CN1963500B - Method for determining dissociation amylase content of pneumonia streptococcus capsular polysaccharide combo - Google Patents

Abstract

This invention relates to one test method for free polysaccharide content of pneumococcal peritonitis compound in biological technique field, which comprises the following steps: a, fetching same content of the compound and adding ethanol without water and common salt for 70 to 90 hours under -20 degrees then removing upper liquid by anticentripetal and using proper agent to clear deposition to collect upper liquid through filter salt and ethanol; b, using anthranone to test sample one and two to compute free polysaccharide content.

Description

Free measurement of the polysaccharide content method in a kind of streptococcus pneumoniae capsular polysaccharide bond

Technical field:

The invention belongs to biological technical field, more specifically, relate to free measurement of the polysaccharide content method in a kind of streptococcus pneumoniae capsular polysaccharide bond.

Background technology:

Streptococcus pneumonia is the important pathogen of bringing out bacterial pneumonia, meningitis, pleurisy, endocarditis, tympanitis, is the modal cause of disease of developing country's children's bacterial pneumonia.The coccus the infected that dies of pneumonia global every year has 100-200 ten thousand.China's pneumonia incidence of disease is very high always, and over nearly 50 years, the bacteremia mortality ratio is paced up and down between 25-29% always among the pneumococcal infection person.Pneumonia also is to cause one of old man's main causes of death more than 60 years old in addition.Because the streptococcus pneumonia antibody-resistant bacterium is up to more than 96%, common problem has at present become international.

Nineteen eighty-three, the U.S. successfully developed 23 valency polysaccharide vaccines, but the protectiveness that this vaccine is induced in infant below 2 years old is low, no immunological memory reaction.Chemically protein carrier being covalently bound on the streptococcus pneumoniae capsular polysaccharide, being prepared into the streptococcus pneumoniae capsular polysaccharide combined vaccine, is T cell dependence antigen with polysaccharide by T cell dependent/non-dependent antigenic shift.Behind the inoculation polysaccharide-protein combined vaccine, can make infant below 2 years old produce efficient immune, thereby obtain immunoprotection.Preliminary clinical test results shows, security and the immunogenicity of streptococcus pneumoniae capsular polysaccharide combined vaccine in the infant is good, induce the antipolysaccharide antibody of generation to be higher than polysaccharide vaccine, but and the induction of immunity anamnestic response, so the development of streptococcus pneumoniae capsular polysaccharide combined vaccine has great social significance and value.

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 based on the difference of the electrically charged character of molecule and distinguish in conjunction with polysaccharide and free polysaccharide, also be vulnerable to influence with the impurity of the electrically charged similar performance of polysaccharide, streptococcus pneumoniae polysaccharides does not have strong light absorption at ultraviolet region in addition, can't measure the dissociation amylase content of streptococcus pneumoniae polysaccharides bond in the high performance liquid chromatogram system of configuration UV-detector.Still there is not at present the report that utilizes ethanol precipitation, ultrafiltration, anthrone method to measure free polyoses content in the streptococcus pneumoniae capsular polysaccharide bond.

Summary of the invention:

Method of the present invention has overcome the deficiency that existing method can't accurately be measured streptococcus pneumoniae capsular polysaccharide bond dissociation amylase content, and this method can be measured the content of free polysaccharide in the streptococcus pneumoniae capsular polysaccharide bond accurately, simply.

Technical scheme of the present invention and step:

(1) getting streptococcus pneumoniae capsular polysaccharide bond to be measured is sample 1; The derivative solution of streptococcus pneumoniae capsular polysaccharide bond correspondence to be measured is a sample 3;

(2) get streptococcus pneumoniae capsular polysaccharide bond 4.5ml in addition, place the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumoniae capsular polysaccharide bond correspondence, place another 50ml centrifuge tube; Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70～90 hours; In centrifugal 60～90 minutes of 5 ℃, 4500～6500rpm, abandon supernatant;

(3) add 0.75ml 0～60% ethanolic solution to every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add identical ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40～60 minutes of 15 ℃, 4500～6500rpm, every centrifuge tube was collected supernatant separately;

(4) with the bond supernatant with the ultrafiltration of 3～100kD ultrafiltration cup, collect liquid after the ultrafiltration, ultrafiltration cup cleaning fluid, and add water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with the ultrafiltration of 3～100K ultrafiltration cup, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid, and add water for injection and be sample 4 to 3ml;

(5) with the polysaccharide concentration of anthrone method mensuration sample 1, sample 2, sample 3, sample 4, be respectively A1, A2, A3, A4;

(6) according to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery should be at 80-120%;

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

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ P is the recovery in the formula.

Streptococcus pneumonia wherein can for following any: 1 type, 2 types, 3 types, 4 types, 5 types, 6B type, 7F type, 8 types, 9N type, 9V type, 10A type, 11A type, 12F type, 14 types, 15B type, 17F type, 18C type, 19A type, 19F type, 20 types, 22F type, 23F type, 33F type; Used ultrafiltration cup molecular cut off can be 5～50kD.

The invention has the beneficial effects as follows: utilize streptococcus pneumoniae capsular polysaccharide and streptococcus pneumoniae capsular polysaccharide bond different settling characters in conjunction with polysaccharide and free polysaccharide under ethanol, strong electrolyte existence condition, with free polysaccharide with combine separation of polysaccharides, remove salt and the ethanol that disturbs anthrone method to measure by the ultrafiltration of ultrafiltration cup then, measure the polysaccharide concentration of each sample then by anthrone method, and then calculate the percentage composition of free polysaccharide.The invention provides the method for free polyoses content in a kind of accurate mensuration streptococcus pneumoniae capsular polysaccharide bond, thereby estimate quality of item.

Embodiment:

Embodiment 1

Getting streptococcus pneumonia 1 type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 1 type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 1 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 1 type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70 hours; In centrifugal 60 minutes of 5 ℃, 4500rpm, abandon supernatant.

Add 0.75ml water for injection (promptly containing ethanol 0%) in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; Add water for injection 2.25ml then, vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 4500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 3kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 3kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=29.37 μ g/ml, A2=5.54 μ g/ml, A3=36.53 μ g/ml, A4=53.03 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 96.78%.

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 12.99% in the streptococcus pneumonia 1 type capsular polysaccharide bond stoste.

Embodiment 2

Getting streptococcus pneumonia 2 type capsular polysaccharide bonds is sample 1, and the derivative solution of getting streptococcus pneumonia 2 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 2 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 2 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 90 hours; In centrifugal 90 minutes of 5 ℃, 6500rpm, abandon supernatant.

Add 0.75ml 60% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 60% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 60 minutes of 15 ℃, 6500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 100kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 100kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4.A1＝18.54μg/ml，A2＝4.92μg/ml，A3＝27.62μg/ml，A4＝40.82μg/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.53%.

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 17.95% in the streptococcus pneumonia 2 type capsular polysaccharide bond stostes.

Embodiment 3

Getting streptococcus pneumonia 3 type capsular polysaccharide bonds is sample 1, and the derivative solution of getting streptococcus pneumonia 3 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 3 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 3 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube. add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 90 hours; In centrifugal 70 minutes of 5 ℃, 5500rpm, abandon supernatant.

Add 0.75ml 40% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 40% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4.A1＝28.35μg/ml，A2＝5.78μg/ml，A3＝26.72μg/ml，A4＝40.21μg/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 100.32%.

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 13.55% in the streptococcus pneumonia 3 type capsular polysaccharide bond stostes.

Embodiment 4

Getting streptococcus pneumonia 4 type capsular polysaccharide bonds is sample 1, and the derivative solution of getting streptococcus pneumonia 4 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 4 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 4 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 90 hours; In centrifugal 90 minutes of 5 ℃, 6500rpm, abandon supernatant.

Add 0.75ml 60% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 60% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 60 minutes of 15 ℃, 6500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4.A1＝18.89μg/ml，A2＝5.05μg/ml，A3＝28.62μg/ml，A4＝41.41μg/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 96.46%.

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 18.48% in the streptococcus pneumonia 4 type capsular polysaccharide bond stostes.

Embodiment 5

Getting streptococcus pneumonia 5 type capsular polysaccharide bonds is sample 1; The derivative solution of getting streptococcus pneumonia 5 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 5 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 5 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5500rpm, abandon supernatant.

Add 0.75ml 45% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 45% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=32.37 μ g/ml, A2=7.54 μ g/ml, A3=24.53 μ g/ml, A4=37.03 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 100.64%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 15.43% in the streptococcus pneumonia 5 type capsular polysaccharide bond stostes.

Embodiment 6

Getting streptococcus pneumoniae 6B type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumoniae 6B type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumoniae 6B type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumoniae 6B type capsular polysaccharide bond correspondence, place the 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 85 hours; In centrifugal 60 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=42.26 μ g/ml, A2=11.42 μ g/ml, A3=26.53 μ g/ml, A4=38.73 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ Calculate recovery rate is 97.32%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 18.51% in the streptococcus pneumoniae 6B type capsular polysaccharide bond stoste.

Embodiment 7

Getting streptococcus pneumonia 7F type capsular polysaccharide bond is sample 1, and the derivative solution of getting streptococcus pneumonia 7F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 7F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 7F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 90 hours; In centrifugal 90 minutes of 5 ℃, 6500rpm, abandon supernatant.

Add 0.75ml 45% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 45% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 60 minutes of 15 ℃, 6500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4.A1＝21.89μg/ml，A2＝7.36μg/ml，A3＝28.12μg/ml，A4＝41.25μg/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 97.80%.

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 22.92% in the streptococcus pneumonia 7F type capsular polysaccharide bond stoste.

Embodiment 8

Getting streptococcus pneumonia 8 type capsular polysaccharide bonds is sample 1; The derivative solution of getting streptococcus pneumonia 8 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 8 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 8 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5500rpm, abandon supernatant.

Add 0.75ml 55% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 55% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=30.57 μ g/ml, A2=6.54 μ g/ml, A3=26.53 μ g/ml, A4=39.03 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.08%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 14.54% in the streptococcus pneumonia 8 type capsular polysaccharide bond stostes.

Embodiment 9

Getting streptococcus pneumonia 9N type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 9N type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 9N type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 9N type capsular polysaccharide bond correspondence, place the 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 85 hours; In centrifugal 60 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=40.26 μ g/ml, A2=9.42 μ g/ml, A3=25.53 μ g/ml, A4=37.23 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ Calculate recovery rate is 97.22%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 16.04% in the streptococcus pneumonia 9N type capsular polysaccharide bond stoste.

Embodiment 10

Getting streptococcus pneumonia 9V type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 9V type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 9V type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 9V type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4. to 3ml

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=28.02 μ g/ml, A2=2.47 μ g/ml, A3=31.20 μ g/ml, A4=46.08 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.5%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 6.0% in the streptococcus pneumonia 9V type capsular polysaccharide bond stoste.

Embodiment 11

Getting streptococcus pneumonia 10A type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 10A type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 10A type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 10A type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5500rpm, abandon supernatant.

Add 0.75ml 55% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 55% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5500rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=32.57 μ g/ml, A2=7.34 μ g/ml, A3=28.53 μ g/ml, A4=42.23 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.68%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 15.23% in the streptococcus pneumonia 10A type capsular polysaccharide bond stoste.

Embodiment 12

Getting streptococcus pneumonia 11A type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 11A type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 11A type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 11A type capsular polysaccharide bond correspondence, place the 50ml centrifuge tube. add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 85 hours; In centrifugal 60 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=38.52 μ g/ml, A2=6.42 μ g/ml, A3=26.53 μ g/ml, A4=38.76 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ Calculate recovery rate is 97.40%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 11.41% in the streptococcus pneumonia 11A type capsular polysaccharide bond stoste.

Embodiment 13

Getting streptococcus pneumonia 12F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 12F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 12F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 12F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=29.52 μ g/ml, A2=5.48 μ g/ml, A3=32.20 μ g/ml, A4=47.38 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.10%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 12.62% in the streptococcus pneumonia 12F type capsular polysaccharide bond stoste.

Embodiment 14

Getting streptococcus pneumonia 14 type capsular polysaccharide bonds is sample 1; The derivative solution of getting streptococcus pneumonia 14 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 14 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 14 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 75 hours; In centrifugal 80 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 55% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 55% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=26.65 μ g/ml, A2=5.62 μ g/ml, A3=23.61 μ g/ml, A4=34.33 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 96.94%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Calculating the polyoses content that dissociates in the streptococcus pneumonia 14 type capsular polysaccharide bond stostes is 14.5%.

Embodiment 15

Getting streptococcus pneumonia 15B type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 15B type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 15B type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 15B type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 50kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=28.52 μ g/ml, A2=5.48 μ g/ml, A3=31.20 μ g/ml, A4=46.38 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 99.10%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 12.93% in the streptococcus pneumonia 15B type capsular polysaccharide bond stoste.

Embodiment 16

Getting streptococcus pneumonia 17F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 17F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 17F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 17F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 75 hours; In centrifugal 80 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 55% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 55% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=27.35 μ g/ml, A2=6.52 μ g/ml, A3=24.61 μ g/ml, A4=36.33 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.42%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Calculating the polyoses content that dissociates in the streptococcus pneumonia 17F type capsular polysaccharide bond stoste is 16.15%.

Embodiment 17

Getting streptococcus pneumonia 18C type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 18C type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 18C type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 18C type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 60 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 30kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=28.65 μ g/ml, A2=4.32 μ g/ml, A3=30.42 μ g/ml, A4=45.92 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 100.64%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Calculating the polyoses content that dissociates in the streptococcus pneumonia 18C type capsular polysaccharide bond stoste is 9.98%.

Embodiment 18

Getting streptococcus pneumonia 19A type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 19A type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 19A type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 19A type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 80 hours; In centrifugal 70 minutes of 5 ℃, 5500rpm, abandon supernatant.

Add 0.75ml 45% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 45% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=24.85 μ g/ml, A2=3.82 μ g/ml, A3=32.42 μ g/ml, A4=46.82 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 96.28%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Calculating the polyoses content that dissociates in the streptococcus pneumonia 19A type capsular polysaccharide bond stoste is 10.64%.

Embodiment 19

Getting streptococcus pneumonia 19F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 19F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 19F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 19F type capsular polysaccharide bond correspondence, place the 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70 hours; In centrifugal 60 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 60% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 60% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4. to 3ml

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=31.65 μ g/ml, A2=5.12 μ g/ml, A3=28.62 μ g/ml, A4=40.92 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 95.32%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 11.31% in the streptococcus pneumonia 19F type capsular polysaccharide bond stoste.

Embodiment 20

Getting streptococcus pneumonia 20 type capsular polysaccharide bonds is sample 1; The derivative solution of getting streptococcus pneumonia 20 type capsular polysaccharide bond correspondences to be measured is a sample 3.

Other gets streptococcus pneumonia 20 type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 20 type capsular polysaccharide bond correspondences, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70 hours; In centrifugal 80 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 40% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 40% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=19.77 μ g/ml, A2=2.54 μ g/ml, A3=29.53 μ g/ml, A4=41.03 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 92.6%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 9.2% in the streptococcus pneumonia 20 type capsular polysaccharide bond stostes.

Embodiment 21

Getting streptococcus pneumonia 22F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 22F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 22F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 22F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70 hours; In centrifugal 80 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=29.57 μ g/ml, A2=6.24 μ g/ml, A3=28.53 μ g/ml, A4=42.03 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.21%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 14.32% in the streptococcus pneumonia 22F type capsular polysaccharide bond stoste.

Embodiment 22

Getting streptococcus pneumonia 23F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 23F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 23F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 23F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70 hours; In centrifugal 80 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml water for injection (containing ethanol 0%) in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add water for injection 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 5kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=31.71 μ g/ml, A2=7.27 μ g/ml, A3=27.36 μ g/ml, A4=38.50 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 93.8%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 16.3% in the streptococcus pneumonia 23F type capsular polysaccharide bond stoste.

Embodiment 23

Getting streptococcus pneumonia 33F type capsular polysaccharide bond is sample 1; The derivative solution of getting streptococcus pneumonia 33F type capsular polysaccharide bond correspondence to be measured is a sample 3.

Other gets streptococcus pneumonia 33F type capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumonia 33F type capsular polysaccharide bond correspondence, place another 50ml centrifuge tube.Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 75 hours; In centrifugal 70 minutes of 5 ℃, 5000rpm, abandon supernatant.

Add 0.75ml 50% ethanolic solution in every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add 50% ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 50 minutes of 15 ℃, 5000rpm, every centrifuge tube was collected supernatant separately.

The bond supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with water for injection ultrafiltration 3 times, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid with 10kD ultrafiltration cup, and adds water for injection and be sample 4 to 3ml.

Polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4; A1=23.45 μ g/ml, A2=4.31 μ g/ml, A3=26.78 μ g/ml, A4=39.69 μ g/ml

According to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is 98.8%;

Calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ Free polyoses content is 12.4% in the streptococcus pneumonia 33F type capsular polysaccharide bond stoste.

Claims (3)

1. free measurement of the polysaccharide content method in the streptococcus pneumoniae capsular polysaccharide bond the steps include:

(1). getting streptococcus pneumoniae capsular polysaccharide bond to be measured is sample 1; The derivative solution of streptococcus pneumoniae capsular polysaccharide bond correspondence to be measured is a sample 3;

(2). other gets streptococcus pneumoniae capsular polysaccharide bond 4.5ml, places the 50ml centrifuge tube; Get the derivative solution 4.5ml of streptococcus pneumoniae capsular polysaccharide bond correspondence, place another 50ml centrifuge tube; Add 5mol/L sodium chloride solution 1.125ml to every centrifuge tube, the vibration mixing adds absolute ethyl alcohol 22.5ml again, the vibration mixing, put-20 ℃ 70～90 hours; In centrifugal 60～90 minutes of 5 ℃, 4500～6500rpm, abandon supernatant;

(3). add 0.75ml 0～60% ethanolic solution to every centrifuge tube, vibration mixes, and room temperature left standstill 1 hour; And then add identical ethanolic solution 2.25ml, and vibration mixes, and room temperature left standstill 2 hours; In centrifugal 40～60 minutes of 15 ℃, 4500～6500rpm, every centrifuge tube was collected supernatant separately;

(4). the bond supernatant with the ultrafiltration of 3～100kD ultrafiltration cup, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid, and add water for injection and be sample 2 to 3ml; Corresponding derivant supernatant with the ultrafiltration of 3～100KD ultrafiltration cup, is collected liquid after the ultrafiltration, ultrafiltration cup cleaning fluid, and add water for injection and be sample 4 to 3ml;

(5). the polysaccharide concentration with anthrone method mensuration sample 1, sample 2, sample 3, sample 4 is respectively A1, A2, A3, A4;

(6). according to formula calculate recovery rate P:

$P=\frac{A4}{A3\×1.5}\×100\%,$ The recovery is at 80-120%;

(7). calculate dissociation amylase content H according to formula:

$H=\frac{A2}{A1\×1.5}\÷P\×100\%,$ P is the recovery in the formula.

2. free measurement of the polysaccharide content method in a kind of streptococcus pneumoniae capsular polysaccharide bond according to claim 1 is characterized by: described streptococcus pneumonia be following any one: 1 type, 2 types, 3 types, 4 types, 5 types, 6B type, 7F type, 8 types, 9N type, 9V type, 10A type, 11A type, 12F type, 14 types, 15B type, 17F type, 18C type, 19A type, 19F type, 20 types, 22F type, 23F type, 33F type.

3. free measurement of the polysaccharide content method in a kind of streptococcus pneumoniae capsular polysaccharide bond according to claim 1, it is characterized by: described ultrafiltration cup molecular cut off is 5～50kD.