CN112034028B - Method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method and application thereof - Google Patents

Abstract

The invention provides a method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis and application thereof. The method comprises the following steps: and (2) feeding the water-phase sample of the foot-and-mouth disease vaccine into a capillary by adopting pressure sample injection, carrying out electrophoretic separation, detecting and recording the characteristic peak of the 146S antigen in the water-phase sample, then integrating to obtain the peak area of the characteristic peak, and then obtaining the concentration of the 146S antigen according to a quantitative standard curve. The method provided by the invention can be used for simultaneously carrying out quantitative detection on various serotype 146S antigens, can be used for detecting univalent, bivalent or more than trivalent foot-and-mouth disease vaccines, has the advantages of low sample volume, high sensitivity, high detection speed and the like, has important significance for realizing rapid spot detection of market vaccines and improving market detection efficiency, and simultaneously has great application value in the aspects of product research and development and quality supervision of multivalent foot-and-mouth disease vaccines.

Description

Method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method and application thereof

Technical Field

The invention belongs to the technical field of antigen quantitative detection, and particularly relates to a method for quantitatively detecting different serotype 146S antigens in univalent, bivalent and multivalent foot-and-mouth disease oil emulsion inactivated vaccines based on a capillary electrophoresis method.

Background

Foot-and-mouth disease (FMD) is used as a type of virulent infectious disease, has the advantages of high transmission speed, wide range and large harm, and brings huge economic loss to the livestock industry in China. At present, the foot-and-mouth disease is prevented and controlled mainly by inactivated vaccine immunization in China, and the used inactivated vaccine is mainly dual-phase oil emulsion vaccine. The effective antigen that plays the most major protective role in foot and mouth disease vaccines is its complete virus 146S. 146S contains seven serotypes, such as A type, O type, C type, south Africa type 1 (SAT type 1), south Africa type 2 (SAT type 2), south Africa type 3 (SAT type 3) and Asia type 1 (Asia type 1), and the different serotypes have no cross protection, and the subtypes of the same serotype have only partial cross immunity, so that the commercial vaccine is mostly developed by developing a multivalent vaccine to improve the immune effect and the protection range of the vaccine.

However, 146S is extremely unstable and is easily cleaved into 12S without immunological activity during production and transportation, thereby affecting the immunological effect. In order to ensure the quality of vaccine products and avoid serious economic loss caused by immune failure, the accurate determination of the 146S content in the foot-and-mouth disease inactivated virus vaccine is a great challenge for the production and quality control of the foot-and-mouth disease vaccine. Therefore, quantitative determination of 146S of different serotypes in the multivalent vaccine is the key of multivalent vaccine quality evaluation and is of great importance for improvement of the quality of the foot-and-mouth disease vaccine. At present, the foot-and-mouth disease vaccine antigen quantitative detection method mainly comprises an ELISA method, an ultracentrifugation method and a high performance liquid size exclusion chromatography. Among them, the ELISA method can distinguish different serotypes, but it is often difficult to completely distinguish 146S and the cleavage product 12S, and therefore, it is difficult to accurately measure the effective antigen amount.

CN101655452A discloses a method for detecting foot-and-mouth disease antigen 146S content by sucrose density gradient ultraviolet light quantitative method, firstly, sucrose density gradient ultracentrifugation is carried out on a virus concentrated solution to be detected, and then, an ultraviolet light spectrophotometer is used for continuously detecting OD of each fraction_259nmA value; then calculating the peak area of the OD value of the detected sample. However, the ultracentrifugation method has the disadvantages of poor accuracy and repeatability, complex operation, long time consumption and the like; meanwhile, the ultracentrifugation method cannot distinguish the 146S of different serotypes because the 146S of different serotypes have similar sedimentation coefficients.

CN104634891A discloses a method for determining foot-and-mouth disease vaccine antigen 146S by adopting high performance liquid size exclusion chromatography. The method adopts a molecular weight separation range of 2 × 10⁴～1×10⁷Da size exclusion high performance liquid chromatography column, and performing chromatographic separation on the detected sample on a high performance liquid chromatograph. And detecting an optical signal of the effluent at an outlet of the size exclusion high performance liquid chromatography column by adopting an ultraviolet detector and a laser detector, and analyzing the area of the effluent peak of the sample by a computer software system of the high performance liquid chromatography. Tong (Chinese character of 'tong')And establishing a standard curve of the absorption peak area and the 146S concentration according to the relation between the ultraviolet absorption peak of the 146S standard substance with different concentrations and the concentration of the ultraviolet absorption peak. And then carrying out chromatographic separation on the detected sample through a size exclusion high performance liquid chromatography column. Although the high performance liquid size exclusion chromatography has the advantages of high detection speed, high sensitivity, low sample volume, good repeatability and the like, the size exclusion chromatography is used for separation according to the size of an analyzed sample, and the multivalent vaccine sample has similar sizes of different serotype 146S virus particles, so that typing detection cannot be realized.

Therefore, it is important to the art to provide a method capable of realizing rapid typing of multivalent foot-and-mouth disease vaccines.

Disclosure of Invention

In view of the problems in the prior art, the invention provides a method for detecting 146S antigen in foot-and-mouth disease vaccine based on Capillary Electrophoresis (CE) and application thereof. The method has the advantages of simple operation, rapidness, accuracy, good repeatability, low sensitivity, low sample loss and the like, and can realize the rapid typing of the multivalent vaccine.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis, which comprises the following steps:

feeding a water-phase sample of the foot-and-mouth disease vaccine into a capillary by adopting pressure sampling, carrying out electrophoretic separation, detecting and recording a characteristic peak of the 146S antigen in the water-phase sample, then integrating to obtain a peak area of the characteristic peak, and obtaining the concentration of the 146S antigen according to a quantitative standard curve;

wherein the foot-and-mouth disease vaccine comprises serotype 146S antigen of any one or at least two of A type, O type, Asia1 type (Asia 1 type), C type, south Africa 1 type (SAT 1 type), south Africa 2 type (SAT 2 type) or south Africa 3 type (SAT 3 type). The 146S antigen of different serotypes has different Zeta potentials, so that when capillary electrophoresis is used, absorption peaks generated by different serotypes are different in positions.

The detection method provided by the invention can detect 146S antigens in various types of foot-and-mouth disease vaccines, including monovalent, bivalent or more than trivalent foot-and-mouth disease vaccines, by adopting a Capillary Electrophoresis method, specifically a Capillary Zone Electrophoresis (CZE) technology according to the charge heterogeneity of different serotype 146S particles, wherein the bivalent or more than trivalent foot-and-mouth disease vaccines contain different serotype 146S antigens, such as monovalent foot-and-mouth disease vaccine containing A type 146S antigen, bivalent foot-and-mouth disease vaccine detecting A type and O type, or trivalent foot-and-mouth disease vaccine detecting A type, O type and Asia1 type. The method avoids structural damage and adsorption loss of 146S in the detection process, improves the detection accuracy, has the advantages of rapidness, accuracy, good repeatability, low sample loss and the like, can realize rapid typing of the multivalent vaccine, and has important significance for realizing rapid spot inspection of the vaccine in the market and improving the market detection efficiency.

Also, by way of typical but non-limiting example, the method may also detect monovalent foot and mouth disease vaccines of type O, Asia1, C, SAT 1, SAT 2 or SAT 3; bivalent vaccines can also be detected, e.g., a combination of type a and type O, a combination of type a and type C, a combination of type a and type Asia 1; trivalent vaccines, such as combinations of type a, O, C, can also be detected; or combinations of tetravalent vaccines, e.g., type a, type O, Asia type 1 (Asia type 1), type C; or a severential vaccine comprising a combination of seven antigens of type A, type O, type Asia1, type C, type SAT 1, type SAT 2, or type AT 3.

In the capillary electrophoresis detection process, conditions such as buffer salt type, salt concentration, pH, additives and the like of an electrophoresis buffer solution can influence the migration time, signal intensity, separation degree and the like of a sample to be detected; meanwhile, the sample introduction volume, the type of the detector, the detection wavelength and the like directly influence the signal intensity, the peak shape and the like of the detection. Therefore, in order to make the migration time of different serotype virus particles obviously different and realize the separation of different serotype antigens, the parameters of the capillary electrophoresis are selected in the invention.

Preferably, the electrophoresis buffer comprises any one of or a combination of at least two of phosphate buffer, Tris-HCl buffer or HEPES buffer, preferably phosphate buffer.

Preferably, the phosphate buffer has a molar concentration of 10 to 150mM, and may be, for example, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, or 140mM, and preferably 10 to 50 mM.

Preferably, the pH of the electrophoresis buffer is 7.5 to 9.0, for example, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9, and preferably 8.0. On one hand, the pH of the electrophoresis buffer can influence the stability of 146S, and 146S is extremely unstable and can be cracked in a solution environment with the pH of less than 7.0; on the other hand, pH also affects the migration rate of electrophoresis and thus the degree of separation between different serotype virions.

As a preferable technical scheme of the invention, the electrophoresis buffer solution also comprises an additive.

Preferably, the additive comprises any one of tween 20, glycerol, PEG6000, sodium deoxycholate or sodium lauryl sulfate or a combination of at least two of the same.

Preferably, the additives include tween 20, glycerol and PEG 6000.

Preferably, the mass fraction of tween 20 is 0.01 to 0.3%, and may be, for example, 0.02%, 0.05%, 0.1%, 0.2%, or 0.3%.

Preferably, the volume fraction of glycerol is 0.5-5%, such as 0.6%, 0.8%, 1%, 1.2%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5%.

Preferably, the mass fraction of the PEG6000 is 0.05 to 0.1%, and may be, for example, 0.06%, 0.07%, 0.08%, 0.09%, or the like.

In a preferred embodiment of the present invention, the voltage during the electrophoretic separation is 10 to 50kV, for example, 12kV, 15kV, 18kV, 20kV, 22kV, 24kV, 25kV, 28kV, 30kV, 32kV, 35kV, 40kV or 45kV, preferably 15 to 30kV, and more preferably 20 kV. The separation voltage directly affects the separation degree and detection efficiency of the sample. The high-voltage separation is short in time and high in efficiency, but the separation degree is reduced; low voltage detection can improve the detection separation degree, but is accompanied by peak broadening; meanwhile, the separation voltage may affect the structure of the sample, the stability of the baseline, and the like, and further affect the detection result.

Preferably, the time of the electrophoretic separation is 10-30 min, for example, 12min, 14min, 15min, 16min, 18min, 20min, 22min, 25min, 26min or 28 min.

Preferably, the temperature of the capillary column during the electrophoretic separation is 10 to 30 ℃, and may be, for example, 12 ℃, 14 ℃, 15 ℃, 16 ℃, 18 ℃, 20 ℃, 22 ℃, 25 ℃, 26 ℃ or 28 ℃.

The pressure adopted during the pressure sample injection is 0.5-3.0 psi, such as 0.6psi, 0.8psi, 1psi, 1.2psi, 1.5psi, 1.8psi, 2psi, 2.4psi, 2.5psi, 2.6psi or 2.8 psi; the time is 5 to 25s, and may be, for example, 6s, 8s, 10s, 12s, 15s, 16s, 18s, 20s, 22s, 23s, or 24 s. The sample feeding amount of capillary electrophoresis is influenced by the sample feeding pressure and the sample feeding time. The larger the pressure and the longer the time are during sample injection, the larger the sample injection volume is. The larger the sample injection amount is, the stronger the signal is, but the broadening of the peak shape is increased, and simultaneously, the separation buffer solution system has large change and the baseline is easier to drift; however, insufficient sample introduction amount can lead to weak detection signal, which is not favorable for low concentration sample detection. Preferably, the pressure adopted during the pressure sample injection is 0.5-2.0 psi and the time is 5-15 s.

Preferably, the capillary comprises a fused silica capillary.

Preferably, the inner diameter of the capillary is 25 to 75 μm, and may be, for example, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, or 70 μm.

Preferably, the capillary has a length of 20 to 80cm, for example, 25cm, 30cm, 35cm, 40cm, 45cm, 50cm, 55cm, 60cm, 65cm, 70cm, or 75 cm.

In a preferred embodiment of the present invention, the detector used for the detection includes a laser induced fluorescence detector (LIF) and/or an ultraviolet detector.

Preferably, the ultraviolet detection wavelength is 205 to 280nm, such as 208nm, 210nm, 212nm, 214nm, 220nm, 225nm, 228nm, 230nm, 235nm, 240nm, 245nm, 250nm, 255nm, 260nm, 265nm or 270nm, preferably 214 to 259nm when detected by an ultraviolet detector.

As a preferable technical scheme of the invention, before pressure sample injection, the method also comprises the operation of demulsification of the foot-and-mouth disease vaccine. The demulsification operation aims at the finished product or semi-finished product of the foot-and-mouth disease vaccine, and an adjuvant added in the preparation process of the vaccine.

Preferably, the method of demulsifying comprises: and adding a demulsifier into the foot-and-mouth disease vaccine, mixing, centrifuging, and removing an oil phase to obtain a water phase sample of the foot-and-mouth disease vaccine.

Preferably, the demulsifier comprises n-pentanol.

Preferably, the volume ratio of the n-pentanol to the foot-and-mouth disease vaccine is 1 (5-12), and the volume ratio may be 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11 or 1:11.5, and the like, and is preferably 1 (8-10).

As a preferable technical scheme of the invention, the method further comprises the operation of pretreating the foot-and-mouth disease vaccine before pressure sample injection. The pretreatment operation aims at crude vaccine which is not purified, and the crude vaccine is low in antigen content, contains more impurities and is not beneficial to detection.

Preferably, the method of purification comprises any one of ultracentrifugation, ultrafiltration or PEG precipitation or a combination of at least two.

The purification method can adopt the following three modes:

(1) taking a virus culture solution before purification, ultracentrifuging, removing a supernatant, redissolving by using a sodium phosphate buffer solution (pH8.0), and then carrying out CZE detection;

(2) and (3) taking the virus culture solution before purification, centrifuging, transferring the supernatant into an ultrafiltration concentration tube with the molecular weight cutoff of 100KD, concentrating under the centrifugal condition, and carrying out CZE detection.

(3) Adding 10% of PEG6000 into the virus culture solution before purification, standing, centrifuging, removing the supernatant, re-dissolving with sodium phosphate buffer solution (pH8.0), and performing CZE detection.

As a preferred technical scheme of the invention, the method comprises the following steps:

(1) preparation of aqueous phase samples of foot and mouth disease vaccine: adding n-amyl alcohol into the foot-and-mouth disease vaccine, wherein the volume ratio of the n-amyl alcohol to the foot-and-mouth disease vaccine is 1 (8-10), mixing, centrifuging, and removing an oil phase to obtain a water phase sample of the foot-and-mouth disease vaccine;

(2) enabling a water phase sample of the foot-and-mouth disease vaccine to enter a capillary tube with the sample introduction time of 5-15 s and the sample introduction pressure of 0.5-2.0 psi, wherein the capillary tube is a fused quartz capillary tube, the inner diameter of the capillary tube is 25-75 mu m, and the length of the capillary tube is 20-80 cm;

(3) carrying out electrophoretic separation at a voltage of 15-30 kV for 10-30 min, wherein the temperature of a capillary column is 10-30 ℃;

the electrophoresis buffer solution used in the electrophoresis separation is 10-50 mM phosphate buffer solution with pH of 7.5-9.0, and the phosphate buffer solution further comprises Tween 20 with the mass fraction of 0.01-0.3%, glycerol with the volume fraction of 0.5-5% and PEG6000 with the mass fraction of 0.05-0.1%;

(4) and detecting and recording characteristic peaks by using a laser-induced fluorescence detector and/or an ultraviolet detector, then integrating to obtain peak areas of the characteristic peaks, and then obtaining the concentrations of the different serotype 146S antigens according to a quantitative standard curve.

In a second aspect, the present invention also provides the use of a method as described in the first aspect in the product development or quality monitoring of a foot and mouth disease vaccine.

The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) the detection method provided by the invention can be used for quantitative detection of the foot-and-mouth disease virus 146S antigens of different serotypes in the univalent, bivalent or more than trivalent foot-and-mouth disease vaccine; according to the method, by analyzing the charge heterogeneity of different serotype 146S particles, a capillary zone electrophoresis technology is adopted, the migration time of different serotype virus particles is obviously different by optimizing detection conditions, the separation of different serotype antigens is realized, meanwhile, the structural damage and adsorption loss of 146S in the detection process are avoided, and the detection accuracy is improved; the aim of quantitative detection of virus particles is achieved by establishing a standard curve of the peak area of an antigen ultraviolet absorption peak or the peak area of a fluorescence signal and the concentration of 146S;

(2) the method provided by the invention solves the problem that the prior art is difficult to quantitatively detect multiple serotype 146S complete virus particles simultaneously, has the advantages of simplicity in operation, rapidness, accuracy, good repeatability, low sensitivity, low sample loss and the like, can realize rapid typing of multivalent vaccines, has important significance for realizing rapid spot inspection of market vaccines and improving market detection efficiency, and provides reference for establishing a rapid and efficient quality evaluation method of the foot-and-mouth disease oil emulsion inactivated vaccines.

Drawings

FIG. 1 is an electrophoretic image of a bivalent vaccine against the type A and type O146S antigens of example 3.

FIG. 2 is a graph of the linear relationship between the absorption peak area and the concentration of the type A pure 146S antigen in example 4.

FIG. 3 is a graph of the linear relationship between the absorption peak area and the concentration of O-type pure 146S antigen in example 4.

FIG. 4 is a graph of the linear dependence of the absorption peak area of the pure 146S antigen form Asia1 on concentration in example 4.

Detailed Description

The technical solutions of the present invention are further described in the following embodiments with reference to the drawings, but the following examples are only simple examples of the present invention and do not represent or limit the scope of the present invention, which is defined by the claims.

Unless otherwise specified, the experimental procedures used in the following examples were carried out by those skilled in the art. In the following examples, the reagent materials used are available from manufacturers conventional in the art unless otherwise specified.

Example 1

The embodiment provides a method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis, which comprises the following specific steps:

(1) preparation of a sample injection sample:

a. preparing a water phase sample of the finished foot-and-mouth disease vaccine:

because the adjuvant is added into the vaccine finished product, demulsification is required to be carried out before measurement; the demulsification method comprises the following steps: adding n-amyl alcohol according to the volume of a vaccine stock solution, wherein the volume ratio of the stock solution to the n-amyl alcohol is 9:1, uniformly mixing, standing for 1h at 4 ℃, centrifuging for 2min at 5000rpm, and collecting a bottom layer solution containing an antigen as a water phase sample for sample injection;

b. preparing a water phase sample of the semi-finished foot-and-mouth disease vaccine:

the semi-finished aftosa vaccine is not added with an adjuvant, and can be directly sampled to be used as a sample injection sample;

(2) electrophoresis: the sample was pumped into a capillary electrophoresis system using a pressure of 1psi for 10 seconds, the electrophoresis buffer was 20mM phosphate buffer (pH8.0) containing 0.2% Tween 20, 0.4% glycerol and 0.2% PEG6000, and the 146S peak absorption area at 214nm was detected at a separation voltage of 20 kV.

Wherein the capillary used is a fused silica capillary; the pipe diameter is 50 mu m; the length is 60 cm.

Example 2

This example serves to demonstrate the feasibility of using capillary zone electrophoresis to detect different serotype 146S antigens. The method comprises the following steps of carrying out charge heterogeneity analysis on different serotype antigens:

pure A-type, O-type and Asia 1-type foot-and-mouth disease 146S products with the concentration of 100 mu g/mL are taken, dynamic light scattering is utilized to detect the particle size and the Zeta potential of three different types of serotype viruses 146S, three parallel tests are respectively carried out, and the determination results are shown in Table 1.

TABLE 1

Serotype	Particle size (nm, n ═ 3)	Zeta potential (mV, n ═ 3)
			Type A	27.3±0.2	-5.2±0.3
O type	26.9±0.3	-10.8±0.5
			Asia1 type	27.1±0.2	-13.9±0.5

The results in the table show that the particle sizes of the serotype viruses 146S of different types are similar and are about 27nm, but the Zeta potential difference is large, which indicates that the serotype typing of the three serotypes 146S by capillary electrophoresis has theoretical feasibility.

Similarly, although the Zeta potentials of the 146S antigens of type C, type south africa 1, type south africa 2 and type south africa 3 were not detected in this example, the several different antigens described above also have different amino acid sequences compared to type a, type O and type asia1, and therefore, the Zeta potentials of type C, type south africa 1, type south africa 2 and type south africa 3 are also greatly different. Therefore, the method provided by the invention can also be used for detecting the foot-and-mouth disease vaccine containing the 146S antigen of C type, south Africa type 1, south Africa type 2 and south Africa type 3.

Example 3

The purpose of this example was to investigate the effect of background buffer salt systems, detection conditions, and additives on the results of capillary electrophoresis.

(1) Influence of background buffer salt type on detection results

1. A buffer system: selecting three different types of buffer systems, namely 20mM phosphate buffer solution, 20mM Tris-HCl buffer solution and 20mM HEPES buffer solution, and inspecting the influence of different buffer system types on the detection of the three serotypes 146S by taking the separation degree and the characteristic peak height as parameters.

2. pH of the buffer: the pH investigation range is 7.5-9.0, and the pH investigation range is specifically set to be 7.5, 8.0, 8.5 and 9.0; the setting reason is as follows: the pH can influence the stability of 146S, and 146S is extremely unstable and cracks in a solution environment with the pH being less than 7.0; meanwhile, pH also affects migration rate of electrophoresis, and further affects the separation degree among different serotype virus particles.

The A, O bivalent vaccine was used as a research object to examine the separation degree and peak height (reflecting signal intensity) of two kinds of virus particles under different electrophoresis buffers, and the specific results are shown in table 2:

TABLE 2

As can be seen from Table 2, the phosphate buffer solution at pH8.0 had the best signal intensity and resolution for detection, which were 7.02mAU and 7.37mAU, respectively, and the A and O resolution was 1.10. The reason for the lower peak height of detection due to too high or too low pH in other buffers of the same type may be analyzed as follows: weak lysis of 146S in the hostile solution environment resulted in a decrease in the detected 146S content.

(2) Influence of different detection conditions on detection results

1. Detection wavelength: different detection wavelengths have different signal intensities, and the peak heights at the wavelengths of 214nm, 254nm, 259nm and 280nm are respectively considered in the embodiment;

the A, O bivalent vaccine is used as a research object, and under the same sample and detection conditions, the detection signal at 214nm is strongest, and is respectively 3.4 times, 3.6 times and 5 times of the peak height under the conditions of 254nm, 259nm and 280 nm.

2. Separation voltage: under the conditions of sample introduction of 0.5psi 10s and detection wavelength of 214nm, respectively selecting four detection voltages of 15kV, 20kV, 25kV and 30kV in 20mM phosphate buffer solution (pH8.0) for detection, and inspecting the separation degree, theoretical plate number and peak height under different voltages;

a, O bivalent vaccine was used as the subject, and the test results are shown in Table 3.

TABLE 3

As can be seen from the above table, when the detection voltage is 20kV, the three parameters of the separation degree, the theoretical plate number and the signal intensity of the detection result are relatively optimal.

3. Sample introduction amount: the influence of the sample amount was examined under the conditions that the detection wavelength was 214nm, the separation voltage was 20kV, and 20mM phosphate buffer (pH8.0) was used as the electrophoresis buffer;

wherein, the pressure is selected to be 0.5psi and 1psi, the sample injection time is 5s, 10s and 15s, A, O bivalent vaccine is taken as a research object, and the result is shown in Table 4.

TABLE 4

As can be seen from the above table, the signal intensity and the theoretical plate number obtained by comprehensive comparison of the detection result show that the sample injection amount achieved under the sample injection condition of 1psi 10s has the optimal effect during the detection.

(3) Effect of different additives on the test results

1. Additives, including SDS, Tween 20, PEG6000 and glycerol, were added to the electrophoresis buffer to study the effect of the additives on the assay results. The absorption peak at 214nm of A, O, Asia1 trivalent vaccine was detected under the conditions of 0.5psi 10s injection, 20mM phosphate buffer (pH8.0) electrophoresis buffer, and 20kV voltage, and the specific results are shown in Table 5.

TABLE 5

As can be seen from the above table, 0.1-0.5 mM SDS can improve the repeatability of migration time, but too high concentration can cause the lysis of 146S antigen; tween 20, glycerol and PEG6000 can obviously reduce the relative deviation of characteristic peak areas and improve the peak area repeatability.

2. Under the same other detection conditions, 0.2% of Tween 20, 0.4% of glycerol and 0.2% of PEG6000 are simultaneously added into the electrophoresis buffer solution to detect A, O bivalent vaccine, and the effects of several additives on improving the repeatability and the separation degree of CZE detection are examined.

TABLE 6

After tween, glycerol and PEG6000 are added into the electrophoresis buffer solution, a CZE detection spectrum of the A, O bivalent sample is shown in figure 1, and the characteristic peaks of the A-type antigen and the O-type antigen can be obviously distinguished, wherein the peak-off time of the O-type antigen is 7.7 min; the peak time of the type A antigen was 11.3 min.

As can be seen from Table 6, the separation degree and the repeatability of 146S can be remarkably improved by the background solution containing 0.2% of Tween 20, 0.4% of glycerol and 0.2% of PEG6000, the separation degree is more than 1.5, the peak area and the migration time relative deviation are within 3%, and the requirements of serotype typing and quantification are met.

Example 4

In this example, a 146S antigen quantification standard curve was plotted using the method provided in example 1.

(1) Firstly, preparing a pure 146S antigen:

sequentially using ammonium sulfate and sodium hydroxide solution to respectively adjust the foot-and-mouth disease inactivated virus culture solution to the conductivity of 120mS/cm and the pH value of 8.0;

feeding to a Butyl Sepharose 4FF hydrophobic chromatography column previously equilibrated with ammonium sulfate to a sodium phosphate buffer (pH8.0) of conductivity 100 mS/cm; after feeding, continuously washing, eluting by using a 5mS/cm sodium phosphate buffer solution (pH8.0) and collecting an elution peak;

6mL of the hydrophobic chromatography eluate was fed to a Superdex 200 gel filtration column (GE Healthcare, 60 cm. times.1.6 cm I.D.) with a mobile phase of 20mM sodium phosphate buffer (pH8.0) containing 0.15M sodium chloride;

according to the collection of 146S by the ultraviolet signal, the purity of 146S obtained by detection is over 99 percent, and the method can be used for preparing a standard curve.

(2) The detection method of the CZE detection is as follows:

for 146S antigen pure products with different concentrations, a sample is pumped into a capillary electrophoresis system by adopting the pressure of 1psi for 10S, the electrophoresis buffer solution is 20mM phosphate buffer solution (pH8.0) containing 0.2% of Tween 20 by mass fraction, 0.4% of glycerol by volume fraction and 0.2% of PEG6000 by mass fraction, electrophoresis is carried out under the voltage of 20kV, the detection wavelength is 214nm, and the absorption peak of 146S is recorded and integrated to obtain the peak area.

And (3) detection results:

1. standard curve for type a 146S antigen:

respectively carrying out gradient dilution on the pure A-type 146S antigen with the concentration of 400 mu g/mL by using electrolyte solution; pumping the sample into a capillary electrophoresis system, and detecting the 146S absorption peak area at 214 nm;

as shown in fig. 2, the absorption peaks of the antigens at different concentrations are integrated to obtain peak areas, and the peak areas are used as abscissa and the concentration of the 146S pure product is used as ordinate to obtain a linear response curve:

C＝0.0032PA；

wherein C is the concentration of 146S in the unit of mug/mL, PA is the peak area of the absorption peak in the unit of mAU multiplied by S, and R of the obtained standard curve²＝0.9981。

2. Standard curve for O-type 146S antigen:

respectively carrying out gradient dilution on the pure O-type 146S antigen with the concentration of 350 mu g/mL by using a background electrolyte solution; the sample was pumped into a capillary electrophoresis system and the 146S absorption peak area at 214nm was detected.

As shown in fig. 3, the absorption peaks of the antigens at different concentrations are integrated to obtain peak areas, and the peak areas are used as abscissa and the concentration of the 146S pure product is used as ordinate to obtain a linear response curve:

C＝0.0025PA；

wherein C is the concentration of 146S in the unit of mug/mL, PA is the peak area of the absorption peak in the unit of mAU multiplied by S, and R of the obtained standard curve²＝0.9988。

3. Standard curve for Asia1 type 146S antigen:

respectively carrying out gradient dilution on the pure Asia1 type 146S antigen with the concentration of 400 mu g/mL by using a background electrolyte solution; the sample was pumped into a capillary electrophoresis system and the 146S absorption peak area at 214nm was detected.

As shown in fig. 4, the absorption peaks of the antigens at different concentrations are integrated to obtain peak areas, and the peak areas are used as abscissa and the concentration of the 146S pure product is used as ordinate to obtain a linear response curve:

C＝0.0035PA；

wherein C is the concentration of 146S in the unit of mug/mL, PA is the peak area of the absorption peak in the unit of mAU multiplied by S, and R of the obtained standard curve²＝0.9905。

The quantitative standard curves of the three serotype virus particles show that the method provided by the invention has higher accuracy and repeatability.

Example 5

In the embodiment, capillary zone electrophoresis is adopted to determine the content of the 146S antigen in the finished products of the foot-and-mouth disease vaccines of different serotypes.

Selecting finished products of the foot-and-mouth disease oil emulsion inactivated vaccines of 3 different types in 4 enterprises, measuring 12 vaccines in total, numbering the vaccines according to different types, and respectively recording the number as:

a represents the O-type inactivated vaccine of the pig foot-and-mouth disease;

b represents bivalent inactivated vaccine of O type and A type of foot-and-mouth disease;

c represents trivalent inactivated vaccine of O type, A type and Asia1 type of foot and mouth disease.

(1) Pretreatment of vaccine finished products: because the adjuvant is added into the vaccine finished product, demulsification is required before measurement;

the demulsification method comprises the following steps: adding n-amyl alcohol according to the volume of the vaccine stock solution, wherein the volume ratio of the stock solution to the n-amyl alcohol is 9:1, uniformly mixing, standing for 1h at 4 ℃, centrifuging for 2min at 5000rpm, and collecting a bottom layer solution containing the antigen;

(2) antigen detection: the detection of the bottom layer 146S solution was performed according to the capillary electrophoresis conditions provided in example 1, and after the peak area was obtained by integration, the 146S concentration was obtained according to the corresponding standard curve in example 4, and the measurement was repeated three times, and the content measurement results are shown in table 7.

TABLE 7

As can be seen from Table 7, the CZE is suitable for the typing and quantitative detection of antigens in univalent, bivalent and multivalent foot-and-mouth disease vaccines, and the detection repeatability is good.

Example 6

This example is used to compare the differences between the high performance size exclusion chromatography and the capillary electrophoresis in detecting the inactivated vaccine of oil emulsion of foot-and-mouth disease.

(1) Standard Curve for High Performance Size Exclusion Chromatography (HPSEC)

The high performance liquid chromatography system is purchased from Agilent company, and the high performance liquid gel filtration column TSK G4000SWXL is purchased from Tosohaas company;

the 146S pure antigen was subjected to gradient dilution by the method described in CN104634891A to prepare a standard curve for high performance liquid chromatography quantitative determination of 146S:

C＝0.0158PA；

wherein C is the concentration of 146S antigen in the unit of mug/mL, PA is the peak area of an absorption peak in the unit of mAU multiplied by S, and R of the obtained standard curve²＝0.9991。

(2) Standard curve for capillary electrophoresis:

the standard curve for the 146S antigen was determined according to the capillary electrophoresis conditions described in example 4.

(3) Selecting three different types of foot-and-mouth disease oil emulsion inactivated vaccine finished products of a certain manufacturer for determination,

wherein:

a represents the O-type inactivated vaccine of the pig foot-and-mouth disease;

b represents bivalent inactivated vaccine of O type and A type of foot-and-mouth disease;

c represents the trivalent inactivated vaccine of foot-and-mouth disease type O, type A and Asia 1.

(4) Pretreatment of vaccine finished products:

emulsion breaking is needed before measurement because emulsion adjuvant is added into a vaccine finished product; the demulsification method comprises the following steps: adding n-amyl alcohol according to the volume of the vaccine stock solution, wherein the volume ratio of the stock solution to the n-amyl alcohol is 9:1, uniformly mixing, standing for 1h at 4 ℃, centrifuging for 2min at 5000rpm, and collecting a bottom layer solution containing the antigen;

(5) antigen detection:

the antigen phase obtained after pretreatment is quantitatively detected by using two methods of high performance liquid chromatography and capillary electrophoresis, the detection is repeated three times and the detection effect is compared, and the detection result is shown in table 8.

TABLE 8

As can be seen from the above table, HPSEC and CZE have good reproducibility in the detection of the total content of the finished vaccine 146S, and although there is a numerical difference between the results of the two detection methods when determining the total content of 146S, the difference is within the allowable range.

CZE represents a significant advantage over HPSEC in terms of good reproducibility in serotype typing and quantification of multivalent vaccines.

Example 7

The 146S antigen was quantitatively detected in this example in combination with a laser-induced fluorescence detector.

(1) Detection method of CZE detection:

the sample was pumped into a capillary electrophoresis system at 1psi for 10s in 20mM phosphate buffer (pH8.0) and detected at 20kV separation voltage.

(2) Standard curve for type a 146S antigen:

respectively carrying out gradient dilution on the 146S antigen pure product with the concentration of 400 mug/mL by using an electrophoresis buffer solution;

after mixing FITC (fluorescein isothiocyanate) and 146S, the mixture was incubated at 4 ℃ overnight with shaking for 12 hours. The sample was pumped into a capillary electrophoresis system and the fluorescence signal of FMDV signature peaks was detected using a laser induced fluorescence detector (LIF) at 20kV separation voltage.

Integrating the fluorescence signals of the antigens with different concentrations to obtain peak areas, and taking the peak areas as abscissa and the concentration of the 146S pure product as ordinate to obtain a linear response curve:

C＝4.5×10^-6PA

wherein C is the concentration of 146S in μ g/mL, PA is the peak area of 146S in mAU × S, and R of the obtained standard curve²＝0.9972。

(2) Standard curve for O-type 146S antigen:

respectively carrying out gradient dilution on the 146S antigen pure product with the concentration of 350 mu g/mL by using a background electrolyte solution; after FITC and FMDV are mixed uniformly, shaking and incubating for 12h at 4 ℃ overnight; and pumping the sample into a capillary electrophoresis system, and detecting the fluorescence signal of the FMDV characteristic peak by LIF under the separation voltage of 20 kV.

Integrating the fluorescence signals of the antigens with different concentrations to obtain peak areas, and taking the peak areas as abscissa and the concentration of the 146S pure product as ordinate to obtain a linear response curve:

C＝3.9×10^-6PA

wherein C is the concentration of 146S in μ g/mL, PA is the peak area of 146S in mAU × S, and R of the obtained standard curve²＝0.9956。

(3) Standard curve for Asia1 type 146S antigen:

respectively carrying out gradient dilution on the 146S antigen pure product with the concentration of 400 mu g/mL by using a background electrolyte solution; after FITC and FMDV are mixed uniformly, shaking and incubating for 12h at 4 ℃ overnight; and pumping the sample into a capillary electrophoresis system, and detecting the fluorescence signal of the FMDV characteristic peak by LIF under the separation voltage of 20 kV.

Integrating the fluorescence signals of the antigens with different concentrations to obtain peak areas, and taking the peak areas as abscissa and the concentration of the 146S pure product as ordinate to obtain a linear response curve:

C＝4.8×10^-6PA

wherein C is the concentration of 146S in μ g/mL, PA is the peak area of 146S in mAU × S, and R of the obtained standard curve²＝0.9933。

The quantitative standard curves of the three serotype virus particles show that the method has higher accuracy and repeatability.

Example 8

In this example, quantitative detection of HPSEC and CZE was carried out on the foot-and-mouth disease vaccine containing nucleic acid impurities.

Because a part of vaccine water phase contains a large amount of nucleic acid macromolecular impurities, the detection process can be interfered. In order to remove the influence of the impurities, nuclease digestion treatment is additionally added, and the total antigen concentration before and after the treatment is compared to obtain a quantitative result.

Selecting three different types of foot-and-mouth disease oil emulsion inactivated vaccine finished products. Wherein a represents the O-type inactivated vaccine of the pig foot-and-mouth disease; b represents bivalent inactivated vaccine of O type and A type of foot-and-mouth disease; c represents trivalent inactivated vaccine of O type, A type and Asia I type of foot and mouth disease. HPSEC and CZE quantitative determinations were performed separately.

The demulsification operation described in example 6 was followed to obtain a 146S-containing aqueous phase.

The enzyme digestion treatment method comprises the following steps: 200 μ L of the antigen aqueous phase solution was added with 500U/mL nuclease (Benzonase), reacted at 25 ℃ for 1 hour, and then subjected to HPSEC and CZE detection, the detection results of which are shown in Table 9.

TABLE 9

As can be seen from Table 9, nucleic acid impurities had no significant effect on the CZE assay, and detection of aqueous phase antigens was achieved without the need for enzyme treatment. However, HPSEC is interfered by nucleic acids and cannot be used for direct antigen quantification.

Example 9

In this example, the CZE assay was performed on low concentration samples or samples to be purified.

Since the vaccine may contain a large amount of impurities such as proteins and nucleic acids released during cell culture before purification, and the concentration is too low for detection, a pretreatment step is added in the present embodiment, and the CZE detection method described in example 1 is used for detection.

The pretreatment method is divided into three types, and specifically comprises the following steps:

the pretreatment method comprises the following steps:

10mL of the virus culture medium before purification was subjected to ultracentrifugation at 30000rpm for 4 hours, and the supernatant was discarded, and then reconstituted with 200mM sodium phosphate buffer (pH8.0) to 2.5mL for CZE assay.

The concentration of 146S before purification was 7.2. mu.g/mL.

And a second pretreatment method:

respectively taking 500 mu L of virus culture solution before purification, centrifuging at 10000rpm/min for 5min, transferring the supernatant into an ultrafiltration concentration tube with the molecular weight cutoff of 100KD, concentrating to 100 mu L under the centrifugation condition of 6000rpm/min, and then carrying out CZE detection.

The concentration of 146S before purification was 7.5. mu.g/mL.

And a third pretreatment method:

respectively taking 500 mu L of virus culture solution before purification, adding 10% of PEG6000 by mass fraction, standing for 10h, centrifuging at 8000rpm/min for 20min, discarding supernatant, re-dissolving to 100 mu L with 200mmol/L sodium phosphate buffer solution (pH8.0), and performing CZE detection.

The concentration of 146S before purification was 6.8. mu.g/mL.

Since the vaccine may have a large amount of impurities such as protein and nucleic acid released during cell culture before purification, and the detection is not facilitated due to too low concentration, the application range of the detection can be improved by adding the pretreatment step. Therefore, the method of the invention can not only measure the content of the 146S antigen in the purified vaccine, but also measure the content of the 146S antigen in an impure or low-concentration vaccine intermediate product.

In conclusion, the method provided by the invention establishes a standard curve of the peak area of an antigen ultraviolet absorption peak or the peak area of a fluorescence signal and the concentration of 146S by using the capillary zone electrophoresis technology through the charge heterogeneity of different serotype 146S particles, realizes the quantitative detection of virus particles, has the advantages of simple operation, rapidness, accuracy, good repeatability, low sensitivity, low sample loss and the like, and can realize the rapid typing of multivalent vaccines.

The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are within the scope and disclosure of the present invention.

Claims (21)

1. A method for detecting 146S antigen in foot-and-mouth disease vaccine based on capillary electrophoresis method is characterized by comprising the following steps:

feeding a water-phase sample of the foot-and-mouth disease vaccine into a capillary by adopting pressure sampling, carrying out electrophoretic separation, detecting and recording a characteristic peak of the 146S antigen in the water-phase sample, then integrating to obtain a peak area of the characteristic peak, and obtaining the concentration of the 146S antigen according to a quantitative standard curve;

the foot-and-mouth disease vaccine comprises serotype 146S antigens of any one or at least two of A type, O type, Asia1 type, C type, south Africa 1 type, south Africa 2 type or south Africa 3 type;

the capillary comprises a fused silica capillary;

in the step of electrophoretic separation, the separation voltage is 10-50 kV, the time is 10-30 min, and the column temperature of the capillary is 10-30 ℃;

the electrophoresis separation step adopts an electrophoresis buffer solution with the molar concentration of 10-150 mM and the pH of 7.5-9.0, and the electrophoresis buffer solution also comprises Tween 20 with the mass fraction of 0.01-0.3%, glycerol with the volume fraction of 0.5-5% and PEG6000 with the mass fraction of 0.05-0.1%.

2. The method according to claim 1, wherein the phosphate buffer solution has a molarity of 10 to 50 mM.

3. The method of claim 1, wherein the electrophoresis buffer has a pH of 8.0.

4. The method according to claim 1, wherein the voltage for the electrophoretic separation is 15 to 30 kV.

5. The method according to claim 4, wherein the voltage for the electrophoretic separation is 20 kV.

6. The method according to claim 1, wherein the pressure used for the sample injection is 0.5-3.0 psi for 5-25 s.

7. The method according to claim 6, wherein the pressure used for the sample injection is 0.5-2.0 psi for 5-15 s.

8. The method of claim 1, wherein the capillary has an inner diameter of 25 to 75 μm.

9. The method of claim 1, wherein the capillary has a length of 20 to 80 cm.

10. The method of claim 1, wherein the detector used in the detecting comprises a laser-induced fluorescence detector and/or an ultraviolet detector.

11. The method of claim 10, wherein the ultraviolet detection wavelength is 205-280 nm when the ultraviolet detector is used for detection.

12. The method of claim 11, wherein the ultraviolet detection wavelength is 214-259 nm.

13. The method of claim 1, wherein before the pressure injection, the method further comprises the step of demulsifying the foot-and-mouth disease vaccine.

14. The method of claim 13, wherein the method of demulsifying comprises: and adding a demulsifier into the foot-and-mouth disease vaccine, mixing, centrifuging, and removing an oil phase to obtain a water phase sample of the foot-and-mouth disease vaccine.

15. The method of claim 14, wherein the demulsifier comprises n-pentanol.

16. The method according to claim 15, wherein the volume ratio of n-pentanol to aftosa vaccine is 1 (5-12).

17. The method according to claim 16, wherein the volume ratio of n-pentanol to aftosa vaccine is 1 (8-10).

18. The method of claim 1, wherein the pressure injection step further comprises a step of pre-treating the aftosa vaccine.

19. The method of claim 18, wherein the method of pretreatment comprises any one or a combination of at least two of ultracentrifugation, ultrafiltration, or PEG precipitation.

20. Method according to claim 1, characterized in that it comprises the following steps:

(1) preparation of aqueous phase samples of foot and mouth disease vaccine: adding n-amyl alcohol into the foot-and-mouth disease vaccine, wherein the volume ratio of the n-amyl alcohol to the foot-and-mouth disease vaccine is 1 (8-10), mixing, centrifuging, and removing an oil phase to obtain a water phase sample of the foot-and-mouth disease vaccine;

(2) enabling a water phase sample of the foot-and-mouth disease vaccine to enter a capillary tube with the sample introduction time of 5-15 s and the sample introduction pressure of 0.5-2.0 psi, wherein the capillary tube is a fused quartz capillary tube, the inner diameter of the capillary tube is 25-75 mu m, and the length of the capillary tube is 20-80 cm;

(3) carrying out electrophoretic separation at a voltage of 15-30 kV for 10-30 min, wherein the temperature of a capillary column is 10-30 ℃;

the electrophoresis buffer solution used in the electrophoresis separation is 10-50 mM phosphate buffer solution with pH of 7.5-9.0, and the phosphate buffer solution further comprises Tween 20 with the mass fraction of 0.01-0.3%, glycerol with the volume fraction of 0.5-5% and PEG6000 with the mass fraction of 0.05-0.1%;

(4) and detecting and recording characteristic peaks by using a laser-induced fluorescence detector and/or an ultraviolet detector, then integrating to obtain peak areas of the characteristic peaks, and then obtaining the concentrations of the different serotype 146S antigens according to a quantitative standard curve.

21. Use of a method according to any one of claims 1 to 20 in the product development or quality monitoring of a foot and mouth disease vaccine.

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