CN102175841A - Method for measuring content of free polysaccharides in meningococcal polysaccharide combo - Google Patents

Abstract

The invention provides a method for measuring the content of free polysaccharides in A, C, W135 and Y cluster meningococcal polysaccharide combo, wherein the method comprises the following steps: 1) sampling: respectively taking 1ml of a meningococcal polysaccharide combo to be measured and a corresponding derivative solution of the meningococcal polysaccharide combo to be measured as a sample 1 and a sample 3; 2) centrifuging: additionally placing the meningococcal polysaccharide combo and the corresponding derivative solution of the meningococcal polysaccharide combo to be measured in centrifuge tubes; adding a sodium deoxycholate solution into each centrifuge tube, performing ice bath for 30 minutes and adding a hydrochloric acid solution; fully oscillating, centrifuging and collecting the supernatant of each centrifuge tube as a sample 2 and a sample 4; 3) calculating the concentration of the polysaccharides; and 4) calculating the content of the free polysaccharides. The method provided by the invention can measure the content of free polysaccharides in A, C, W135 and Y cluster meningococcal polysaccharide combo accurately and simply, so that the quality of products is evaluated.

Description

Free measurement of the polysaccharide content method in a kind of meningococcal polysacharide bond

Technical field

The present invention relates to a kind of assay method of free polysaccharide, be specifically related to free measurement of the polysaccharide content method in a kind of A, C, W135, the Y group meningitis cocci polysaccharide conjugate.

Background technology

Meningococcal infection is the primary pathogenic bacteria that cause children's meningitis and explosive septicemia.According to statistics, global annual epidemic meningitis number of the infected is about 500,000, and especially developing country's incidence of disease is higher.In the world wide, nearly all meningitis disease is caused by A group, B group, C group, Y group and W135 group meningitis cocci that all vaccine is prevention and controls meningitic ideal measure and inoculate safely and effectively.

At home and abroad the meningitis vaccines of listing mainly contains polysaccharide vaccine and polysaccharide conjugate vaccine two big classes at present, wherein the antigen of polysaccharide vaccine is haptens, be T-independent antigen, its immune response of inducing does not have the T lymphocyte to participate in, and can not produce immunological memory.So WHO advised once that the epidemic meningitis polysaccharide vaccine generally was not used in baby below 2 years old, child's inoculation.The epidemic meningitis polysaccharide conjugate vaccine then is meningococcal capsular polysaccharide and the protein carrier be combined into that will extract, its antigen is T cell dependence antigen, its immune response of inducing has the T lymphocyte to participate in, can stimulate body to produce high-level antibody, enhancing is to infant's immunological memory, thus the raising immune effect.

The content of free polysaccharide is one of key index of polysaccharide conjugate vaccine quality control in the polysaccharide conjugate vaccine, and dissociation amylase content surpasses prescribed limit can produce adverse influence to the vaccine clinical effectiveness.Therefore dissociation amylase content is the important indicator of reflection combined vaccine quality good or not.Have in the foreign literature report and adopt hydrophobic chromatography, HPLC method to carry out the mensuration of free polysaccharide in the polysaccharide conjugate, but because of being vulnerable to the influence of the close impurity of molecular size in the chromatography process, the measurement result error is bigger.

People such as domestic Zhu Wei have introduced a kind of method of utilizing ethanol precipitation to measure A group meningitis cocci polysaccharide-tetanus toxoid bond free polysaccharide in " foundation of free polysaccharide determination method in A group meningitis cocci polysaccharide-tetanus toxoid bond ", but this method operation is more loaded down with trivial details, a sample needs many days ability to obtain the result, may have certain error to the lower sample of dissociation amylase content simultaneously;

People such as state Neihuang County town disclose " assay method of free polysaccharide in a kind of A, the C group meningitis cocci polysaccharide conjugate " in Chinese patent 200610048876.9, the method needs to add cold phenol solution and shakes in sample, but cold phenol toxicity is stronger, skin, mucous membrane there is strong corrosive attack, long-term contact can suppress nervous centralis or infringement liver, renal function, sucks phenol and also can cause headache, dizzy, weak, blurred vision, pulmonary edema etc.

Bigger owing to different polysaccharide conjugate nature differences in addition, so the mensuration of free polysaccharide is a technical barrier always in the polysaccharide conjugate vaccine, does not still have the report that utilizes the NaTDC extracting process to measure free polyoses content in the polysaccharide conjugate at present.

Summary of the invention

Method of the present invention has overcome the deficiency of existing mensuration dissociation amylase content method, can accurately measure the content of free polysaccharide in A, C, W135, the Y group meningitis cocci polysaccharide conjugate, the method for operating simple and fast.

The method of free polysaccharide in a kind of meningococcal polysacharide bond of mensuration provided by the invention, sampling, centrifugal, calculate polysaccharide concentration, calculate dissociation amylase content, in the derivant of meningococcal polysacharide bond and meningococcal polysacharide bond correspondence to be measured, add the deoxycholic acid sodium solution when wherein centrifugal.

The method of free polysaccharide is applicable to A, C, W135, Y group meningitis cocci polysaccharide conjugate in a kind of meningococcal polysacharide bond of mensuration provided by the invention.

The method of measuring free polysaccharide in A, C, W135, the Y group meningitis cocci polysaccharide conjugate provided by the invention may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; DOC (NaTDC) solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M HCl solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: A group meningitis cocci polysaccharide conjugate is measured phosphorus content, calculates the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 by phosphorus content; C group, W135 group, Y group meningitis cocci polysaccharide conjugate are measured sialic acid content, and by the polysaccharide concentration of sialic acid content calculating sample 1, sample 2, sample 3, sample 4, the polysaccharide concentration of sample 1, sample 2, sample 3, sample 4 is respectively C1, C2, C3, C4.

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

Free measurement of the polysaccharide content method in a kind of A group meningitis cocci polysaccharide conjugate provided by the invention may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: A group meningitis cocci polysaccharide conjugate is measured phosphorus content, according to " A group's polysaccharide concentration=phosphorus content/0.08 ", calculate 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 C1, C2, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

Free measurement of the polysaccharide content method in a kind of C group meningitis cocci polysaccharide conjugate provided by the invention may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: C group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " C group's polysaccharide concentration=sialic acid content/0.8 ", calculate 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 C1, C2, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

Free measurement of the polysaccharide content method in a kind of W135 group meningitis cocci polysaccharide conjugate provided by the invention may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: W135 group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " W135 group's polysaccharide concentration=sialic acid content/0.56 ", calculate 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 C1, C2, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

Free measurement of the polysaccharide content method in a kind of Y group meningitis cocci polysaccharide conjugate provided by the invention may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: Y group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " Y group's polysaccharide concentration=sialic acid content/0.56 ", calculate 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 C1, C2, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

Wherein

The concentration unit of described DOC is 1% (1g/100ml);

The concentration unit of described hydrochloric acid is mol/L, is abbreviated as M;

Recovery P is higher than 95% and shows the accuracy height between 80-120% among the present invention.

The invention has the beneficial effects as follows:

1, the invention provides a kind of accurately, the method for free polyoses content in fast measuring A group, C group, W135 group and the Y group meningitis cocci polysaccharide conjugate, thereby estimate quality of item.

2, NaTDC, molecular formula: C ₂₄H ₄₀O ₄Na, molecular weight: 414.56, proterties: white crystalline powder.Similar bile smell has strong bitter taste.Easily moisture absorption.Soluble in water, be slightly soluble in absolute alcohol, be insoluble to ether.Low toxicity, median lethal dose (rat, per os) 1370mg/kg.Pungency is arranged.

The present invention utilizes the characteristic of DOC (NaTDC) precipitable protein under acid condition, sample through DOC handle and high speed centrifugation after, bond in the combined vaccine is precipitated and polysaccharide is still stayed in the supernatant under similarity condition, to separate in conjunction with polysaccharide and free polysaccharide with this, by measuring the polyoses content of centrifuged supernatant and stoste respectively, can calculate the content of free polysaccharide.

3, compare with patent 200610048876.9, though the recovery is more or less the same, but overcome cold phenol to big this unfavorable factor of human body harm, this method is not only applicable to the mensuration of A group, C group meningitis cocci polysaccharide conjugate dissociation amylase content simultaneously, can measure free polyoses content in W135, the Y group meningitis cocci polysaccharide conjugate quickly and accurately yet.

Embodiment

Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.

Embodiment 1

1, sampling: get A group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this A group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this A group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution lml that gets this A group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4.

3, calculate polysaccharide concentration: measure the phosphorus content of sample 1, sample 2, sample 3, sample 4, calculate polysaccharide concentration C1, C2, C3 and the C4 of sample 1, sample 2, sample 3, sample 4 according to formula " A group's polysaccharide concentration=phosphorus content/0.08 ".

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus A group polysaccharide conjugate samples, and the result sees table 1 for details.

Table 1:3 criticizes meningococcus A group polysaccharide conjugate sample determination result

The result of table 1 shows: this method can accurately be measured free polyoses content in the meningococcus A group polysaccharide conjugate.

Embodiment 2

1, sampling: get C group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this C group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this C group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets this C group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3, calculate polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, calculate sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4 according to formula " C group's polysaccharide concentration=sialic acid content/0.8 ";

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus C group polysaccharide conjugate samples, and the result sees table 2 for details:

Table 2:3 criticizes meningococcus C group polysaccharide conjugate sample determination result

The result of table 2 shows: this method can accurately be measured free polyoses content in the meningococcus C group polysaccharide conjugate.

Embodiment 3

1, sampling: get W135 group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this W135 group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this W135 group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets this W135 group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3, calculate polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, calculate sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4 according to formula " W135 group's polysaccharide concentration=sialic acid content/0.56 ";

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus W135 group polysaccharide conjugate samples, and the result sees table 3 for details:

Table 3:3 criticizes meningococcus W135 group polysaccharide conjugate sample determination result

20100407	233.7	22.9	196.3	189.5	96.5	10.2
							20100412	286.5	26.0	225.6	218.9	97.0	9.4

The result of table 3 shows: this method can accurately be measured free polyoses content in the meningococcus W135 group polysaccharide conjugate.

Embodiment 4

1, sampling: get Y group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this Y group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this Y group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets this Y group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3, calculate polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, calculate sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4 according to formula " Y group's polysaccharide concentration=sialic acid content/0.56 ";

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus Y group polysaccharide conjugate samples, and the result sees table 4 for details:

Table 4:3 criticizes meningococcus Y group polysaccharide conjugate sample determination result

The result of table 4 shows: this method can accurately be measured free polyoses content in the meningococcus Y group polysaccharide conjugate.

Embodiment 5

1, sampling: get W135 group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this W135 group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this W135 group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets this W135 group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 6000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3, calculate polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, calculate sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4 according to formula " W135 group's polysaccharide concentration=sialic acid content/0.56 ";

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus W135 group polysaccharide conjugate samples, and the result sees table 3 for details:

Table 5:3 criticizes meningococcus W135 group polysaccharide conjugate sample determination result

The result of table 3 shows: this method can accurately be measured free polyoses content in the meningococcus W135 group polysaccharide conjugate.

Embodiment 6

1, sampling: get Y group meningitis cocci polysaccharide conjugate 1ml to be checked as sample 1, the derivative solution of getting this Y group meningitis cocci polysaccharide conjugate correspondence is as sample 3;

2, centrifugal: other gets this Y group meningitis cocci polysaccharide conjugate 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets this Y group meningitis cocci polysaccharide conjugate correspondence to be checked places another 1.5ml centrifuge tube; Add the DOC solution of 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, and the back adds 0.5M HCL solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 6000rpm, every centrifuge tube is collected supernatant 1ml separately, wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3, calculate polysaccharide concentration: measure the sialic acid content of sample 1, sample 2, sample 3, sample 4, calculate sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4 according to formula " Y group's polysaccharide concentration=sialic acid content/0.56 ";

4, calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5, utilization this method is measured 3 batches of meningococcus Y group polysaccharide conjugate samples, and the result sees table 4 for details:

Table 6:3 criticizes meningococcus Y group polysaccharide conjugate sample determination result

The result of table 4 shows: this method can accurately be measured free polyoses content in the meningococcus Y group polysaccharide conjugate.

Though, above used general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (6)

1. free measurement of the polysaccharide content method in the meningococcal polysacharide bond, comprise sampling, centrifugal, calculate polysaccharide concentration, calculate dissociation amylase content, it is characterized in that, in the derivant of meningococcal polysacharide bond and meningococcal polysacharide bond correspondence to be measured, add the deoxycholic acid sodium solution when centrifugal.

2. assay method according to claim 1 is characterized in that, this meningococcal polysacharide bond is selected from A group, C group, W135 group or Y group.

3. free measurement of the polysaccharide content method in the A group meningitis cocci polysaccharide conjugate according to claim 2 may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: A group meningitis cocci polysaccharide conjugate is measured phosphorus content, according to " A group's polysaccharide concentration=phosphorus content/0.08 ", calculates sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

4. free measurement of the polysaccharide content method in the C group meningitis cocci polysaccharide conjugate according to claim 2 may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: C group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " C group's polysaccharide concentration=sialic acid content/0.8 ", calculates sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

5. free measurement of the polysaccharide content method in the W135 group meningitis cocci polysaccharide conjugate according to claim 2 may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: W135 group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " W135 group's polysaccharide concentration=sialic acid content/0.56 ", calculates sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$

6. free measurement of the polysaccharide content method in the Y group meningitis cocci polysaccharide conjugate according to claim 2 may further comprise the steps:

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

2) centrifugal: other gets meningococcal polysacharide bond 1ml and places the 1.5ml centrifuge tube, and the derivative solution 1ml that gets meningococcal polysacharide bond correspondence to be checked places another 1.5ml centrifuge tube; The deoxycholic acid sodium solution that adds 100 μ l 1% to every centrifuge tube, ice bath kept 30 minutes, the back adds 0.5M hydrochloric acid solution 25 μ l, fully vibration, centrifugal 15～20 minutes of 5000～6000rpm, every centrifuge tube is collected supernatant 1ml separately, and wherein the bond supernatant is a sample 2, and the derivant supernatant is a sample 4;

3) calculate polysaccharide concentration: Y group meningitis cocci polysaccharide conjugate is measured sialic acid content, according to " Y group's polysaccharide concentration=sialic acid content/0.56 ", calculates sample 1, sample 2, sample 3, polysaccharide concentration C1, the C2 of sample 4, C3, C4;

4) calculate dissociation amylase content: wherein P is the recovery, and H is a dissociation amylase content

$P=\frac{C4}{C3}\×100\%,$ $H=\frac{C2}{C1}\÷P\×100\%.$