ℃ for 1.5 h.

6. The indirect ELISA detection method of any one of claims 1 to 5 wherein the time for development is between 5min and 20 min.

7. The indirect ELISA detection method of any one of claims 1 to 6 wherein the time for development is 15 min.

8. The indirect ELISA detection method of any one of claims 1 to 7 wherein the Cut-off value is the negative control serum OD₄₅₀Mean × 2.1.

9. A method for determining a candidate vaccine of nipah virus, wherein an antibody induced after a mouse is immunized with the candidate vaccine is used as a primary antibody, and the detection is performed according to the indirect ELISA detection method of any one of claims 1 to 7.

10. Method for determining antibodies against nipah virus G protein in a test serum, characterized in that the test serum is used as a primary antibody and the detection is carried out according to the indirect ELISA method of any one of claims 1 to 7.