CN100578217C - Method for detecting content of free polysaccharides in group A and C meningococcal polysaccharide conjugate - Google Patents

Abstract

A detecting method for the content of free polysaccharides in the A, C cluster parameningococcus polysaccharide conjugate is disclosed that belongs to the field of biotechnology. The steps are: obtains the A, C cluster parameningococcus polysaccharide conjugate as the sample 1; obtains a mount of derivant liquor corresponding to the A, C cluster parameningococcus polysaccharide conjugate as the sample 3; obtains a mount of A, C cluster parameningococcus polysaccharide conjugate, adds cooling phenol liquor, vibrates fully, and centrifugally collects the supernatant as the sample 2; obtains a mount of derivant liquor corresponding to A, C cluster parameningococcus polysaccharide conjugate, adds cooling phenol liquor, vibrates fully, and centrifugally collects the supernatant as the sample 4. The A cluster parameningococcus polysaccharide conjugate measures the phosphorus content, the C cluster parameningococcus polysaccharide conjugate measures sialic acid content, calculates the polysaccharide concentration of the sample 1, sample 2, sample 3, sample 4, and calculates the content of free polysaccharides by formula. The invention could detect the content of free polysaccharides in conjugate quickly, fast and easily, and evaluates the quality of item.

Description

Free measurement of the polysaccharide content method in a kind of A, the C group meningitis cocci polysaccharide conjugate

Technical field:

The invention belongs to biological technical field, more specifically, relate to free measurement of the polysaccharide content method in a kind of A, the C group meningitis cocci polysaccharide conjugate.

Background technology:

Meningococcal infection be cause the common disease of children's meningitis because of, case fatality rate is higher.Because the incidence of disease has been controlled in the use of meningococcal capsular polysaccharide vaccine effectively.But capsular polysaccharide belongs to T cell dependent/non-dependent antigen, and immunity can not be reinforced once more, and can not induce children below 2 years old to produce immune response, and certain limitation is arranged.Chemically protein carrier is covalently bound on the capsular polysaccharide, can makes it to become T cell dependent type antigen, thereby have the immunological memory reaction, therefore provide immunoprotection infant below 5 years old.

Studies show that, A group meningitis cocci polysaccharide-tetanus toxoid bond, C group meningitis cocci polysaccharide-tetanus toxoid bond have very strong immunogenicity in mouse, can stimulate mouse to produce high-caliber IgG, and independent capsular polysaccharide can not stimulate mouse to produce antibody or only have low-level antibody to produce.What as seen really work in the bond is the polysaccharide that combines with tetanus toxoid.The limit that dissociation amylase content in the polysaccharide conjugate vaccine surpasses regulation can produce adverse influence to the effect of the clinical use of vaccine, therefore free determination of polysaccharide is one of key index in the polysaccharide conjugate vaccine quality control in the GL-PP bond, also is one of the leading indicator of the quality of reflection combined vaccine quality.

Because different bacterium capsular polysaccharide and different polysaccharide conjugate nature difference are bigger, be technological difficulties so the free polysaccharide in the polysaccharide conjugate vaccine is measured, still there is not the universal method of free determination of polysaccharide in the polysaccharide conjugate at present.The foreign literature report adopts high performance liquid chromatography to carry out free polysaccharide determination in the bacterial polysaccharides bond, and a kind of employing efficient gel filters, a kind of employing high performance anion exchange chromatography.The efficient gel filtration method is distinguished in conjunction with polysaccharide and free polysaccharide based on molecular size difference, is vulnerable to the influence of the close impurity of molecular size, and the measurement result error is bigger; High performance anion exchange chromatography is distinguished in conjunction with polysaccharide and free polysaccharide based on the difference of the electrically charged character of molecule, also is vulnerable to the influence with the impurity of the electrically charged similar performance of polysaccharide.Domestic Zhu is, (make progress by the microbiology immunology in " foundation of free polysaccharide determination method in A group meningitis cocci polysaccharide-tetanus toxoid bond " for Li Fengxiang, 2002,30 the 4th phases of volume) introduced a kind of method of utilizing ethanol precipitation to measure A group meningitis cocci polysaccharide-tetanus toxoid bond free polysaccharide on, but this method step is various, and a sample needs 5 days ability to obtain the result.But still there is not the report that utilizes cold phenol solution extracting process to measure free polysaccharide in A, the C group meningitis cocci polysaccharide conjugate at present.

Summary of the invention:

Method of the present invention has overcome the deficiency that existing method is measured dissociation amylase content, and this method can accurately be measured the content of free polysaccharide in A, the C group meningitis cocci polysaccharide conjugate, and method is easy, quick.

Technical scheme of the present invention and step:

(1) get meningococcal polysacharide bond 2ml to be checked as sample 1, the derivative solution 2ml that gets this meningococcal polysacharide bond correspondence to be checked is as sample 3;

(2) get this meningococcal polysacharide bond 2ml in addition and place the 15ml centrifuge tube, the derivative solution 2ml that gets this meningococcal polysacharide bond correspondence to be checked places another 15ml centrifuge tube; Add the cold phenol solution of 2～10ml to every centrifuge tube, vibration mixing 10～30 minutes was put ice bath 5～20 minutes, centrifugal 10～30 minutes of 5000～10000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

(3) A group meningitis cocci polysaccharide conjugate is measured phosphorus content, calculates the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4; C group meningitis cocci polysaccharide conjugate is measured sialic acid content, calculates the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4; The polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 is respectively p1, p2, p3, p4;

(4) according to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery should be 80～120%;

(5) calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ P is the recovery in the formula.

Wherein said cold phenol solution can be the ratio preparation that adds 40ml 1/10 saturated neutral sodium acetate solution dissolving according to every 100g phenol, is chilled to 4 ℃ then in advance and is prepared from.

The invention has the beneficial effects as follows: utilize the extremely strong characteristics that cause protein denaturation of phenol, in the phenol existence and behind high speed centrifugation, in conjunction with polysaccharide because of the irreversible sedimentation of the albuminous degeneration of coupling, free polysaccharide is still in supernatant, separate in conjunction with polysaccharide and free polysaccharide like this, measure the polyoses content of centrifuged supernatant and stoste respectively, can calculate dissociation amylase content.The invention provides a kind of accurately, the method for free polyoses content among the fast measuring A, C group meningitis cocci polysaccharide conjugate, set up evaluation meningococcal polysacharide combined vaccine method for quality.

Embodiment:

Embodiment 1

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get A group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence is as sample 3.Other gets this A group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube.Add the cold phenol solution of 2ml to every centrifuge tube, vibration mixing 10 minutes was put ice bath 5 minutes, centrifugal 10 minutes of 5000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the phosphorus content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " A group's polysaccharide concentration=phosphorus content/0.08 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝140.0μg/ml，p2＝12.0μg/ml，p3＝88.9μg/ml，p4＝85.1μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 95.7%;

Calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 9.0% in the A group meningitis cocci polysaccharide conjugate.

Embodiment 2

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get A group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence is as sample 3; Other gets this A group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube; Add the cold phenol solution of 10ml to every centrifuge tube, vibration mixing 30 minutes was put ice bath 20 minutes, centrifugal 30 minutes of 10000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the phosphorus content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " A group's polysaccharide concentration=phosphorus content/0.08 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝143.2μg/ml，p2＝14.1μg/ml，p3＝75.2μg/ml，p4＝72.4μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 96.3%;

Calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 10.2% in the A group meningitis cocci polysaccharide conjugate.

Embodiment 3

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get A group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence is as sample 3; Other gets this A group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this A group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube.Add the cold phenol solution of 4ml to every centrifuge tube, vibration mixing 20 minutes was put ice bath 15 minutes, centrifugal 20 minutes of 7000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the phosphorus content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " A group's polysaccharide concentration=phosphorus content/0.08 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝94.7μg/ml，p2＝7.6μg/ml，p3＝114.9μg/ml，p4＝112.3μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 97.7%;

Calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 8.2% in the A group meningitis cocci polysaccharide conjugate.

Embodiment 4

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get C group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence is as sample 3; Other gets this C group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube.Add the cold phenol solution of 2ml to every centrifuge tube, vibration mixing 10 minutes was put ice bath 5 minutes, centrifugal 10 minutes of 5000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " C group's polysaccharide concentration=sialic acid content/0.8 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝68.5μg/ml，p2＝7.8μg/ml，p3＝57.2μg/ml，p4＝54.5μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 95.3%;

Calculate dissociation amylase content according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 11.9% in the C group meningitis cocci polysaccharide conjugate.

Embodiment 5

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get C group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence is as sample 3; Other gets this C group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube.Add the cold phenol solution of 10ml to every centrifuge tube, vibration mixing 30 minutes was put ice bath 20 minutes, centrifugal 30 minutes of 10000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " C group's polysaccharide concentration=sialic acid content/0.8 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝116.2μg/ml，p2＝6.6μg/ml，p3＝49.6μg/ml，p4＝47.2μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 95.2%;

Calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 6.0% in the C group meningitis cocci polysaccharide conjugate.

Embodiment 6

500 grams are analyzed pure anhydrous sodium acetate and be dissolved in 800ml water for injection under heating, use 0.45 filtering with microporous membrane while hot, bulk crystallization occurs after the sodium acetate solution cooling after the filtration, and this moment, supernatant was the saturated acetic acid sodium solution.Get this saturated acetic acid sodium solution 100ml, add injection and be diluted with water to 1000ml, regulating pH with 1mol/L hydrochloric acid is 6.9～7.1, is 1/10 saturated neutral sodium acetate solution.Get 500 grams and analyze one bottle in purified petroleum benzin phenol, heating makes thawing under 60 ℃ of water-baths, and the phenol that will melt is poured in the container that 200ml 1/10 saturated neutral sodium acetate solution is housed while hot, and mixing is put 4 ℃ of refrigerators preservations and spent the night rapidly, is cold phenol solution.

Get C group meningitis cocci polysaccharide conjugate 2ml to be checked as sample 1, the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence is as sample 3; Other gets this C group meningitis cocci bond 2ml and places the 15ml centrifuge tube, and the derivative solution 2ml that gets this C group meningitis cocci polysaccharide conjugate correspondence to be checked places another 15ml centrifuge tube.Add the cold phenol solution of 4ml to every centrifuge tube, vibration mixing 20 minutes was put ice bath 15 minutes, centrifugal 20 minutes of 7000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

Measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration according to formula " C group's polysaccharide concentration=sialic acid content/0.8 " calculating sample 1, sample 2, sample 3, sample 4 is respectively:

p1＝50.2μg/ml，p2＝3.1μg/ml，p3＝50.5μg/ml，p4＝48.6μg/ml

According to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery is 96.2%;

Calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ Free polyoses content is 6.4% in the C group meningitis cocci polysaccharide conjugate.

Claims (1)

1, free measurement of the polysaccharide content method in a kind of A, the C group meningitis cocci polysaccharide conjugate the steps include:

(1) get meningococcal polysacharide bond 2ml to be checked as sample 1, the derivative solution 2ml that gets this meningococcal polysacharide bond correspondence to be checked is as sample 3;

(2) get this meningococcal polysacharide bond 2ml in addition and place the 15ml centrifuge tube, the derivative solution 2ml that gets this meningococcal polysacharide bond correspondence to be checked places another 15ml centrifuge tube; Add the cold phenol solution of 2～10ml to every centrifuge tube, vibration mixing 10～30 minutes was put ice bath 5～20 minutes, centrifugal 10～30 minutes of 5000～10000rpm, and every centrifuge tube is collected supernatant separately, and the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

Described cold phenol solution is the ratio preparation that adds the saturated neutral sodium acetate solution dissolving of 40ml1/10 according to every 100g phenol, is chilled to 4 ℃ then in advance and is prepared from;

(3) A group meningitis cocci polysaccharide conjugate is measured phosphorus content, calculates the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4; C group meningitis cocci polysaccharide conjugate is measured sialic acid content, calculates the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4; The polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 is respectively p1, p2, p3, p4;

(4) according to formula calculate recovery rate P:

$P=\frac{p4}{p3}\×100\%,$ The recovery should be 80～120%;

(5) calculate dissociation amylase content H according to formula:

$H=\frac{p2}{p1}\÷P\×100\%,$ P is the recovery in the formula.