CN112798787A - Rabies vaccine antigen content detection method and reagent or kit - Google Patents
Rabies vaccine antigen content detection method and reagent or kit Download PDFInfo
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Abstract
The invention provides a rabies vaccine antigen content detection method and a reagent or a kit, wherein the detection method comprises the following steps: preparing a monoclonal antibody coated ELISA plate, and immobilizing the monoclonal antibody; diluting the vaccine to be detected and the standard vaccine according to a multiple ratio gradient, adding the diluted vaccine and the standard vaccine into the monoclonal antibody coated enzyme label plate, and incubating; and adding a mouse polyclonal antibody as a primary antibody, adding horseradish enzyme labeled goat anti-mouse peroxide with a proper dilution as a secondary antibody, obtaining OD values of the standard vaccine and the vaccine to be detected at the position of 450nm of wavelength, and calculating the concentration of the vaccine to be detected. The humanized monoclonal antibody coated enzyme label plate and the polyclonal antibody are used as primary antibodies to carry out enzyme-linked immunosorbent assay to detect the antigen content of the rabies virus vaccine, and the detection method has the advantages of short detection period, long-term storage of detection raw materials and simple and easily-obtained detection result of the titer of the vaccine.
Description
Technical Field
The invention belongs to the field of biological agents, and particularly relates to a rabies vaccine antigen content detection method and a reagent or a kit.
Background
Rabies is a zoonosis caused by rabies virus, and once the zoonosis occurs, the mortality rate is nearly 100 percent. Rabies virus Glycoprotein (GP) is a main protective antigen of rabies virus, can induce an organism to generate a neutralizing antibody, and is also related to virulence, neurophilicity, transportation, infection and distribution in nerves and brain and the like of the virus. The titer of the rabies vaccine is closely related to the glycoprotein of the virus, and the existing pharmacopoeia recommends an NIH method for in vitro detection as a standard method for evaluating the titer of the rabies vaccine.
The NIH method is to inject vaccine into mouse, attack virus with CVS, and calculate the vaccine potency after comparing with standard antigen. The NIH method requires a large number of mice and each experimental period is about one month, resulting in an extended period of rabies vaccine detection. In addition, in the experimental process, due to the participation of living bodies, the state and the performance of the mice need to be closely observed, so that the detection cost is high, and the titer of the vaccine is complicated to calculate.
Disclosure of Invention
Aiming at the problems, the invention provides a rabies vaccine antigen content detection method and a reagent or a kit.
A rabies vaccine antigen content detection method comprises the following steps:
preparing a monoclonal antibody coated ELISA plate, and immobilizing the monoclonal antibody;
diluting a vaccine to be detected and a standard vaccine respectively according to a multiple ratio gradient, coating the monoclonal antibody on an enzyme label plate, washing the plate, patting the plate dry, adding the vaccine to be detected and the standard vaccine which are diluted in different gradients into the monoclonal antibody coated enzyme label plate respectively, and incubating for 1h at 37 ℃;
washing and drying an ELISA plate containing the vaccine to be detected and the standard vaccine, adding a mouse polyclonal antibody as a primary antibody, sealing the ELISA plate containing the primary antibody, and incubating at 37 ℃ for 1-2 h;
washing and drying the enzyme label plate containing the primary antibody, adding horse radish enzyme labeled goat anti-mouse peroxide with proper dilution as a secondary antibody to obtain an enzyme label plate containing a secondary antibody, and incubating for 1 h-2 h at 37 ℃;
after washing the enzyme label plate containing the secondary antibody, adding a developing solution, reacting for a set time in a dark place at room temperature, and adding a stop solution to terminate the reaction;
and placing the enzyme label plate for terminating the reaction into an enzyme label instrument, obtaining the OD values of the standard vaccine and the vaccine to be detected at the position of 450nm of wavelength, and calculating the concentration of the vaccine to be detected.
Further, the preparation of the monoclonal antibody coated ELISA plate comprises the following steps:
diluting the monoclonal antibody, adding the diluted monoclonal antibody into an enzyme label plate, and incubating overnight at 2-8 ℃;
after the monoclonal antibody is coated on an enzyme label plate washing plate for 3 times, sealing liquid is added to seal the antibody,
and adding phosphate buffer salt solution to carry out solid phase solidification on the monoclonal antibody to prepare the monoclonal antibody coated enzyme label plate.
Further, the monoclonal antibody is a humanized monoclonal antibody aiming at the rabies virus glycoprotein epitope.
Furthermore, the addition amount of the monoclonal antibody in the monoclonal antibody coated ELISA plate is 100 mu l/hole, and the concentration of the monoclonal antibody is 1-20 mu g/ml.
Further, the blocking solution is one of bovine serum albumin, sucrose, trehalose and glucose, and the addition amount of the blocking solution is 300 mul/hole.
Further, the phosphate buffered saline solution contains sucrose and fructose.
Further, the polyclonal antibody is prepared by adopting the rabies virus derived from the mouse brain.
Further, calculating the concentration of the vaccine to be tested comprises:
obtaining OD values of the standard vaccine and the instrument to be measured at the position with the wavelength of 450nm in an enzyme-labeling instrument;
establishing a concentration-OD value standard curve according to the OD value of the standard vaccine at the wavelength of 450 nm;
and substituting the OD value of the vaccine to be detected at 450nm into the standard curve to calculate the concentration of the vaccine to be detected.
Further, the microplate reader is also provided with a wavelength of 630nm for detecting an impurity interference control value.
A rabies vaccine antigen content detection reagent or kit, the reagent or kit comprises: humanized monoclonal antibody, mouse polyclonal antibody, horseradish enzyme labeled goat anti-mouse peroxide, developing solution, stopping solution and medically acceptable auxiliary materials.
According to the invention, the humanized monoclonal antibody coated ELISA plate and the polyclonal antibody are used as primary antibodies to detect the antigen content of the rabies virus vaccine according to the steps of an enzyme-linked immunosorbent assay (ELISA method), the detection period is short, and the detection result can be rapidly obtained; in addition, the detection process does not need living body participation, the vaccine titer result can be obtained, the used detection raw materials can be stored for a long time, and the detection result of the vaccine titer is simple and easy to obtain.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a graph showing the change of OD value of each sample to be tested at different dilution concentrations in the embodiment of the present invention;
FIG. 2A shows the OD curves corresponding to different concentrations of the standard according to the embodiment of the present invention;
FIG. 2B is a graph showing the relationship between the expected value of the standard and the concentration of the standard calculated according to the standard curve;
FIG. 3 is a graph showing the GP content measured by ELISA method and the potency value dispersion measured by NIH method according to the embodiment of the present invention;
FIG. 4 is a graph showing the relationship between the results of ELISA assay and the results of NIH assay titer in the examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The rabies virus glycoprotein is the only virus protein exposed outside a virus envelope, belongs to the I-type transmembrane glycoprotein, is positioned on a rabies virus double-layer lipid membrane in the form of trimeric spinous process (with the length of 8.3nm), and plays an important role in the cell adhesion, membrane fusion and axial plasma transportation processes of the virus. The mature glycoprotein consists of 505 amino acids, divided into 3 regions: amino acids 1-439 in the outer membrane region, 440-461 in the transmembrane region, and 462-505 in the inner membrane region.
There are at least 3 major neutralizing antibody binding sites on glycoproteins: antigenic sites I-III, and in addition, some minor linear epitopes. Wherein the antigenic site I is positioned in the region of 218-240 amino acids and is a minor linear neutralizing epitope, the minimum binding region of the antigenic site I is KLCGVL (226-231 positions), and the core residue is K-CGV-. The site II is a space epitope with high conservation formed by two sites of amino acids from 34 th to 42 th and from 198 th to 200 th through a disulfide bond C35-C207, wherein the amino acids from 34 th to 42 th, 147 th, 184 th and 198 th to 200 th are important. And the position III is positioned at the amino acids 330-357, and is also a space epitope, wherein the amino acid segments 333-338 are the main binding sites of the neutralizing antibody, the a epitope positioned at the positions 342-343 is a minor neutralizing site, and the amino acids 330, 333, 336 and 338 are key residues. Antigenic sites II and III are the main virus neutralizing epitopes, and the two sites are very close in space, wherein some key residue mutations can influence the virulence, the host range or the propagation speed of the virus. Experiments show that 260-267 bits (LHDFRSDE) on glycoprotein are linear neutralizing epitopes, and an anti-rabies antibody can be induced in a mouse body independently or together with a T cell epitope, so that the virus can be effectively resisted for attack.
A humanized monoclonal antibody directed against site I or site II or site III, or a humanized monoclonal antibody directed against the combined I/II site, or combined I/III site, or combined II/III site, or combined I/II/III site is used. The monoclonal antibody can specifically bind to rabies virus active glycoprotein, and effectively avoids interference of inactive or soluble glycoprotein.
According to the specific combination of the rabies virus glycoprotein, the invention provides a rabies vaccine antigen content detection method, which comprises the following steps:
(1) preparing a monoclonal antibody coated ELISA plate, and immobilizing the monoclonal antibody.
The preparation of the monoclonal antibody coated ELISA plate comprises the following steps: diluting the monoclonal antibody to a proper concentration, adding the diluted monoclonal antibody into an enzyme label plate, and incubating overnight at 2-8 ℃;
coating an enzyme label plate with a monoclonal antibody for 3 times, and adding a confining liquid to block the antibody;
and adding phosphate buffer saline solution (PBS solution) to carry out solid phase immobilization on the monoclonal antibody to prepare the monoclonal antibody coated enzyme label plate.
The addition amount of the monoclonal antibody in the monoclonal antibody coated ELISA plate is 100 mu l/hole, and the concentration of the monoclonal antibody is 5 mu g/ml.
The confining liquid is one of bovine serum albumin, sucrose, trehalose and glucose, and the addition amount of the confining liquid is 300 mu l/hole.
When washing the plate, the amount of the washing solution added is 100-.
The monoclonal antibody used was a humanized monoclonal antibody: the humanized monoclonal antibody aiming at the rabies virus Glycoprotein (GP) epitope is adopted to coat the ELISA plate, and compared with the human immunoglobulin with complex components, the specificity is higher because the human immunoglobulin has complex components.
The PBS solution also contains 5% of sucrose and fructose, and the PBS solution containing the sucrose and the fructose is added into the closed ELISA plate to fix the sealing solution, so that the sealing effect of the sealing solution is further improved, and meanwhile, the monoclonal antibody coated ELISA plate is convenient to store after being prefabricated, and has better stability.
(2) Diluting the vaccine to be detected and the standard vaccine respectively according to a multiple ratio gradient, coating the monoclonal antibody on an enzyme label plate, washing the plate, drying the plate, washing the plate with 100-.
(3) The ELISA plate containing the vaccine to be detected and the standard vaccine is patted dry, the washing liquid is 100-: 100-1: 6400 diluted mouse polyclonal antibody is used as primary antibody, the addition amount is 100 mul/hole, the primary antibody-containing target is sealed and then incubated for 1 h-2 h at 37 ℃.
The addition amount of the vaccine to be tested and the standard vaccine is 100 mul/hole.
Wherein the mouse polyclonal antibody is prepared from rabies virus derived from mouse brain. Since polyclonal antibodies can recognize multiple epitopes on any antigen, they have high affinity: because multiple epitopes on the vaccine target protein can bind more than one antibody molecule, polyclonal antibodies can amplify low levels of glycoprotein signal;
the use of polyclonal antibodies has advantages in sensitivity and detection rate compared to monoclonal antibodies.
Polyclonal antibody preparation using murine brain-derived rabies virus: can reduce foreign protein and has high purity of rabies virus antibody. The rabies virus or antigen used for preparing the mouse polyclonal antibody uses host cells (bacteria, yeast, mammalian cells, insect cells and the like) or bovine serum, Bovine Serum Albumin (BSA), human serum albumin and the like in the production process, has complex components, can generate strong immune reaction aiming at foreign protein after being injected into a mouse, and the prepared polyclonal antibody not only has polyclonal antibody aiming at rabies virus, but also has more polyclonal antibodies aiming at foreign protein, has low purity and has poor specificity when detecting virus antigen protein. The virus used in the invention is produced by mouse brain, the impurities mainly comprise mouse brain components, the virus is homologous with the mouse, polyclonal antibodies aiming at Bovine Serum Albumin (BSA) or human serum albumin and other components cannot be generated, and the problem of poor specificity is effectively avoided.
In conclusion, the sensitivity and accuracy of the polyclonal antibody prepared from the murine encephalo-derived rabies virus during detection are fundamentally improved.
(4) The washing plate containing the primary anti-enzyme label plate is patted dry, the washing liquid is 100-.
(5) After washing the plate containing the second antibody enzyme label plate, adding 100 mu l of developing solution, reacting for a proper time in a dark place at room temperature, and adding a stop solution to stop the reaction;
(6) and placing the enzyme label plate for terminating the reaction into an enzyme label instrument, obtaining the OD values of the standard vaccine and the vaccine to be detected at the position of 450nm of wavelength, and calculating the concentration of the vaccine to be detected.
Obtaining OD values of the standard vaccine and the instrument to be measured at the position with the wavelength of 450nm in an enzyme-labeling instrument;
establishing a concentration-OD value standard curve according to the OD value of the standard vaccine at the wavelength of 450 nm;
and substituting the OD value of the vaccine to be detected at 450nm into a standard curve to calculate the concentration of the vaccine to be detected.
The microplate reader is also provided with a wavelength of 630nm, and the OD value at the wavelength of 630nm is used as an impurity interference control value.
The rabies virus glycoprotein is detected by adopting the operation steps of the in-vitro enzyme-linked immunosorbent assay (ELISA method), the operation is simple and convenient, the time consumption is short, the detection personnel do not need special training, and animals do not need to participate in the detection process to meet the welfare protection trend of the animals.
In the combination of the humanized monoclonal antibody and the mouse polyclonal antibody of the present invention, the mouse polyclonal antibody may be replaced with a polyclonal antibody against a different virus strain or a polyclonal antibody against a different species.
The combination of humanized monoclonal antibodies and mouse polyclonal antibodies employed in the present invention can be replaced by a combination of mouse or other species derived monoclonal antibodies and humanized monoclonal antibodies.
The combination of the humanized monoclonal antibody and the mouse polyclonal antibody adopted by the invention can be replaced by the combination of the mouse or other species derived monoclonal antibody and the human immunoglobulin.
The HRP or biotin-labeled monoclonal antibody can be used as a primary antibody in the combination of the invention and can be directly developed.
The invention also relates to an application of the rabies vaccine antigen content detection method, and the application can be used for preparing the rabies vaccine antigen content detection reagent or kit based on the detection method.
The invention also relates to a rabies vaccine antigen content detection reagent or kit, and the detection reagent or kit comprises: humanized monoclonal antibody, mouse polyclonal antibody, horseradish enzyme labeled goat anti-mouse peroxide, developing solution, stopping solution and auxiliary materials acceptable in the quarantine field.
The rabies vaccine antigen content detection reagent or kit can be used for conveniently and quickly detecting the antigen content.
Example rabies vaccine antigen content detection
Experimental materials and sources
The humanized monoclonal antibody is a recombinant CHO cell expression product, and the mouse polyclonal antibody is obtained by purifying a mouse after a mouse is immunized by the mouse encephalotoxin.
An enzyme label plate, purchased from Costar,
a secondary goat anti-mouse antibody labeled with HRP, purchased from Thermo,
the TMB developing solution and the stop solution m were purchased from Soblarbio,
a sample to be tested: rabies virus solution, inactivated solution, concentrated solution, rabies vaccine finished products, human serum albumin and sucrose solution which are cultured by Vero cells are all self-made by the company;
washing liquid: PBST solution, used in this example at 300 ul/well.
Fixing liquid: PBS solution containing 5% sucrose and fructose.
PBST solution and PBS solution according to "molecular cloning" specified method and steps for self-made.
1. Method for detecting sample to be detected
1) Taking a humanized monoclonal antibody with the concentration of 100 mu g/ml, adding phosphate buffer saline solution as coating solution, diluting the humanized monoclonal antibody to 5 mu g/ml by using the coating solution, adding 100 mu l/hole of the diluted monoclonal antibody into an enzyme-labeled strip, setting blank control taking only the addition of the coating solution as detection coated antibody, and standing overnight at 2-8 ℃.
2) Washing the enzyme label plate containing the coated antibody with a washing solution (PBST solution) for 3 times, performing mu patting, adding 300 mu l/hole of a closed solution, and sealing for 1 hour at room temperature; washing the plate for 3 times by using a washing solution, drying by beating, and adding a PBS (phosphate buffer solution) containing 5% of sucrose and fructose for fixation for later use;
3) and taking rabies virus solution, inactivated solution, concentrated solution, rabies vaccine finished product, human serum albumin and sucrose solution which are cultured by Vero cells, diluting in multiple proportions respectively, diluting according to gradient, adding 100 mu l/hole into a micropore plate containing the antibody, and incubating for 1h at 37 ℃.
4) Washing the plate for the washing solution for the microporous plate added with the sample to be detected for 3 times, beating to dry, and then adding 1: 100-1: the murine polyclonal antibody diluted 6400 was used as a primary antibody at 100. mu.l/well and incubated at 37 ℃ for 1 h. The incubation time can be extended to 2h, but in order to reduce the detection time, 1h is selected in this example.
5) Adding 100 mu l/well of secondary HRP enzyme labeled antibody into the microplate in the previous step, and incubating at 37 ℃ for 1h, wherein the incubation time can be prolonged to 2h, but in order to reduce the detection period, 1h is selected in the embodiment.
Then adding TMB color development solution to carry out catalytic reaction under dark condition until color development is achieved, and adding 100 mu l of stop solution (containing sulfuric acid) after color development.
6) Placing the enzyme-labeled plate in a preheated microplate reader, and reading OD values of the enzyme-labeled plate with the stopped reaction at the wavelength of 450nm and the wavelength of 630nm simultaneously.
And (3) feasibility verification of ELISA detection of a sample to be detected: the OD values of the virus harvest liquid, the virus concentrated liquid, the virus inactivated liquid and the vaccine finished product at 450nm are shown in figure 1, and the OD values of all solutions to be detected are in a gradient trend under different dilution times according to figure 1, so that the antigen protein detection can be performed by adopting an ELISA method, and the antigen proteins after different treatments can be subjected to differential reaction, which indicates that the method can be used for detecting the content of the antigen protein in a sample.
2. Establishment of a Standard Curve
Self-made standard products: the self-made standard product is prepared by inoculating the PM rabies virus inoculated cells, purifying and freeze-drying. The homemade standard is self-calibrated by using a rabies virus glycoprotein international standard (07/162) provided by WHO.
The experimental method comprises the following steps:
preparing a closed humanized monoclonal antibody coated enzyme label plate in steps 1) to 2), and diluting glycoprotein standard products according to different concentrations, wherein the final concentrations are 110, 83, 66, 55, 47 and 41mU/ml respectively. And adding standard substances with different concentrations into the single-antibody coated ELISA plate, and incubating and culturing for 1h at 37 ℃. And continuing to perform the steps 4) to 6) in the example 1), detecting the OD values of 450nm at different concentrations, repeating each concentration experiment for 6 times, and drawing a standard curve according to the concentration of the antigen standard substance and the average value of the corresponding OD values.
The experimental results are as follows: the standard curve of the antigen standard is shown in fig. 2, and fig. 2A is the OD curve corresponding to different concentrations of the standard.
FIG. 2B is a graph showing the relationship between the expected value of the standard substance and the concentration of the standard substance calculated according to the standard curve.
As shown in FIG. 2A, the concentration of the standard substance is in positive correlation with the OD value, the linear relation of the ELISA standard curve is good, and R is20.995, a linear equation of the form Y aX + b can be formed.
As can be seen from FIG. 2B, the OD value in the range of 41 to 110mIU/ml was substituted into the calibration curve to calculate the concentration of the standard, and the difference between the actual measured value of the standard and the expected value of the standard, i.e., the prepared concentration, was small, and the coefficient of variation was between 0.030 and 0.076.
Therefore, the OD value of the sample to be measured is substituted into the standard curve, so that the concentration of the sample to be measured can be calculated.
3. Calculation of concentration of sample to be measured
Selecting samples to be detected with high, medium and low concentrations, and detecting the glycoprotein content of the samples. Taking the standard substances with different concentrations as X-axis of abscissa and corresponding OD values as Y-axis of ordinate to obtain a linear regression equation: y 0.011x +0.2743, R20.9988. And substituting the OD value of the sample to be detected into an equation, and obtaining a result of the y value and multiplying the result by the dilution factor of the diluted sample to obtain the content of the sample to be detected. The results are shown in Table 1.
TABLE 1 calculation of the concentration of the samples to be tested
| Sample to be tested | Dilution factor | Average value of OD value | Glycoprotein content(mU/ml) |
| High concentration sample | Original multiple | 1.4955 | 110.02 |
| Final concentration sample | Original multiple | 1.0383 | 69.45 |
| Low concentration sample | Original multiple | 0.8070 | 48.43 |
Storage stability test
Experimental methods
Preparing the enzyme label plate which is sealed according to the steps 1) to 2) in the embodiment, and storing at the temperature of 2-8 ℃. And (3) detecting the glycoprotein by using the coated and sealed enzyme label plate on the 0 th day, the 1 st day, the 7 th day, the 10 th day, the 14 th day and the 28 th day respectively. The glycoprotein standards were diluted to 0.066IU/ml and tested for OD values at different days of storage according to steps 3) to 6) of example 1. The test result concentration was calculated using the standard curve in example 2.
The experimental method is adopted to evaluate whether the storage at the temperature of 2-8 ℃ has influence on the detection result, samples (0.066IU/ml) with known glycoprotein content are respectively subjected to experiment by enzyme label plates placed for different days, each sample is repeatedly measured for 8 times, the detection data is statistically processed, and the variation coefficient is investigated.
The experimental results are as follows:
the glycoprotein values of the samples measured on different days of storage are shown in table 2:
TABLE 2
As can be seen from Table 1, the ELISA plate prepared by the preparation method provided by the embodiment of the invention is used for detecting rabies virus glycoprotein after 28 days when stored at 2-8 ℃, and the variation coefficient is 5.92%, which indicates that the kit is good in stability.
Detection of consistency with detection results of NIH method
Experimental materials and sources:
12-14g Kunming mice, purchased from the laboratory animals center of university of medical, Anhui;
the NIH method rabies vaccine titer standard is purchased from China food and drug testing research institute.
The experimental method comprises the following steps:
according to the current edition of the Chinese pharmacopoeia method (NIH method), the test article and the reference vaccine are respectively mixed according to the ratio of 1: 25. 1: 125. 1: 625 serial dilutions, each dilution immunizing 16 Kunming mice with a weight of 12-14g, each mouse being injected with 0.5ml intraperitoneally, and 1 additional immunization at 1 week intervals. Mice were challenged with a pre-titrated CVS virus (virus titer 10) on day 14 after the 2 nd immunization3-4LD50/ml) was added to each 0.03ml for intracerebral detoxification. After 14 days of observation, the number of dead and diseased mice was counted and the results were calculated.
Reagents required for detection by an ELISA method are prepared according to the experimental method in the example 1, and rabies vaccine finished products are detected according to the experimental steps.
The ELISA method is used for detecting the freeze-dried vaccine sample, and the NIH method is used for detecting the titer of the sample in a mouse body.
The experimental results are as follows:
and (3) detecting the vaccine titer of 19 dead and diseased mice to obtain the potency value of the corresponding vaccine sample measured by an NIH method, and detecting the GP content of the vaccine sample with the same number by an ELISA method. The NIH-based potency values and the ELISA-based GP content values for the 19 vaccine samples are shown in figure 3.
As can be seen from fig. 3, for the vaccine samples with the same number, the efficacy value measured by the NIH method and the GP content value measured by the ELISA method are not completely the same, and in order to find out the relationship between the efficacy value measured by the NIH method and the GP content value measured by the ELISA method, the correlation between the ELISA method detection result and the NIH method titer result needs to be verified.
To verify the correlation between the ELISA assay results and NIH assay potency results, for vaccine samples of the same number: the relationship between the ELISA assay results and the NIH assay titer results is shown in FIG. 4.
As can be seen from fig. 4, 19 ELISA assay results and NIH assay titer results were distributed on or around a straight line, and it was found by analysis that: the Pearson value r between the ELISA and NIH titer was 0.9112, and P was 0.0006. As can be seen from the r value, the ELISA method detection result and the NIH method titer result have strong correlation, namely the ELISA method detection result can replace the NIH method titer result.
The ELISA method provided by the embodiment of the invention can accurately detect the concentration of glycoprotein in the rabies vaccine so as to obtain the titer of the rabies vaccine, the experimental process is simple and convenient, and the prepared detection raw material can be stored for a long time.
Rabies vaccine antigen content detection reagent or kit:
the detection reagent or the reagent box is respectively provided with: humanized monoclonal antibody, mouse polyclonal antibody, horseradish enzyme labeled goat anti-mouse peroxide, developing solution, stopping solution and medically acceptable auxiliary materials. Wherein, the medically acceptable auxiliary materials can be buffer solution, washing solution, sealed package box and the like.
The reagents or substances in the reagent kit are used in sequence according to the steps in the method for detecting the sample to be detected in the embodiment to obtain the OD value of the sample to be detected, and the glycoprotein content of the sample to be detected is obtained through the established standard curve.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A rabies vaccine antigen content detection method is characterized by comprising the following steps:
preparing a monoclonal antibody coated ELISA plate, and immobilizing the monoclonal antibody;
diluting a vaccine to be detected and a standard vaccine respectively according to a multiple ratio gradient, coating the monoclonal antibody on an enzyme label plate, washing the plate, patting the plate dry, adding the vaccine to be detected and the standard vaccine which are diluted in different gradients into the monoclonal antibody coated enzyme label plate respectively, and incubating for 1h at 37 ℃;
washing and drying an ELISA plate containing the vaccine to be detected and the standard vaccine, adding a mouse polyclonal antibody as a primary antibody, sealing the ELISA plate containing the primary antibody, and incubating at 37 ℃ for 1-2 h;
washing and drying the enzyme label plate containing the primary antibody, adding horse radish enzyme labeled goat anti-mouse peroxide with proper dilution as a secondary antibody to obtain an enzyme label plate containing a secondary antibody, and incubating for 1 h-2 h at 37 ℃;
after washing the enzyme label plate containing the secondary antibody, adding a developing solution, reacting for a set time in a dark place at room temperature, and adding a stop solution to terminate the reaction;
and placing the enzyme label plate for terminating the reaction into an enzyme label instrument, obtaining the OD values of the standard vaccine and the vaccine to be detected at the position of 450nm of wavelength, and calculating the concentration of the vaccine to be detected.
2. The detection method according to claim 1, wherein preparing the monoclonal antibody-coated microplate comprises:
diluting the monoclonal antibody, adding the diluted monoclonal antibody into an enzyme label plate, and incubating overnight at 2-8 ℃;
after the monoclonal antibody is coated on an enzyme label plate washing plate for 3 times, sealing liquid is added to seal the antibody,
and adding phosphate buffer salt solution to carry out solid phase solidification on the monoclonal antibody to prepare the monoclonal antibody coated enzyme label plate.
3. The method of claim 1 or 2, wherein the monoclonal antibody is a humanized monoclonal antibody directed against an epitope of rabies virus glycoprotein.
4. The detection method according to claim 1 or 2, wherein the addition amount of the monoclonal antibody in the monoclonal antibody-coated microplate is 100 μ l/well, and the concentration of the monoclonal antibody is 1-20 μ g/ml.
5. The detection method according to claim 2, wherein the blocking solution is one of bovine serum albumin, sucrose, trehalose and glucose, and the amount of the blocking solution added is 300. mu.l/well.
6. The method according to claim 2, wherein the phosphate buffered saline solution contains sucrose and fructose.
7. The method of claim 1 or 2, wherein the polyclonal antibody is prepared from murine encephalo-derived rabies virus.
8. The method of claim 1, wherein calculating the concentration of the test vaccine comprises:
obtaining OD values of the standard vaccine and the instrument to be measured at the position with the wavelength of 450nm in an enzyme-labeling instrument;
establishing a concentration-OD value standard curve according to the OD value of the standard vaccine at the wavelength of 450 nm;
and substituting the OD value of the vaccine to be detected at 450nm into the standard curve to calculate the concentration of the vaccine to be detected.
9. The detection method according to claim 7, wherein the microplate reader is further provided with a wavelength of 630nm for detecting an impurity interference control value.
10. A rabies vaccine antigen content detection reagent or kit, characterized in that the reagent or kit comprises: humanized monoclonal antibody, mouse polyclonal antibody, horseradish enzyme labeled goat anti-mouse peroxide, developing solution, stopping solution and medically acceptable auxiliary materials.
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