CN102998378A - Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system - Google Patents
Method for quantification of 146S content in foot-and-mouth disease antigen by using liquid chromatography detection system Download PDFInfo
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Abstract
The present invention discloses a method for quantification of 146s content in foot-and-mouth disease antigen by using a liquid chromatography detection system. The method comprises: adopting protamine sulfate to purify virus, adopting PEG6000 to concentrate the virus, carrying out sucrose density gradient ultracentrifugation on the concentrated virus, adopting a liquid chromatography detection system to detect an absorption peak of the centrifugated sample at a wavelength of 259 nm, and calculating 146S content according to the following formula: C=FRS/76Wb. The method can be used for determination of the 146S content in various types of foot-and-mouth disease antigens. With quantification of the 146S content in the foot-and-mouth disease antigen, foot-and-mouth disease vaccine preparation can be guided, vaccine efficacy can be indirectly evaluated, and major guiding significances are provided for enhancing quality of the foot-and-mouth disease vaccine in our country.
Description
Technical field
The present invention relates to the detection method of 146S content in a kind of quantitative foot-and-mouth disease antigen, particularly a kind of method of utilizing foot-and-mouth disease antigen 146S content in the liquid chromatographic detection systematic quantification sucrose density gradient centrifugation sample.Belong to field of biological detection.
Background technology
In the foot and mouth disease virus sample, there are 4 kinds of virion that vary in size, maximum is complete virus particle, sedimentation coefficient is 146S, and slightly little a kind of particle is hollow capsid, and sedimentation coefficient is 75S, also has a kind of capsomere that is, sedimentation coefficient is 12S, and a kind of of minimum is VIA antigen, and sedimentation coefficient is 3～4.5S.In animal immune, relevant with sensitized lymphocyte with stimulation body generation antibody mainly is aftosa 146S antigen, and 146S contains all neutralizing sites of foot and mouth disease virus, is the Effective Antigens composition of foot and mouth disease virus.Animal only has the enough 146S antigen of immunity could effectively stimulate the immune system of body, produces good humoral immunity and cellular immunity.The content of 146S is a major criterion estimating the vaccine quality quality, and the foot-and-mouth disease antigen quantitative measurement technology of having reported at present mainly contains: 1. cell median infective dose (TCID50) is measured the quantitative aftosa 146S content 4. protein chromatographic analytic approachs of 2. complement fixation test (CFT)s (CFT) 3.ELISA 5. quantitative fluorescent PCRs and is carried out the quantitative etc. of antigenic content in the vaccine.Method 1 is that viral nucleic acid is detected, and except measuring complete virus particle, has also measured the nucleic acid of 146S pyrolysis product, can not reflect the immunogenicity of virus fully, can not measure viral sample after the deactivation; Method 2 accuracys, susceptibility are bad, and can not well distinguish 146S and 12S; Method 3 needs to screen respectively monoclonal for veriform virus, and the higher cycle of cost is longer, does not have at present commercial product, only limits to academic research; Method 4,5 costs are higher, complicated operation, and practicality is lower in actual production.
Set up a kind of fast and accurately 146S antigen quantify detection method, significant for the production efficiency and the quality control that improve vaccine.China Patent Publication No. CN101655452A, open day is on February 24th, 2010, invention and created name is " sucrose density gradient ultraviolet light quantitatively detects the method for foot-and-mouth disease antigen 146S content ", this application case discloses a kind of method of utilizing the sucrose density gradient centrifugation ultraviolet light quantitatively to detect foot-and-mouth disease antigen 146S content, its weak point is the sample preparation complex process in early stage of at first this invention, and do not have fully to remove foreign protein and nucleic acid in the sample, affect next step quantitative accuracy; Secondly, the computing formula desired parameters is numerous and diverse, do not detect collection of illustrative plates and be aided with support, and continuous stream ultraviolet photometer both domestic and external is difficult to satisfy the demand parameter of this formula, uses limited.
Method of the present invention utilizes the liquid chromatographic detection system at 259nm place continuous detecting sucrose density gradient centrifugation sample, and 146S cubage formula is easy; Compare with existing granted patent, this method adopts protamine sulfate to remove first part foreign protein and nucleic acid, again through concentrated foreign protein and the nucleic acid of further removing the overwhelming majority of PEG6000, both combinations have avoided foreign protein and nucleic acid in the impact on the 146S absorption peak of the uv absorption at 259nm place greatly, and through after the protamine sulfate processing, removed most nucleic acid, avoided further having improved the accuracy of 146S assay because of the resuspended error that does not thoroughly cause of nucleic acid thickness virus; The liquid chromatographic detection system that service precision and accuracy are higher, so that the detection of sample is more accurate and quick behind the ultracentrifugation, according to Lang Bo-Beer law and definite integral principle, draw computing formula, this formula needs four parameters: sample is by flow velocity, detection system sample cell optical path length, 146S absorption peak area and the applied sample amount of sample cell, the sample cell light path is set known, sample all is controlled by flow velocity and the applied sample amount of sample cell, and 146S absorption peak area is generated automatically by chromatographic work station.Adopt this method can measure accurately and fast 146S content in the foot-and-mouth disease antigen sample, can be used for the content of monitor production process viral antigen, instruct vaccine preparation and Indirect evaluation vaccine potency.
Summary of the invention
Technical matters to be solved by this invention provides the method that a kind of foot-and-mouth disease antigen 146S content quantitative detects, and method of the present invention is characterized in that may further comprise the steps:
(1) viral purification: in foot and mouth disease virus sample to be measured, add protamine sulfate, the final concentration that makes the protamine sulfate of adding is 0.5～2.0mg/ml, room temperature effect 0.5～2h, the centrifugal 15min～1h of 5000～10000rpm separates obtaining supernatant;
Described foot and mouth disease virus is hoof-and-mouth disease poison strain or its deactivation strain of any type or hypotype.
In specific embodiments of the invention, described hoof-and-mouth disease poison strain is that (Asia I/JQ strain hollow capsid vaccine is to the mensuration of the different strain immune effects of Asia I type FMDV two strains for ox Asia I/JSL/GSZY/06 poison, Li Zhi is brave etc., the veterinary drug conference of first China---veterinary biological product is learned, veterinary microbiology academic marketplace collection of thesis (2008)), (the foundation (I) of foot and mouth disease virus O type-Asia I type bivalent inactivated vaccine seed culture of viruses seed lot of cattle O type ONXC/92 poison, Xu Chunhe etc., the ten scientific seminar's meeting paper of China's animal and veterinary association's aftosa credit meeting), pig O type O/ZK/93 poison (Schweineseuche O-shaped inactivated vaccine OZK/93 strain and OZK/93 strain+OS/99 strain immune effect contrast test, Xu Xinhong etc., feed and herding scale raise pigs 2010, (9)) buy from the Lanzhou veterinary institute;
(2) PEG6000 concentrating virus: by the quality percent by volume, in the supernatant that obtains, add NaCl, the final concentration that makes NaCl is 2～6%, fully add PEG6000 after the dissolving, the final concentration that makes PEG6000 is to mix 1～16h under 5～10%, 2～8 ℃, centrifugal 1～the 2h of 5000～10000rpm, obtain the foot and mouth disease virus precipitation, use the resuspended virus precipitation of TEN damping fluid, obtain viral concentrate;
(3) SDGU: preparation quality volume ratio (g/ml) is 15%～45% (to be that sucrose concentration is 15～45g/100ml) even linear saccharose gradient liquid, the viral concentrate that step (2) is obtained carries out SDGU, 6～10 ℃, 30000～40000rpm, centrifugal 2.5～3.5h obtains the ultracentrifugation sample;
(4) the liquid chromatographic detection system detects: the ultracentrifugation sample that step (3) is obtained passes through the liquid chromatographic detection system continuously, the ultracentrifugation sample is carried out continuous detecting in the absorption value at wavelength 259nm place, obtain the ultracentrifugation sample at the absorption collection of illustrative plates at wavelength 259nm place;
Preferably, the used liquid chromatographic detection service system of the present invention should use accuracy higher, light stability, the detecting device that accuracy of the wavelength, is high, and supporting corresponding auto Analysis (being chromatographic work station) arranged; Constant flow pump answers working pressure to stablize, degree of accuracy is high, the pressure-stabilizing constant flow pump of elimination pulse.
In a specific embodiment of the present invention, the liquid chromatographic detection system that uses available from LabTech company, formed by liquid chromatogram detector, chromatographic work station and constant flow pump, the liquid chromatogram detector model is UV600 ultraviolet-visible variable-wavelenght detector, and the constant flow pump model is the P600 high pressure constant flow pump.
(5) calculate 146S content: according to the absorption peak area of the measured 146S of liquid chromatography workstation at the 259nm place, calculated the content of 146S by following formula,
C＝F
RS/76Wb
In the above-mentioned formula, C is 146S content in the sample; F
RBe the flow velocity of sample by sample cell; S is the absorption peak area of liquid chromatography workstation 146S in the test sample of wavelength 259nm place; W is the volume that is added in the viral sample on the gradient liquid; B is the optical path length of the sample cell that flows.
According to a preferred embodiment of the present invention, the final concentration that makes the protamine sulfate of adding in the step (1) is 1mg/ml, room temperature effect 1h.
According to a preferred embodiment of the present invention, the centrifugal rotational speed described in the step (1) is 7000rpm, centrifugal 30min.
According to a preferred embodiment of the present invention, in the step (2) by quality percent by volume (g/ml), in the supernatant that obtains, add NaCl, making its final concentration is 4% (being that the NaCl that adds in the 100ml supernatant is 4g), fully add PEG6000 after the dissolving, making its final concentration is 8% (being that the PEG6000 that adds in the 100ml supernatant is 8g), and 4 ℃ mix 2～6h, the centrifugal 1h of 7000rpm obtains the foot and mouth disease virus precipitation.
According to a preferred embodiment of the present invention, the TEN damping fluid described in the step (2) is for containing 0.01MTris, 0.01M EDTA, and the solution of 0.1M NaCl, PH are 7.6～8.0.
According to a preferred embodiment of the present invention, with the resuspended virus precipitation of described TEN damping fluid, the volume that obtains is viral sample volume 1/100-1/50 virus concentrate, more preferably with the resuspended virus precipitation of described TEN damping fluid, the volume that obtains is viral sample volume 1/100 viral concentrate.Described viral sample volume refers to the volume of the foot and mouth disease virus sample to be measured described in the step (1).
According to a preferred embodiment of the present invention, the SDGU described in the step (3) is 8 ℃, 35,000rpm, centrifugal 3.5h.
Among the present invention, used liquid chromatographic detection system is concentration type detection system, by the absorption peak area of the 146S in the liquid chromatography workstation institute test sample at the 259nm place, according to Lang Bo-Beer law and definite integral principle, draws the formula that calculates 146S content.Concrete derivation is as follows:
Lang Bo-Beer law: A=Ebc,
In the formula, c is institute's test sample product concentration, the g/L of unit; A is absorbance; B is the sample liquid layer thickness, and the thickness that is generally sample cell also is optical path length, the cm of unit; E is absorptivity, the L/ of unit (gcm), and absorptivity and incident light wavelength and the material that is passed through by light are relevant, as long as light wavelength is fixed, the same material, absorptivity is just constant.
Derive c=A/Eb,
Sample flow when sample cell a period of time Δ t, the sample volume v=F of the sample cell of flowing through
RΔ t, wherein F
RBe the flow velocity of sample flow through sample cell; Then draw the flow through quality of 146S of sample cell of a period of time Δ t:
m＝AF
RΔt/Eb，
According to the definite integral principle, the gross mass of the 146S of the sample cell of flowing through:
In the method, the absorptivity E of aftosa 146S antigen at the 259nm place is 76; According to the definite integral principle, 146S continuous stream under wavelength 259nm is aftosa 146S at the absorption peak area S of wavelength 259nm place continuous stream through sample cell formation through the definite integral sum of the absorbance (A) of corresponding each time point of sample cell, also is
T1, t2 are respectively 146S absorption peak start and end time.Then derive the quality of total 146S of the sample cell of flowing through:
M＝SF
R/76b
Be the density gradient ultracentrifugation applied sample amount such as W, unit/ml then calculates the content of aftosa 146S in institute's test sample product:
C＝SF
R/76Wb
In the above-mentioned formula, C is 146S content in the sample; S is the absorption peak area of liquid chromatography workstation 146S in the test sample of wavelength 259nm place; F
RBe the flow velocity of sample flow through sample cell; B is the optical path length of the sample cell that flows; W by the applied sample amount of detection viral sample.
Compared to prior art, the invention has the advantages that:
1, adopt protamine sulfate to remove first part foreign protein and nucleic acid, again through concentrated foreign protein and the nucleic acid of further removing the overwhelming majority of PEG6000, both combinations have avoided foreign protein and nucleic acid in the impact on the 146S absorption peak of the uv absorption at 259nm place greatly, and through after the protamine sulfate processing, removed most nucleic acid, avoided further having improved the accuracy of 146S assay because of the resuspended error that does not thoroughly cause of nucleic acid thickness virus.
2, the use of liquid chromatographic detection system is so that the detection of sample is more accurately and quick behind the ultracentrifugation, according to Lang Bo-Beer law and definite integral principle, the computing formula of deriving 146S content is very simple, and only need four parameters: sample is by flow velocity, detection system sample cell optical path length, 146S absorption peak area and the applied sample amount of sample cell.The sample cell light path is set known, and sample all is controlled by flow velocity and the applied sample amount of sample cell, and 146S absorption peak area is generated automatically by chromatographic work station, adopts this method can Fast Measurement 146S content.
3, have good repeatability, batch in and batch between deviation little; Have higher sensitivity, all can measure concentrated 5～100 times of virus liquids; Have preferably applicability, can measure the 146S content of live virus, can measure again the 146S content of inactivation of viruses; Have widely versatility, be suitable for the detection of the various types of aftosa and subtype virus.
Therefore, the inventive method quantitative result is accurate, and good reproducibility is highly sensitive, and convenient, fast, can be used for the mensuration of 146S content in the various foot-and-mouth disease antigen.Can instruct the preparation of aftosa vaccine by the content of 146S in the quantitative foot-and-mouth disease antigen, the effectiveness of Indirect evaluation vaccine, this has great directive significance for promoting China's aftosa vaccine quality.
Description of drawings
Fig. 1 detects collection of illustrative plates for foot and mouth disease virus 146S liquid chromatographic system after concentrated;
Fig. 2 detects collection of illustrative plates for the pure 146S liquid chromatographic system of collecting;
Fig. 3 is that different cycles of concentration foot and mouth disease virus liquid chromatographic systems detect collection of illustrative plates;
Curve is followed successively by concentrated 100 times from top to bottom, and 80 times, 40 times, 20 times, 10 times, 5 times concentrating virus liquid;
Fig. 4 repeats liquid chromatographic system in same cycles of concentration virus liquid is criticized and between criticizing to detect collection of illustrative plates.
Curve is red, blue to be repeated in criticizing, and curve black is repetition between different batches.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
The quantitative detection of 146S antigenic content in embodiment 1 sample
1, material
1.1 test sample: ox Asia I/JSL/GSZY/06 poison, cattle O type ONXC/92 poison, pig O type O/ZK/93-08 poison are bought from the Lanzhou veterinary institute.
1.2 instrument and reagent: the liquid chromatographic detection system is available from LabTech company, and sucrose, protamine sulfate are available from sigma company, and PEG6000 is available from AMRESCO company.
The phase chromatographic detection system is comprised of liquid chromatogram detector, chromatographic work station and constant flow pump, and the liquid chromatogram detector model is UV600 ultraviolet-visible variable-wavelenght detector, and the constant flow pump model is the P600 high pressure constant flow pump.The liquid chromatogram detector light source is deuterium lamp, and spectral bandwidth is 8nm, and wavelength accuracy is ± 0.2nm that wavelength accuracy is ± 1.0nm; The measurement indication range is-1～4AU; Baseline noise is ± 0.5 * 10-5AU (methyl alcohol, non-NULL pond), and baseline wander is 1.0 * 10-4AU/h (methyl alcohol, non-NULL pond).Constant flow pump is the double plunger series connection, and diaphragm type pulse damper and the simultaneously controlled pressure pulsation of electronics pulsation inhibition technology ensure minimum baseline noise, pressure fluctuation＜1.0%, the flow accuracy is ± 1.0% (1.0ml/min, water, room temperature), flow rates is 0.001～9.999ml/min.GLP, GMP, 21CFR laboratory code requirement are followed in the increasingly automated design of chromatographic work station, guarantee desired data tracing ability and data integrity; Workstation has powerful data processing function, and is convenient easy to operate, reads in sampled data by the CDF file that meets AIA (U.S.'s analytics meeting) standard, automatically declares peak, reference area, and can do plus and minus calculation or overlap demonstration between many spectrograms.
2, method:
(1) viral purification: get the viral antigen that suspends and cultivate in the large production: 1. sample lives malicious for ox AsiaI/JSL/GSZY/06,2. sample lives malicious for cattle O type ONXC/92,3. sample is pig O type O/ZK/93-08 poison alive, 4. sample is ox Asia I/JSL/GSZY/06 deactivation poison, 5. sample is cattle O type ONXC/92 deactivation poison, and 6. sample is pig O type O/ZK/93-08 deactivation poison.The preparation of deactivation poison sample can prepare by existing conventional method, and (BEA is cyclized into BEI to the BEI of employing 3mM under the effect of the NaOH of process 0.2N in the present embodiment.BEA is available from sigma company.) 30 ℃ of deactivations carried out inactivation treatment in 28 hours.Add respectively protamine sulfate in each viral sample (being virus stock solution used), so that the final concentration of protamine sulfate in sample solution that adds is 1mg/ml, stirring at room is mixed 1h, and the centrifugal 30min of 7000rpm separates obtaining supernatant.
(2) PEG6000 concentrating virus: calculate by quality percent by volume (g/ml), in each the sample supernatant that obtains, add respectively NaCl, making its final concentration is 4%, fully add PEG6000 after the dissolving, making its final concentration is 8%, 4 ℃ mix 2h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, with TEN damping fluid (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH 7.6) resuspended 1/100,0.45 μ m pin type filter filtration to the provirus sample volume, place 4 ℃ of refrigerators for subsequent use.
(3) SDGU: in 11ml ultracentrifugation pipe, prepare the even linear saccharose gradient liquid 10ml of 15%～45% (W/V), get the above-mentioned concentrate of 1ml and add to the even linear saccharose gradient liquid of 15%～45% (W/V) top for preparing, 8 ℃, 35,000rpm, behind the centrifugal 3.5h, obtain the ultracentrifugation sample 1.～6..
(4) the liquid chromatographic detection system detects: 1.～6. the ultracentrifugation sample that step (3) is obtained passes through respectively the liquid chromatographic detection system continuously, the ultracentrifugation sample is carried out continuous detecting in the absorption value at wavelength 259nm place, obtain respectively the ultracentrifugation sample 1.～6. at the absorption collection of illustrative plates at wavelength 259nm place;
(5) calculating of 146S content: the cubage formula of the 146S that derives according to the present invention calculates,
C＝F
RS/76Wb
In the above-mentioned formula, C is 146S content in the sample; F
RBe the flow velocity of sample by sample cell; S is the absorption peak area of liquid chromatography workstation 146S in the test sample of wavelength 259nm place; W is the volume that is added in the viral sample on the gradient liquid; B is the optical path length of the sample cell that flows.
3, sensitivity and repeatability detect: as stated above with sample 1. ox Asia I/JSL/GSZY/06 poison be concentrated into respectively 100 times, 80 times, 40 times, 20 times, 10 times, 5 times, the calculating of 146S content is carried out in repeating step (3), (4), (5).
4, result
4.1 albumen and dna content are relatively before and after the antigen purification
Get the viral antigen that suspends and cultivate in the large production: 1. sample lives malicious for ox Asia I/JSL, 2. sample lives malicious for cattle O type ONXC/92,3. sample is pig O type O/ZK/93-08 poison alive, 4. sample is ox AsiaI/JSL/GSZY/06 deactivation poison, 5. sample is cattle O type ONXC/92 deactivation poison, and 6. sample is pig O type O/ZK/93-08 deactivation poison.Add protamine sulfate in each sample, so that the final concentration of protamine sulfate in sample solution that adds is 1mg/ml, stirring at room is mixed 1h, the centrifugal 30min of 7000rpm, separate and obtain supernatant, adding NaCl in the supernatant that obtains, to make its final concentration be 4%, fully add PEG6000 after the dissolving, make its final concentration be 8%, 4 ℃ and mix 2h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, filter with resuspended 1/100, the 0.45 μ m pin type filter to the provirus sample volume of TEN damping fluid, sample is purifying front and back albumen 1.～6., the DNA average removal rate relatively sees Table 1, and virus titer relatively saw Table 2 before and after 3. 2. 1. sample processed.
Table 1 sample 1.～6. concentrated front and back of purifying albumen, DNA average removal rate compares
| Sample | Albumen average removal rate (%) | DNA average removal rate (%) |
| Behind the protamine sulfate purifying | 33.9 | 93.9 |
| After PEG6000 is concentrated | 94.8 | 98.6 |
As shown in table 1,1.～6. sample has on average removed 93.9% DNA behind the middle adding protamine sulfate purifying, has removed 33.9% foreign protein; After PEG6000 was concentrated, the albumen average removal rate reached 94.8%, DNA average removal rate and reaches 98.6%.As seen after processing through protamine sulfate and PEG6000, foreign protein and DNA in the sample more than 94% are removed, and have effectively avoided foreign protein and nucleic acid in the impact on the 146S absorption peak of the uv absorption at 259nm place; Removed 98% nucleic acid, avoided being conducive to the accurate quantitative analysis of 146S because of the resuspended error that does not thoroughly cause of nucleic acid thickness virus.
Virus titer relatively before and after 3. 2. 1. table 2 sample processed
As shown in table 2, from the virus titer aspect, 1. sample 2. 3. after protamine sulfate purifying and PEG6000 are concentrated, is compared with virus stock solution used, viral antigen is not loss almost, illustrate that after protamine sulfate purifying and PEG6000 are concentrated 146S does not lose in the viral antigen.
4.2 the liquid chromatographic detection system detects centrifugal sample at the absorption collection of illustrative plates at wavelength 259nm place
The liquid chromatographic detection system detects centrifugal sample at the absorption collection of illustrative plates at wavelength 259nm place.As shown in Figure 1,2 absorption peaks appear in the foot and mouth disease virus density gradient ultracentrifugation behind the purifying, learn that in conjunction with the detection of cell median infective dose wherein first peak is 146S antigen absorption peak, the second peak is other impurity peaks, obtain Fig. 2 after the first peak of collecting re-started sucrose density gradient centrifugation, only has a peak, as seen only there is 146S antigen also can find out from Fig. 1 among Fig. 1 in the first peak, the second peak, also be that impurity peaks is less, illustrate that albumen and nucleic acid are residual few, verified that further sample treatment effect in early stage is fine.
4.3 calculate foot and mouth disease virus 146S content
1. ox Asia I/JSL/GSZY/06 is alive malicious to sample, 2. cattle O type ONXC/92 lives malicious, 3. pig O type O/ZK/93-08 lives malicious, 4. ox Asia I/JSL/GSZY/06 deactivation is malicious, 5. cattle O type ONXC/92 deactivation poison, 6. pig O type O/ZK/93-08 deactivation poison carries out the 146S assay, according to the absorption peak area of the measured 146S of liquid chromatography workstation at the 259nm place, calculated the content of 146S by following formula
C＝F
RS/76Wb
In the above-mentioned formula, C is 146S content in the sample; F
RBe the flow velocity of sample by sample cell; S is the absorption peak area of liquid chromatography workstation 146S in the test sample of wavelength 259nm place; W is the volume that is added in the viral sample on the gradient liquid; B is the optical path length of the sample cell that flows.
Result of calculation is as shown in table 3, and 100 times of concentrated antigen 146S content are converted to stoste namely between 5.39-5.96 μ g/ml between 538.86-595.52 μ g/ml.
Each sample 146S content of table 3
4.4 sensitivity and repeated testing result
As stated above with sample 1. ox Asia I/JSL/GSZY/06 poison be concentrated into respectively 100,80 times, 40 times, 20 times, 10 times, 5 times, repeat step (3), (4), (5) among the embodiment 1, draw table 4 and Fig. 3, collection of illustrative plates shown in Figure 4.Different cycles of concentration ox Asia I/JSL/GSZY/06 poison 146S content are as shown in table 4, different cycles of concentration virus liquid liquid chromatographic systems detect collection of illustrative plates as shown in Figure 3, curve shown in the figure is followed successively by concentrated 100 times from top to bottom, and 80 times, 40 times, 20 times, 10 times, repeat liquid chromatographic system detection collection of illustrative plates in 5 times concentrating virus liquid, same cycles of concentration virus liquid are criticized and between criticizing as shown in Figure 4, yellow, red for criticizing interior the repetition, black is repetition between different batches.
The different cycles of concentration ox of table 4 Asia I/JSL/GSZY/06 poison 146S content
| |
100 |
80 |
40 |
20 |
10 times | 5 times |
| 146S content (μ g/ml) | 595.52 | 475.54 | 238.65 | 120.02 | 60.86 | 31.17 |
Can be found out by table 4 and Fig. 3,4: this method has higher sensitivity, all can measure concentrated 5～100 times of virus liquids; Have good repeatability, batch in and batch between deviation little.
Can find out from embodiment 1 result, the method has preferably applicability, can measure the 146S content of live virus, can measure again the 146S content of inactivation of viruses; Have widely versatility, be suitable for the detection of the various types of aftosa and subtype virus.And sensitivity is higher, and repeatability better.
Take the measured 146S content of method of the present invention as foundation preparation vaccine; aftosa O type in the bivalent vaccine of preparing; corresponding 146S content equates in the Asia I type antigen liquid; " (the aftosa O type that the method for efficacy test is drafted according to Ministry of Agriculture's tissue; Asia I type bivalent inactivated vaccine is made and the check Trial Regulation " carry out; the result is as shown in table 5; the content of 146S described in the table is aftosa O type or Asia I type antigen 1 46S content; the two equates; for example: in the cattle O type ONXC/92-Asia I type divalence inactivation antigen liquid 3; the 146S content of aftosa O type is 17.17 μ g/ml; Asia I type 146S content also is 17.17 μ g/ml, separately half protection (PD
50) value is that 15.59, O type is 12.51 for the Asia I type.
The corresponding half protection of the antigen liquid value (PD of the different 146S content of table 5 (μ g/ml)
50)
Record the result more than inciting somebody to action and carry out statistical study, with PD
50Value is y, and 146S content is x, and the linear equation of ONXC/92 type antigen is y=0.501x+3.5129, coefficient R
2The linear equation of=0.9668, Asia I type antigen is y=0.5152x+3.811, coefficient R
2=0.9498.As can be seen from Table 5, aftosa Asia I type 146S content can reach 3 PD that the Ministry of Agriculture requires at 0.5ug/ml
50, and ONXC/92 type 146S content needs just can reach requirement greater than 0.5ug/ml.
Can be found out by embodiment 2, it is feasible carrying out the vaccine preparation by 146S content, and different strains reaches the needed 146S content difference of immunoprotection efficient.
The optimization of the concentrated condition of embodiment 3 foot and mouth disease virus purifying
1 material
1.1 vaccine strain: Asia I/JSL/GSZY/06 lives the poison purchase from the Lanzhou veterinary institute.
1.2 chemical reagent: protamine sulfate is sigma company product; PEG6000 is available from AMRESCO company.
2 methods
According to the result of documents and materials and preliminary experiment, choosing affects the concentrated principal element of aftosa purifying: protamine sulfate working concentration, temperature and time, PEG6000 working concentration and mixing time are optimized.
2.1 the protamine sulfate working concentration is optimized
Get the Asia I/JSL/GSZY/06 viral antigen that suspends and cultivate in the large production, add protamine sulfate to different final concentrations, be followed successively by 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml and five concentration gradients of 2.5mg/ml, after the room temperature effect 1 hour, the centrifugal 30min of 7000rpm gets supernatant, detects the protein content of supernatant, dna content calculates albumen clearance and DNA clearance.
2.2 the optimization of protamine sulfate operative temperature and time
Get in the large production Asia I viral antigen that suspends and cultivate, add protamine sulfate, making its final concentration is 1mg/ml, acts on 30min respectively under 4 ℃ and room temperature condition, 1.0h, and 1.5h, 2.0h, 3.0h, the centrifugal 30min of 7000rpm gets supernatant; Detect the protein content of supernatant, dna content and TCID
50, calculate albumen clearance and DNA clearance.
2.3PEG6000 the optimization of working concentration
By the quality percent by volume, in being supernatant behind the 1mg/ml protamine sulfate room temperature effect 1h, final concentration adds NaCl, making its final concentration is 4%, fully add PEG6000 after the dissolving, make its final concentration be respectively 4%, 5%, 6%, 7%, 8%, 9%, 10%, 4 ℃ mix 12h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, with TEN damping fluid (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH 7.6) resuspended 1/100,0.45 μ m pin type filter filtration to the provirus sample volume, detect TCID
50, protein content, dna content calculates albumen clearance and DNA clearance.
2.4PEG6000 the optimization of action time
By the quality percent by volume, in being supernatant behind the 1mg/ml protamine sulfate room temperature effect 1h, final concentration adds NaCl, making its final concentration is 4%, fully add PEG6000 after the dissolving, making its final concentration is 8%, 4 ℃ mix respectively 1h, 2h, 4h, 6h, 8h, 12h, 16h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, with TEN damping fluid (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH 7.6) resuspended 1/100,0.45 μ m pin type filter filtration to the provirus sample volume, detect TCID
50, protein content, dna content calculate albumen clearance and DNA clearance.
3 results
3.1 the protamine sulfate working concentration is optimized
Get the Asia I viral antigen that suspends and cultivate in the large production, add protamine sulfate to different final concentrations, be followed successively by 0.5mg/ml, 1.0mg/ml, 1.5mg/ml, 2.0mg/ml and five concentration gradients of 2.5mg/ml, after the room temperature effect 1 hour, the centrifugal 30min of 7000rpm gets supernatant, detects the protein content of supernatant, dna content calculates albumen clearance and DNA clearance.
The result is as shown in table 6.
Table 6 protamine sulfate working concentration is optimized
According to the result of table 6, remove the factors such as the effect of host cell foreign protein and nucleic acid and cost at comprehensive protamine sulfate after, the best working concentration of determining protamine sulfate is 1mg/ml.
3.2 the optimization of protamine sulfate operative temperature and time
Get in the large production Asia I viral antigen that suspends and cultivate, add protamine sulfate, making its final concentration is 1mg/ml, acts on 30min respectively under 4 ℃ and room temperature condition, 1.0h, and 1.5h, 2.0h, 3.0h, the centrifugal 30min of 7000rpm gets supernatant; Detect the protein content of supernatant, dna content and TCID
50, calculate albumen clearance and DNA clearance.The result is as shown in table 7.
The optimization of table 7 protamine sulfate operative temperature and time
Result according to table 7, remove the effect of host cell foreign protein and nucleic acid at comprehensive protamine sulfate, on after the impact of virus titer and processing the factor such as simplifications, the best use of temperature and time of determining protamine sulfate is that room temperature (25 ℃) acts on 1.0h.
3.3PEG6000 the optimization of working concentration
By the quality percent by volume, add NaCl in the supernatant behind the final concentration 1mg/ml protamine sulfate room temperature effect 1h, making its final concentration is 4%, fully add PEG6000 after the dissolving, make its final concentration be respectively 4%, 5%, 6%, 7%, 8%, 9%, 10%, 4 ℃ mix 12h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, with TEN damping fluid (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH 7.6) resuspended 1/100,0.45 μ m pin type filter filtration to the provirus sample volume, detect TCID
50, protein content, dna content calculate albumen clearance and DNA clearance, and the result is as shown in table 8.
Table 8PEG6000 working concentration is optimized
According to the result of table 8, remove the effect of host cell foreign protein and nucleic acid at comprehensive PEG6000, on factors such as the impact of virus titer and costs after, the best use of concentration of determining PEG6000 is 8%.
3.4PEG6000 the optimization of action time
By the quality percent by volume, add NaCl in the supernatant behind the final concentration 1mg/ml protamine sulfate room temperature effect 1h, making its final concentration is 4%, fully add PEG6000 after the dissolving, making its final concentration is 8%, 4 ℃ mix respectively 1h, 2h, 4h, 6h, 8h, 12h, 16h, the centrifugal 1h of 7000rpm, obtain the foot and mouth disease virus precipitation, with TEN damping fluid (0.01M Tris, 0.01M EDTA, 0.1M NaCl, PH 7.6) resuspended 1/100,0.45 μ m pin type filter filtration to the provirus sample volume, detect TCID
50, protein content, dna content calculate albumen clearance and DNA clearance, and the result is as shown in table 9.
Show the 9PEG6000 optimization of action time
Result according to table 9, it is longer action time that 2h begins PEG6000, albumen and DNA clearance are lower, and after comprehensive PEG6000 removed the effect of host cell foreign protein and nucleic acid, factors such as impact on virus titer, the best use of time of determining PEG6000 was 2h.
Claims (8)
1. method of utilizing the liquid chromatographic detection systematic quantification to measure 146S content in the foot-and-mouth disease antigen is characterized in that may further comprise the steps:
(1) viral purification: in foot and mouth disease virus sample to be measured, add protamine sulfate, the final concentration that makes the protamine sulfate of adding is 0.5～2.0mg/ml, room temperature effect 0.5～2h, the centrifugal 15min～1h of 5000～10000rpm separates obtaining supernatant;
(2) PEG6000 concentrating virus: by the quality percent by volume, in the supernatant that obtains, add NaCl, the final concentration that makes NaCl is 2～6%, fully add PEG6000 after the dissolving, the final concentration that makes PEG6000 is to mix 1～16h under 5～10%, 2～8 ℃, centrifugal 1～the 2h of 5000～10000rpm, obtain the foot and mouth disease virus precipitation, use the resuspended virus precipitation of TEN damping fluid, obtain viral concentrate;
(3) SDGU: the preparation quality volume ratio is 15%～45% even linear saccharose gradient liquid, the viral concentrate that step (2) is obtained carries out SDGU, 6～10 ℃, 30000～40000rpm, centrifugal 2.5～3.5h obtains the ultracentrifugation sample;
(4) the liquid chromatographic detection system detects: the ultracentrifugation sample that step (3) is obtained passes through the liquid chromatographic detection system continuously, the ultracentrifugation sample is carried out continuous detecting in the absorption value at wavelength 259nm place, obtain the ultracentrifugation sample at the absorption collection of illustrative plates at wavelength 259nm place;
(5) calculate 146S content: according to the absorption peak area of the measured 146S of liquid chromatography workstation at the 259nm place, calculated the content of 146S by following formula,
C＝F
RS/76Wb
In the above-mentioned formula, C is 146S content in the sample; F
RBe the flow velocity of sample by sample cell; S is the absorption peak area of liquid chromatography workstation 146S in the test sample of wavelength 259nm place; W is the volume that is added in the viral sample on the gradient liquid; B is the optical path length of the sample cell that flows.
2. method according to claim 1 is characterized in that making the final concentration of the protamine sulfate of adding in the step (1) is 1mg/ml, room temperature effect 1h.
3. method according to claim 1 is characterized in that the centrifugal rotational speed described in the step (1) is 7000rpm, centrifugal 30min.
4. method according to claim 1, it is characterized in that in the step (2) by the quality percent by volume, adding NaCl in the supernatant that obtains, to make its final concentration be 4%, it is 8% that the rear adding of abundant dissolving PEG6000 makes its final concentration, 4 ℃ mix 2～6h, the centrifugal 1h of 7000rpm obtains the foot and mouth disease virus precipitation.
5. method according to claim 1 is characterized in that the TEN damping fluid described in the step (2) for containing 0.01M Tris, 0.01M EDTA, and the solution of 0.1M NaCl, PH are 7.6～8.0.
6. method according to claim 1 or 5 is characterized in that obtaining volume is the viral concentrate of viral sample volume 1/100～1/50 with the resuspended virus precipitation of described TEN damping fluid.
7. method according to claim 6 is characterized in that precipitating with the resuspended virus of described TEN damping fluid in the step (2), and the volume that obtains is the viral concentrate of provirus liquid long-pending 1/100.
8. method according to claim 1 is characterized in that the SDGU described in the step (3) is 8 ℃, 35,000rpm, centrifugal 3.5h.
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