CN109001198A - The method that the NaTDC precipitation method detect free polysaccharide in Hib combined vaccine - Google Patents
The method that the NaTDC precipitation method detect free polysaccharide in Hib combined vaccine Download PDFInfo
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- CN109001198A CN109001198A CN201811020769.4A CN201811020769A CN109001198A CN 109001198 A CN109001198 A CN 109001198A CN 201811020769 A CN201811020769 A CN 201811020769A CN 109001198 A CN109001198 A CN 109001198A
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Abstract
The invention discloses the methods of free polysaccharide in a kind of NaTDC precipitation method detection Hib combined vaccine, belong to vaccine detection technique field, its key points of the technical solution are that, including test sample pretreatment, the collection of free polysaccharide solution, the measurement of ribose concentration and the operating procedure for calculating the content of polyoses content and free polysaccharide in test sample, it removes sucrose first with ultrafiltration, recycle NaTDC precipitate polysaccharides albumen knot object, to accurately be measured to the dissociation amylase content in Hib combined vaccine, the production cost of Hib combined vaccine is reduced.
Description
Technical field
The invention belongs to vaccine detection technique field, in particular to a kind of NaTDC precipitation method detect Hib combination epidemic disease
The method of free polysaccharide in seedling.
Background technique
Haemophilus influenzae is a kind of Gram-negative dialister bacterium, can according to capsular polysaccharide form and antigenicity be divided into a,
B, c, d, e, f type, b type haemophilus influenzae (Haemophilus influenzzae Type b, Hib) pathogenicity is most strong, with
Most of serious local infections are related with invasive infection.
Hib combined vaccine is the Hib capsular polysaccharide using purifying and polysaccharide egg made of tetanus toxoid covalent bond
White conjugate, the polysaccharide protein conjugate can cause body thymus-dependent immune response, successfully develop as prevention such as brain
The invasion Hib disease such as film inflammation, pneumonia provides important tool.
In Hib combined vaccine production process, free polysaccharide based on Hib capsular polysaccharide is non-dependent as T cell because of it
Property antigen, not only cannot make body inducing immunological memory response when being immunized again, but also can be to the stabilization of Hib combined vaccine
Property and effect impact, therefore the free polysaccharide is removed usually as impurity, and the stoste of Hib combined vaccine and at
Using the measurement of dissociation amylase content as important quality Con trolling index in product examine survey, produced in order to monitor Hib combined vaccine
The removal effect of free polysaccharide in the process.
Currently, measurement dissociation amylase content method it is more, wherein NaTDC (Sodium desoxycholate,
DOC) precipitation method have many advantages, such as that easy to operate, favorable reproducibility, result is reliable and is widely used.Its working principle is that: deoxidation
Sodium taurocholate forms micelle in acid condition, can be precipitated protein substance, and then reaches the polysaccharide in Hib combined vaccine
The purpose that protein conjugates are separated with free polysaccharide；Finally recycle Hib combined vaccine in free polysaccharide in acid condition
Facile hydrolysis generates the characteristics of ribose, and the content of the free polysaccharide is measured by detecting its resulting Ribose concentration of hydrolysis.
However, Hib combined vaccine is also added with the excipient such as sucrose in the production process that finished product is made in conjunction with stoste, with
A possibility that polysaccharide protein conjugate is destroyed in freeze-drying is reduced, and sucrose is by the hemiacetal hydroxyl of a molecule glucose and one
The hemiacetal hydroxyl of molecule fructose is condensed each other to be dehydrated, and is easy to generate the glucose with reproducibility and fruit by hydrolysis
Sugar, and then have stronger interference to the measurement of free polysaccharide, the dissociation amylase content for causing detection to obtain is higher than practical, to lead
Cause Hib combined vaccine be during the preparation process reach examination criteria and spend more manpower and material resources so that its production cost compared with
It is high.Therefore, it is badly in need of a kind of method for detecting free polysaccharide in Hib combined vaccine that detection accuracy is high at present.
Summary of the invention
For the defect of the above prior art, the object of the present invention is to provide a kind of detection Hib combination epidemic diseases of pinpoint accuracy
The method of free polysaccharide in seedling, wherein the present inventor it is creative before carrying out NaTDC processing, first use ultrafiltration
Method removes sucrose, and the content of sucrose in Hib combined vaccine is reduced with this, recycles NaTDC precipitate polysaccharides albumen knot object,
To accurately be measured to the dissociation amylase content in Hib combined vaccine, the production cost of Hib combined vaccine is reduced.
Above-mentioned technical purpose of the invention has the technical scheme that
A kind of method that the NaTDC precipitation method detect free polysaccharide in Hib combined vaccine, which is characterized in that including following behaviour
Make step:
One, test sample pre-processes:
1. being pre-processed in conjunction with stoste: appropriate stoste sterilized water for injection being taken to dilute by labelled amount to get combination stoste for examination
Product solution；2. finished product pre-processes: taking appropriate finished product to be dissolved with sterilized water for injection by labelled amount, take 4.0ml in ultra-filtration centrifuge tube
In, ultrafiltration is centrifuged 8-12min under conditions of temperature is 4 DEG C, revolving speed is 7000-9000r/min, collects concentrate, mends sterilizing
Water for injection to total volume is 4ml, repeats the 8-10 above-mentioned operation that sterilized water for injection is mended after ultrafiltration centrifugation, and piping and druming mixes
The test solution got product afterwards；
Two, the collection of free polysaccharide solution: the test solution of the combination stoste or finished product in step 1 is measured, deoxidation gallbladder is added
Acid sodium solution mixes well rear ice bath 20-40min, adds hydrochloric acid solution, low-temperature centrifugation 20-40min after mixing, in collection
Clear liquid is free polysaccharide solution；
Three, the test solution and free polysaccharide in conjunction with stoste or finished product the measurement of ribose concentration: are measured respectively using chemical method
Ribose concentration in solution, and the ribose concentration that test solution measures is denoted as C1, ribose that free polysaccharide solution is measured
Concentration is denoted as C2；
Four, calculations incorporated stoste or the content of polyoses content and free polysaccharide in finished product:
Polyoses content (μ g/ml)=C1*n1/0.41
Wherein, n1For the extension rate of test sample；0.41 is the conversion coefficient of Ribose concentration and polyoses content；
Dissociation amylase content (%)=(C2/C1)×n2× 100%
Wherein, n2Multiple for test solution through being diluted in NaTDC treatment process.
A kind of embodiment according to the present invention, wherein the accurate test solution measured in step 1 in step 2
1.00ml is added 1% NaTDC, 500 μ l, mixes well rear ice bath 30min, add 300 μ of 0.1mol/L hydrochloric acid solution
L is centrifuged 30min under conditions of temperature is 4 DEG C, revolving speed is 8000-12000r/min after mixing, it is free more for collecting supernatant
Sugar juice.
The present invention is by the measurement in conjunction with free polysaccharide in stoste and finished product, in process of production convenient for Hib combined vaccine
Monitoring to free polysaccharide removal effect.Wherein, stoste and dissolution are combined using sterilized water for injection dilution in step 1
Finished product ties measurement to reduce microorganism a possibility that reduction while reducing test sample concentration by its microorganism infection
The influence of fruit.Polysaccharide binding protein is easy to happen denaturation at high temperature and precipitates, under conditions of 4 DEG C, 7000-9000r/min
Ultrafiltration centrifugation can effectively remove sucrose, while reduce the protein-bonded precipitating of polysaccharide.It would generally be in concentrate in ultra-filtration process
The a small amount of sucrose of middle residual, repeated several times supplement the operation of sterilized water for injection after ultrafiltration centrifugation, can be in test sample
Sucrose clean, reduce test solution in interference free polysaccharide measurement sucrose amount so that measuring the polysaccharide material in test sample
Matter is polysaccharide protein conjugate and free polysaccharide summation；
The hydrochloric acid of 0.1mol/L provides an acidic environment for NaTDC, so that NaTDC is forming micelle shape, it is more
Glycoprotein binding object forms sediment in conjunction with NaTDC, is centrifuged under conditions of 4 DEG C, 8000-12000r/min
30min can effectively remove the polysaccharide protein conjugate of precipitating, so that the polysaccharide material in supernatant is only free polysaccharide, most
Afterwards by the way that the content of free polysaccharide in test sample is calculated；
To sum up, the present invention reduces sucrose and polysaccharide protein conjugate to free polysaccharide by ultrafiltration and the NaTDC precipitation method
The influence of assay improves the detection accuracy to dissociation amylase content in Hib combined vaccine.
A kind of embodiment according to the present invention, wherein the polysaccharide protein conjugate in the test sample of the combination stoste
Content is 60-100 μ g/ml.
Combine the polysaccharide protein conjugate concentration in stoste usually higher obtained in Hib combined vaccine production process, it will
It is diluted to 60-100 μ g/ml, can accurately be measured to the dissociation amylase content in combination stoste.
A kind of embodiment according to the present invention, wherein the finished product is that Hib combined vaccine is freeze-dried, and specification is
0.5ml/ bottles, and the Hib b containing purifying is not less than 10 μ g in every bottle.
Pharmacopeia is 0.5ml/ bottles to the standard specification of Hib combined vaccine finished product, and the bloodthirsty bar of b type influenza containing purifying in every bottle
Bacterium capsular polysaccharide is not less than 10 μ g, with this by being measured to qualified Hib combined vaccine, can reduce rejected product detection
The waste of caused reagent.
A kind of embodiment according to the present invention, wherein the chemical method in step 3 is orcin method, including is operated as follows
Step:
A, 1.0ml sterilized water for injection is measured, ferric trichloride hydrochloric acid solution and orcin ethanol solution is sequentially added, mixes well
After obtain blank control liquid；
B, the ribose standard solution for measuring several pieces 1.0ml various concentration, sequentially adds ferric trichloride hydrochloric acid solution and orcin
Ethanol solution obtains standard control liquid after mixing well；
C, test solution or free polysaccharide solution ribose concentration is diluted to then to measure lower than 25 μ g/ml as sample solution
The 1.0ml sample solution is taken, ferric trichloride hydrochloric acid solution and orcin ethanol solution are sequentially added, obtains sample after mixing well
Measure liquid；
D, the blank control liquid in step a, the sample measurement liquid in the standard control liquid and step c in step b are first placed in
Water-bath 4-6min in boiling water, then it is placed in water-bath 25-35min in ice water, it is then calibrated, is surveyed with the blank control liquid after ice-water bath
Determine the absorbance of each group standard control liquid and sample measurement liquid at wavelength 670nm；
E, it is made using the absorbance that each group standard control liquid measures in step d with the concentration of ribose standard solution to ribose standard
The corresponding absorbance of solution makees regression beeline equation；
F, the absorbance that sample measurement liquid measures in step d is substituted into regression beeline equation and obtains corresponding C1Or C2。
A kind of embodiment according to the present invention, wherein the mass fraction of the ferric trichloride hydrochloric acid solution is 0.1%,
Its additive amount is 5ml；The mass fraction of the orcin ethanol solution is 0.1%, additive amount 0.4ml.
A kind of embodiment according to the present invention, wherein the ribose standard solution of various concentration is 25 by concentration in step b
The D-ribose standard solution of μ g/ml configures, configuration method be successively measure 0.10ml, 0.20ml, 0.40ml,
The D-ribose standard solution of 0.60ml, 0.80ml, 1.00ml, then it is corresponding be added 0.90ml, 0.80ml, 0.60ml, 0.40ml,
The sterilized water for injection of 0.20ml, 0.00ml, mix well to obtain the final product.
Blank control liquid is used for calibration instrument in standard control liquid detection process, so that the extinction that standard control liquid measures
It spends more accurate；In addition, standard control liquid is configured by the ribose dilute solution of multiple isoconcentration gradients, so that returning
Linear equation can have better representativeness, to bring the concentration that test solution and free polysaccharide solution measure into recurrence
Accuracy higher ribose concentration can be obtained when in linear equation, reduced and missed because operating brought by different experiments personnel detection
Difference.
A kind of embodiment according to the present invention, wherein the finished product in step 1 is added with O/W type micro emulsion in dissolution
Liquid.
A kind of embodiment according to the present invention, wherein the O/W type microemulsion is according to the mass fraction, different by 4-6 parts
Octane, 4-6 parts of Triton X-100,4-6 parts of n-butanol and 80-90 parts of sterilized water for injection composition.
A kind of embodiment according to the present invention, wherein the O/W type microemulsion is according to the mass fraction, different pungent by 5 parts
Alkane, 5 parts of Triton X-100,5 parts of n-butanol and 85 parts of sterilized water for injection composition.
Test sample has a small amount of sucrose and occurs hydrolysis in course of dissolution, O/W type microemulsion mainly by oil, water,
Sucrose, and can be coated in microemulsion droplets, reduce sucrose and sterilizing by surfactant and cosurfactant composition
The contact of water for injection, and then inhibit the hydrolysis of sucrose.
Above-mentioned oil can be one of heptane, isooctane, nonane etc. or a variety of mixtures, be preferably different pungent in the present invention
Alkane.Above-mentioned surfactant can be Triton X-100, lauryl sodium sulfate, cetyl trimethylammonium bromide
Deng one of or a variety of mixed liquors, the present invention in preferably Triton X-100.Above-mentioned surfactant adjuvant
It can be one of n-butanol, 2- amylalcohol, 2- methyl -2- butanol etc. or a variety of mixtures, be preferably positive fourth in the present invention
Alcohol.
Wherein, contain oxyethylene group in Triton X-100 molecule, it can be with the hydroxyl in sucrose molecule structure
It is combined by intermolecular hydrogen bond action, n-butanol makes the polar group of Triton X-100 the palisade layer of micelle
Polarity reduces, so that more sucrose enters in O/W type microemulsion droplets, so that the hydrolysis of sucrose is pressed down
System is finally centrifuged in conjunction with ultrafiltration, can quickly and effectively be removed sucrose and one step of O/W type microemulsion, so as to improve sugarcane
The removal efficiency of sugar.
It is quickly scattered in addition, Triton X-100 can also accelerate the polysaccharide protein conjugate in test sample
In sterilized water for injection, while a possibility that polysaccharide protein conjugate precipitates in ultra-filtration process is reduced, thus raising pair
The accuracy of determination of polysaccharide in Hib combined vaccine.
In addition, proving through research experiment, the microemulsion that the addition present invention is set in ultra-filtration process also can increase free more
The stability of sugar facilitates the accuracy for further increasing dissociation amylase content measurement.
In conclusion the invention has the following advantages:
In the NaTDC precipitation method provided by the invention detection Hib combined vaccine the method for free polysaccharide have combine stoste and
For finished product with surveying, detecting accuracy height, advantage easy to operate, can save Hib combined vaccine during the preparation process is to reach inspection
The manpower and material resources that mark is quasi- and spends, to reduce the production cost of Hib combined vaccine.
Detailed description of the invention
Fig. 1 is the operating procedure that the NaTDC precipitation method detect the method for free polysaccharide in Hib combined vaccine.
Specific embodiment
The present invention is described in further detail below in conjunction with the drawings and specific embodiments.
1, detection uses equipment and material
1.1, apparatus: liquid-transfering gun (specification 1-5ml, 100~1000 μ l, 20~200 μ l) is purchased from Ai Bende company of Germany；
1.2, instrument and equipment: Biofuge Primo R desk centrifuge is purchased from U.S. Thermo company；Ultraviolet point of TU-1810
Light photometer is purchased from Beijing Xi Pu company；
1.3, material: chemical reagent according to the present invention is purchased from Sigma public affairs in addition to NaTDC, D-ribose and orcin
Product is taken charge of, other reagents are that domestic analysis is pure.
2, the configuration of material
2.1, mass fraction is 1% sodium deoxycholate solution: weighing NaTDC 1g, adds sterilized water for injection, is settled to
100ml；
2.2, concentration is the hydrochloric acid solution of 0.1mol/L: measuring the concentrated hydrochloric acid that the mass fraction of 8.3ml is 36%, adds sterile injection
With water, it is settled to 1000ml；
2.3, the ferric trichloride hydrochloric acid solution that mass fraction is 0.1%: ferric trichloride (FeCl is weighed3·6H2O) 0.1g adds matter
The concentrated hydrochloric acid that score is 36% is measured, 100ml is settled to；
2.4, orcin (3,5-2 methyl resorcinol) ethanol solution that mass fraction is 0.1%: orcin 1g is weighed, matter is added
The ethyl alcohol that score is 95% is measured, 10ml is settled to；
2.5, concentration is the D-ribose standard solution of 25 μ g/ml: weighing D-ribose 1.25g, adds sterilized water for injection, is settled to
50ml。
3, a kind of method that NaTDC precipitation method detect free polysaccharide in Hib combined vaccine, referring to Fig. 1, including with
Lower operating procedure:
One, test sample pre-processes
1., in conjunction with the pretreatment of stoste: measure the combination stoste of appropriate Hib combined vaccine, with sterilized water for injection dilute combine
Stoste to its polysaccharide protein conjugate concentration is 60-100 μ g/ml, and piping and druming mixes to combine the test solution of stoste；
2., the pretreatment of finished product: taking 36 bottles of specifications is to purify Hib b in 0.5ml/ bottle, every bottle to contain
The finished product of Hib combined vaccine of the amount not less than 10 μ g is added 9ml sterilized water for injection and redissolves, the finished product for taking 2 parts of 4.0ml to redissolve
In ultra-filtration centrifuge tube, ultrafiltration is centrifuged 8-12min under conditions of temperature is 4 DEG C, revolving speed is 7000-9000r/min, collects dense
Contracting liquid, mending sterilized water for injection to total volume is 4ml, is repeated 9 times the above-mentioned operation that sterilized water for injection is mended after ultrafiltration centrifugation,
Last time blows and beats the test solution mixed to get finished product after mending sterilized water for injection to 4ml；
Two, the collection of free polysaccharide solution
Precision measures the test solution 1.00ml in step 1, and 1% NaTDC, 500 μ l is added, mixes well rear ice bath
30min adds 300 μ l of 0.1mol/L hydrochloric acid solution, in the item that temperature is 4 DEG C, revolving speed is 8000-12000r/min after mixing
20-40min is centrifuged under part, collection supernatant is free polysaccharide solution；
Three, the measurement of ribose concentration:
A, blank control liquid is prepared: precision measures 1.0ml sterilized water for injection, sequentially adds three that 5ml mass fraction is 0.1%
Iron chloride salt acid solution mixes, and adds the orcin ethanol solution that 0.4ml mass fraction is 0.1%, obtains after mixing well
Blank control liquid；
B, standard control liquid is prepared: successively precision measures 0.10ml, 0.20ml, 0.40ml, 0.60ml, 0.80ml, 1.00ml
Concentration is the D-ribose standard solution of 25 μ g/ml, then it is corresponding be added 0.90ml, 0.80ml, 0.60ml, 0.40ml, 0.20ml,
The sterilized water for injection of 0.00ml obtains the ribose standard solution of 6 parts of various concentrations, respectively from 6 parts of ribose standard solution
Precision measures 1.0ml, sequentially adds the ferric trichloride hydrochloric acid solution that 5ml mass fraction is 0.1%, mixes, adds 0.4ml matter
The orcin ethanol solution that score is 0.1% is measured, the standard control liquid of 6 parts of different D-ribose concentration is obtained after mixing well；
C, sample measurement liquid is prepared: test solution or free polysaccharide solution are diluted to ribose concentration and are lower than 25 μ g/ml, as
Sample solution then measures the 1.0ml sample solution, and it is molten to sequentially add the ferric trichloride hydrochloric acid that 5ml mass fraction is 0.1%
Liquid mixes, and adds the orcin ethanol solution that 0.4ml mass fraction is 0.1%, and sample measurement liquid is obtained after mixing well；
D, absorbance measurement: the blank control liquid in step a, the sample in the standard control liquid and step c in step b are measured
Liquid is first placed in water-bath 4-6min in boiling water, then is placed in water-bath 25-35min in ice water, then with the blank control after ice-water bath
Liquid is calibrated, and the absorbance of each group standard control liquid and sample measurement liquid at wavelength 670nm is measured；
E, regression beeline equation is established: being made using the absorbance that each group standard control liquid measures in step d molten with ribose standard
The concentration of liquid makees regression beeline equation to the corresponding absorbance of ribose standard solution；
F, ribose concentration calculation: the absorbance that sample measurement liquid measures in step d is substituted into regression beeline equation, test sample is molten
The ribose concentration that liquid measures is denoted as C1, the ribose concentration that free polysaccharide solution measures is denoted as C2；
Four, the content of polyoses content and free polysaccharide in test sample is calculated:
Polyoses content (μ g/ml)=C1*n1/0.41
Wherein, n1For the extension rate of test sample；0.41 is the conversion coefficient of Ribose concentration and polyoses content；
Dissociation amylase content (%)=(C2/C1) × 1.8 × 100%
Wherein, 1.8 be multiple of the test solution through being diluted in NaTDC treatment process.
4, embodiment
4.1, embodiment 1- embodiment 7
Embodiment 1- embodiment 7 on the basis of above-mentioned detection method, makes adjustment to the parameter in step, specifically adjusts feelings
Condition is as shown in the table.
4.2, embodiment 8- embodiment 12
On the basis of the method for embodiment 1, the test sample in step 1 is added with embodiment 8- embodiment 12 in dissolution
The O/W type microemulsion of 1ml, and the O/W type microemulsion mass fraction meter that embodiment 8- embodiment 12 is added, it is different including 4-6 parts
Octane, 4-6 parts of Triton X-100,4-6 parts of n-butanol and 80-90 parts of sterilized water for injection, it is specific each
The proportion of component is referring to following table.
4.3, comparative example 1
Be in place of the difference of embodiment 1, one the step of this comparative example in test sample pretreatment are as follows:
1., in conjunction with the pretreatment of stoste: measure the combination stoste of appropriate Hib combined vaccine, with sterilized water for injection dilute combine
Stoste to its polysaccharide protein conjugate concentration is 60-100 μ g/ml, and piping and druming mixes to combine the test solution of stoste；
2., the pretreatment of finished product: taking 36 bottles of specifications is to purify Hib b in 0.5ml/ bottle, every bottle to contain
The finished product of Hib combined vaccine of the amount not less than 10 μ g is added 9ml sterilized water for injection and redissolves, the confession that piping and druming mixes to get finished product
Test sample solution.
5, the detection performance of each embodiment and comparative example compares
5.1, polyoses content and dissociation amylase content measurement
The combination stoste and finished product for choosing the Hib combined vaccine of same product batch number are respectively as test sample, by this two kinds for examination
Product are successively detected according to the detection method in embodiment 1 to embodiment 7 and comparative example 1, and testing result is as shown in the table.
Note: detecting the content in resulting polyoses content comprising free polysaccharide, and dissociation amylase content removal amount is Hib knot
Close the practical content removed of vaccine free polysaccharide during generation.
5.2, the detection of sucrose removal effect
Using the test solution in embodiment 8- embodiment 12 as test sample, test solution in embodiment 1 as pair
Product in the same old way, the test solution in comparative example 1 is as blank sample.
Make fluorescence probe with the pyrene that concentration is 3.5 μm of ol/L, the PF-540 fluorescence spectrophotometer sold using Japanese Shimadzu Corporation
Photometer measures pyrene in above-mentioned test sample, control sample and blank under conditions of 25 ± 0.1 DEG C, excitation wavelength 338nm
Stabilization fluorescence spectrum in sample, setting pyrene are strong in the fluorescence of the first emission peak (373nm) and third emission peak (384nm) nearby
Ratio (the I of degree1/I3), the as polar size of microenvironment locating for pyrene.
Due to sucrose in Triton X-100 the palisade layer of micelle between n-butanol and pyrene so that pyrene is to poly-
Ethylene glycol octyl phenyl ether the palisade layer of micelle medial movement, and sucrose concentration is higher, the mobile degree of pyrene is bigger, the pyrene institute of measurement
The polarity for locating microenvironment is also just smaller.
Specific testing result is as follows:
5.3, Analysis of test results
Embodiment 1 to embodiment 7 and comparative example 1 are subjected to testing result comparison, remaining stable in conjunction with stoste detection data
Under the conditions of, by embodiment 1, polyoses content measured by test method combines finished product closer to it any one of into embodiment 7
Polyoses content in stoste, then relative reduction, free polysaccharide removal amount increase dissociation amylase content relatively；
In addition, pyrene is combining stoste or finished product to be formed by microenvironment, any one confession into embodiment 7 by embodiment 1
Polarity measured by test product pretreatment mode is greater than through polarity measured by comparative example 1；
And then it is available, provided detection method carries out ultrafiltration centrifugation to finished product through the invention, can effectively tie Bib
The sucrose removal in vaccine is closed, the influence that sucrose measures dissociation amylase content is reduced, to improve in detection Hib combined vaccine
Dissociation amylase content accuracy, while make the Hib combined vaccine produce when actual needs removal dissociation amylase content
It reduces, reducing Hib combined vaccine with this is the production cost for reaching examination criteria and spending.
Embodiment 8 to embodiment 12 and embodiment 1, comparative example 1 are subjected to testing result comparison, it is available, it is combining
Stoste detection data maintains under conditions of stablizing, and finished product by embodiment 8, surveyed by any one test method into embodiment 12
Polyoses content combine the polyoses content in stoste closer to it than embodiment 1 and comparative example 1, dissociation amylase content then phase
To reduction, free polysaccharide removal amount is opposite to be increased；
In addition, pyrene is combining stoste or finished product to be formed by microenvironment, any one confession into embodiment 12 by embodiment 8
Polarity measured by test product pretreatment mode is greater than through polarity measured by comparative example 1；
And then it is available, O/W type microemulsion is added in the dissolution of Hib combined vaccine test sample, Hib knot can be further increased
The dissociation amylase content of actual needs removal when closing the removal effect of sucrose in vaccine, while the Hib combined vaccine being produced
It reduces, further reducing Hib combined vaccine with this is the production cost for reaching examination criteria and spending；And work as O/W type micro emulsion
Isooctane in liquid, Triton X-100, n-butanol and sterilized water for injection proportion when being 5:5:5:85 sucrose remove
Effect reaches best；
In addition, addition O/W type microemulsion enables to polysaccharide protein conjugate to be rapidly and uniformly scattered in sterilized water for injection,
A possibility that polysaccharide protein conjugate precipitates in ultra-filtration process is reduced, detection Hib can be further increased with this and combined
The accuracy of polyoses content and dissociation amylase content in vaccine.
6, specificity detects
Specificity detection is and the determination detection using the combination stoste of known dissociation amylase content and finished product as test sample
Without sucrose in sample, the dissociation amylase content in the test sample is measured again using the present invention, then by comparing using this
The dissociation amylase content and actual dissociation amylase content that invention measures, the calculating rate of recovery (%)=(present invention measures free
Polyoses content/actual dissociation amylase content) * 100%, the wherein rate of recovery and 100% closer, then it represents that the present invention is to free
The accuracy of determination of polysaccharide is higher, therefore, it is determined that exclusive performance of the invention.
Its concrete operations are as follows: taking 3 parts of free polysaccharide actual contents is respectively 17.65%, 18.21%, 18.52% combination
Stoste and 3 parts of free polysaccharide actual contents distinguish 16.82%, 17.27%, 18.13% finished product, and implementation is respectively adopted
The detection method of example 1 and embodiment 10 measures the content of its free polysaccharide, calculates the rate of recovery, and testing result is as shown in the table.
By upper Biao Ke get, the rate of recovery using present invention detection free polysaccharide is stablized between 97.8-101.1%, is met
The requirement of standard can accurately measure the content of free polysaccharide to prove that invention has good specificity；In addition, adding
Stablize added with O/W its rate of recovery of type microemulsion detection method in 99.4-100.1%, so as to increase the stabilization of free polysaccharide
Property, facilitate the accuracy for further increasing dissociation amylase content measurement.
7, minimum detection limit detects
Minimum detection limit detection be using the combination stoste of known dissociation amylase content and finished product as test sample, and determination this
Without sucrose in test sample, the dissociation amylase content in the test sample is measured again using the present invention, is then adopted by comparing
The dissociation amylase content and actual dissociation amylase content measured with the present invention, the calculating rate of recovery (%)=(present invention measures
Dissociation amylase content/actual dissociation amylase content) * 100%, the wherein rate of recovery and 100% closer, then it represents that the present invention couple
The accuracy of dissociation amylase content measurement is higher, so that it is determined that the present invention detects the minimum concentration of free polysaccharide.
Its concrete operations are as follows: taking 6 parts of free polysaccharide actual contents is respectively 1.02 μ g/ml, 1.75 μ g/ml, 2.56 μ g/
Ml, 3.74 μ g/ml, the combination stoste of 5.26 μ g/ml and 6 parts of free polysaccharide actual contents distinguish 0.98 μ g/ml, 1.22 μ g/
The finished product of ml, 1.95 μ g/ml, 2.37 μ g/ml, 3.96 μ g/ml, are respectively adopted the detection side of embodiment 1 and embodiment 10
Method measures the content of its free polysaccharide, calculates the rate of recovery, and testing result is as shown in the table.
By upper Biao Ke get, when detecting the free polysaccharide in Hib binding protein using the present invention, the rate of recovery symbol of free polysaccharide
The requirement of standardization.Wherein, the lowest detection when dissociation amylase content in stoste is combined to be limited to 1.75 μ using present invention detection
The rate of recovery of g/ml, free polysaccharide are stablized in 93.1-104.3%；The free polysaccharide in stoste is combined using present invention detection
Lowest detection when content is limited to 1.22 μ g/ml, and the rate of recovery is stablized in 94.3-102.5%；In addition, the present invention is tied in dilution
Addition O/W type microemulsion can effectively improve the rate of recovery of free polysaccharide when closing stoste or dissolving finished product.
To sum up, the method detection accuracy with higher provided by the invention for detecting free polysaccharide in Hib combined vaccine,
Accurately the dissociation amylase content in Hib combined vaccine can be measured, reduce the production cost of Hib combined vaccine.
This specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
Member can according to need the modification that not creative contribution is made to the present embodiment after reading this specification, but as long as at this
All by the protection of Patent Law in the scope of the claims of invention.
Claims (10)
1. a kind of method of free polysaccharide in NaTDC precipitation method detection Hib combined vaccine, which is characterized in that including following
Operating procedure:
One, test sample pre-processes:
1. being pre-processed in conjunction with stoste: taking and dilute in right amount in conjunction with stoste sterilized water for injection by labelled amount to get stoste is combined
Test solution；
2. finished product pre-processes: it takes appropriate finished product to be dissolved with sterilized water for injection by labelled amount, takes 4.0ml in ultra-filtration centrifuge tube,
Ultrafiltration is centrifuged 8-12min under conditions of temperature is 4 DEG C, revolving speed is 7000-9000r/min, collects concentrate, mends sterile injection
It is 4ml with water to total volume, repeats the 8-10 above-mentioned operation for mending sterilized water for injection after ultrafiltration centrifugation, after piping and druming mixing i.e.
Obtain the test solution of finished product；
Two, the collection of free polysaccharide solution: the test solution of the combination stoste or finished product in step 1 is measured, deoxidation gallbladder is added
Acid sodium solution mixes well rear ice bath 20-40min, adds hydrochloric acid solution, low-temperature centrifugation 20-40min after mixing, in collection
Clear liquid is free polysaccharide solution；
Three, the test solution and free polysaccharide in conjunction with stoste or finished product the measurement of ribose concentration: are measured respectively using chemical method
Ribose concentration in solution, and the ribose concentration that test solution measures is denoted as C1, ribose that free polysaccharide solution is measured
Concentration is denoted as C2；
Four, calculations incorporated stoste or the content of polyoses content and free polysaccharide in finished product:
Polyoses content (μ g/ml)=C1*n1/0.41
Wherein, n1For the extension rate of test sample；0.41 is the conversion coefficient of Ribose concentration and polyoses content；
Dissociation amylase content (%)=(C2/C1)×n2× 100%
Wherein, n2Multiple for test solution through being diluted in NaTDC treatment process.
2. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 1
Sign is that the accurate test solution 1.00ml measured in step 1, is added 1% NaTDC, 500 μ l, sufficiently in step 2
Ice bath 30min after mixing adds 300 μ l of 0.1mol/L hydrochloric acid solution, after mixing temperature be 4 DEG C, revolving speed 8000-
30min is centrifuged under conditions of 12000r/min, collection supernatant is free polysaccharide solution.
3. the method for free polysaccharide in NaTDC precipitation method detection Hib combined vaccine according to claim 1 or 2,
It is characterized in that, the polysaccharide protein conjugate content in the test sample of the combination stoste is 60-100 μ g/ml.
4. the method for free polysaccharide in NaTDC precipitation method detection Hib combined vaccine according to claim 1 or 2,
It is characterized in that, the finished product is that Hib combined vaccine is freeze-dried, specification is 0.5ml/ bottles, and the b type influenza containing purifying in every bottle
Haemophilus capsular polysaccharide is not less than 10 μ g.
5. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 1
Sign is that the chemical method in step 3 is orcin method, including following operating procedure:
A, 1.0ml sterilized water for injection is measured, ferric trichloride hydrochloric acid solution and orcin ethanol solution is sequentially added, mixes well
After obtain blank control liquid；
B, the ribose standard solution for measuring several pieces 1.0ml various concentration, sequentially adds ferric trichloride hydrochloric acid solution and orcin
Ethanol solution obtains standard control liquid after mixing well；
C, test solution or free polysaccharide solution ribose concentration is diluted to then to measure lower than 25 μ g/ml as sample solution
The 1.0ml sample solution is taken, ferric trichloride hydrochloric acid solution and orcin ethanol solution are sequentially added, obtains sample after mixing well
Measure liquid；
D, the blank control liquid in step a, the sample measurement liquid in the standard control liquid and step c in step b are first placed in
Water-bath 4-6min in boiling water, then it is placed in water-bath 25-35min in ice water, it is then calibrated, is surveyed with the blank control liquid after ice-water bath
Determine the absorbance of each group standard control liquid and sample measurement liquid at wavelength 670nm；
E, it is made using the absorbance that each group standard control liquid measures in step d with the concentration of ribose standard solution to ribose standard
The corresponding absorbance of solution makees regression beeline equation；
F, the absorbance that sample measurement liquid measures in step d is substituted into regression beeline equation and obtains corresponding C1Or C2。
6. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 5
Sign is that the mass fraction of the ferric trichloride hydrochloric acid solution is 0.1%, additive amount 5ml；The orcin ethanol solution
Mass fraction be 0.1%, additive amount 0.4ml.
7. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 6
Sign is that the ribose standard solution of various concentration is configured by the D-ribose standard solution that concentration is 25 μ g/ml in step b,
Its configuration method is that the D-ribose standard of successively measurement 0.10ml, 0.20ml, 0.40ml, 0.60ml, 0.80ml, 1.00ml is molten
Liquid, then the corresponding sterilized water for injection that 0.90ml, 0.80ml, 0.60ml, 0.40ml, 0.20ml, 0.00ml is added, sufficiently mixed
It is even to obtain the final product.
8. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 1
Sign is that the finished product in step 1 is added with O/W type microemulsion in dissolution.
9. the method for free polysaccharide, special in NaTDC precipitation method detection Hib combined vaccine according to claim 8
Sign is, the O/W type microemulsion according to the mass fraction, by 4-6 parts of isooctane, 4-6 parts of Triton X-100,
The sterilized water for injection composition of 4-6 parts of n-butanol and 80-90 part.
10. the method for free polysaccharide in NaTDC precipitation method detection Hib combined vaccine according to claim 9,
Be characterized in that, the O/W type microemulsion according to the mass fraction, by 5 parts of isooctane, 5 parts of Triton X-100,5
The n-butanol of part and 85 parts of sterilized water for injection composition.
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| CN114544841A (en) * | 2020-11-24 | 2022-05-27 | 湘潭智联技术转移促进有限责任公司 | Method for determining residual amount of DMAP (dimethyl acetamide) in pneumococcal polysaccharide-protein combination vaccine by adopting high performance liquid chromatography |
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