CN103837671A - Method for measuring free polysaccharide content in polysaccharide protein conjugate - Google Patents
Method for measuring free polysaccharide content in polysaccharide protein conjugate Download PDFInfo
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 63
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 63
- 150000004676 glycans Chemical class 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 62
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 32
- 239000006228 supernatant Substances 0.000 claims abstract description 37
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000000126 substance Substances 0.000 claims abstract description 11
- 230000001900 immune effect Effects 0.000 claims abstract description 10
- 239000000523 sample Substances 0.000 claims description 76
- 239000004382 Amylase Substances 0.000 claims description 35
- 102000013142 Amylases Human genes 0.000 claims description 35
- 108010065511 Amylases Proteins 0.000 claims description 35
- 235000019418 amylase Nutrition 0.000 claims description 35
- 238000010494 dissociation reaction Methods 0.000 claims description 35
- 230000005593 dissociations Effects 0.000 claims description 35
- 239000005457 ice water Substances 0.000 claims description 17
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 claims description 17
- 230000003068 static effect Effects 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 15
- 238000003556 assay Methods 0.000 claims description 13
- 241000193998 Streptococcus pneumoniae Species 0.000 claims description 9
- MYKOKMFESWKQRX-UHFFFAOYSA-N 10h-anthracen-9-one;sulfuric acid Chemical compound OS(O)(=O)=O.C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 MYKOKMFESWKQRX-UHFFFAOYSA-N 0.000 claims description 7
- 241000606768 Haemophilus influenzae Species 0.000 claims description 7
- 229940047650 haemophilus influenzae Drugs 0.000 claims description 7
- 229940031000 streptococcus pneumoniae Drugs 0.000 claims description 7
- 238000005259 measurement Methods 0.000 claims description 6
- 238000002965 ELISA Methods 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 4
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 4
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 4
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 4
- 239000011574 phosphorus Substances 0.000 claims description 4
- 229910052698 phosphorus Inorganic materials 0.000 claims description 4
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 3
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 3
- 201000005010 Streptococcus pneumonia Diseases 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims description 2
- 239000013595 supernatant sample Substances 0.000 claims description 2
- 238000004879 turbidimetry Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 abstract description 34
- 229960003964 deoxycholic acid Drugs 0.000 abstract description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 abstract description 2
- 239000011550 stock solution Substances 0.000 abstract 2
- 238000011084 recovery Methods 0.000 description 22
- 239000000203 mixture Substances 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 201000009906 Meningitis Diseases 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229920002306 Glycocalyx Polymers 0.000 description 3
- 210000004517 glycocalyx Anatomy 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
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- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 238000012869 ethanol precipitation Methods 0.000 description 1
- IXCSERBJSXMMFS-UHFFFAOYSA-N hcl hcl Chemical compound Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229940031937 polysaccharide vaccine Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4722—Proteoglycans, e.g. aggreccan
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Abstract
The invention relates to a method for measuring free polysaccharide content in polysaccharide protein conjugate, which comprises the following steps: under ice bath conditions, adding a sodium deoxycholate solution into a conjugate stock solution with a certain concentration, mixing, standing, adding a trichloroacetic acid solution, evenly mixing, centrifugating, and taking the supernatant, wherein the supernatant is a sample A to be measured, and the conjugate stock solution to be measured is a sample B to be detected; respectively measuring polysaccharide contents a and b in the sample A and sample B by a chemical method or immunologic method; and calculating the free polysaccharide content in the conjugate according to the formula free polysaccharide content=a/b*100%.
Description
Technical field:
The present invention relates to the detection of vaccine, particularly free measurement of the polysaccharide content in a kind of GL-PP bond.
Background technology:
The use of various polysaccharide vaccines, has reduced the incidence of disease of corresponding infectious disease effectively.But because capsular polysaccharide belongs to T-independent antigen, do not possess immunological memory reaction, and can not produce protection antibody to 2 years old following children, therefore, its use has certain limitation.Research shows, by chemical method, protein carrier is covalently bonded in and on capsular polysaccharide, forms GL-PP bond and can make it to be converted into T cell dependent type antigen, thereby the protection to 5 years old Infants Below can be provided.
Experiment shows, in GL-PP bond, the too high meeting of dissociation amylase content causes in vaccine effectively antigenic content to reduce, and clinical effectiveness is had a negative impact.According to the requirement of European Pharmacopoeia, also need the content of the free polysaccharide in GL-PP bond to measure in addition.Therefore, dissociation amylase content is a key factor for the quality control of product.
Due to the combination difference of the purifying mode of bond in addition of polysaccharide kind and polysaccharide and albumen, the universal method of also not measuring about dissociation amylase content at present.We prove by experiment, the method for some mensuration dissociation amylase contents that exist at present, but there is respectively certain limitation, can not measure the content of free polysaccharide in all bonds.There is bibliographical information abroad, can add after deoxycholic acid, then add hydrochloric acid (HCl) to measure the free polysaccharide in the GL-PP bond of haemophilus influenzae.We measure the dissociation amylase content in pneumonia GL-PP bond stoste by the method, and effect is bad, and main cause is albumen can not be precipitated completely after adding HCl, therefore causes free sugar assay result to have certain deviation.Also have bibliographical information to utilize ethanol precipitation to measure the method for A group meningitis cocci polysaccharide-tetanus toxoid bond free polysaccharide, this method length consuming time, and step is various, and the sample size needing is larger.According to documents and materials, there is no at present the report that utilizes trichloroacetic acid precipitation method to measure dissociation amylase content in GL-PP bond stoste.
Summary of the invention:
The present invention discloses a kind of free measurement of the polysaccharide content method in GL-PP bond, and the method step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, add deoxycholic acid sodium solution, fully mix, static; Add trichloroacetic acid solution, mix rear low-temperature centrifugation;
2) supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize chemical method or immunological method respectively the polyoses content in working sample A and sample B be a and b;
3) in GL-PP bond, the percentage composition of free polysaccharide is a/b × 100%.
Preferably, assay method of the present invention, step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, add 0.3%～3% deoxycholic acid sodium solution, addition is 5%～15% of bond stoste volume, fully mixes static 20min～40min; Then add 100% trichloroacetic acid solution, addition is the 0.1%-1% of bond stoste volume, mixes latter 4～8 DEG C, 8000rpm～12000rpm, centrifugal 15～20min;
2) supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize chemical method or immunological method respectively the polyoses content in working sample A and sample B be a and b;
3) in GL-PP bond, dissociation amylase content is a/b × 100%.
Particularly preferred, assay method of the present invention, step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, 1% deoxycholic acid sodium solution, addition is 10% of bond stoste volume, fully mixes static 20min～40min; Then add 100% trichloroacetic acid solution, addition is 0.5% of bond stoste volume, mixes latter 4 DEG C, 12000rpm, centrifugal 15min;
2) getting supernatant is working sample A, and getting bond stoste to be measured is testing sample B, and utilizing the polyoses content in chemical method or immunological method difference working sample A and sample B is a and b;
3) in GL-PP bond, dissociation amylase content is a/b × 100%.
Wherein, described GL-PP bond is any one GL-PP combined vaccine, preferably haemophilus influenzae GL-PP bond, streptococcus pneumoniae polysaccharides protein conjugates, or meningococcal polysacharide protein conjugates.
Wherein, in described GL-PP bond stoste, polysaccharide concentration is 50-150ug/ml, and preferred concentration is 100ug/ml.
The GL-PP bond that described streptococcus pneumoniae polysaccharides protein conjugates is following any one streptococcus pneumonia: 1 type, 2 types, 3 types, 4 types, 5 types, 6A type, 6B type, 7F type, 8 types, 9V type, 9N type, 10A type, 12F type, 14 types, 15B type, 17F type, 18C type, 19A type, 19F type, 20 types, 22F type, 23F, 33F type.
Wherein, the chemical method described in step 2 comprises sulfuric acid anthrone method, ribose determination method, phosphorus detection method or Application of Sialic Acid Measurement method etc., belongs to prior art.
Immunological method described in step 2 is enzyme linked immunosorbent assay or immune turbidimetry, belongs to prior art.
In addition, GL-PP bond of the present invention, obtains according to the method for step 1 of the present invention
Protein content c in supernatant sample A, and protein content d in GL-PP bond stoste B, the result of utilizing Lowry method or BCA method to measure is; Wherein c/d<0.01.
Described in the application, measure the comparison of method and the conventional method of polysaccharide
The invention has the advantages that:
Applicability of the present invention is strong, can measure the content of free polysaccharide in bonds such as comprising haemophilus influenzae GL-PP bond, streptococcus pneumoniae polysaccharides protein conjugates and meningococcal polysacharide protein conjugates.Compared with prior art, the present invention can measure and comprise that (type comprises: 1 type, 2 types, 3 types for haemophilus influenzae (Hib) polysaccharide conjugate, streptococcus pneumoniae polysaccharides bond, 4 types, 5 types, 6A type, 6B type, 7F type, 8 types, 9V type, 9N type, 10A type, 12F type, 14 types, 15B type, 17F type, 18C type, 19A type, 19F type, 20 types, 22F type, 23F, 33F type etc.) and the free measurement of the polysaccharide content in interior GL-PP bond of meningococcal polysacharide bond.Applied range.
Obtain through screening at condition determination of the present invention, with this understanding, albumen is completely precipitated, and free polysaccharide can be not precipitated, and polysaccharide can not be degraded.
Principle of the present invention is, trichloroacetic acid can make albumen generation sex change and separate out, and in conjunction with polysaccharide, because the albuminous degeneration of coupling precipitates, and free polysaccharide is still present in supernatant.By centrifugal, free polysaccharide and bond are separated, then measure respectively the sugared content in upper cleer and peaceful stoste, can try to achieve dissociation amylase content.The invention provides a kind of rapid and convenient and measure the assay method of free sugar content in GL-PP bond.
Whether the result that dissociation amylase content is measured is true, should meet condition below: protein recovery=c/d × 100% should be less than 1%, and under this condition, polysaccharide can not be degraded, and under this condition, the recovery of free polysaccharide is greater than 95%, and the present invention has met above-mentioned condition.
Compared with prior art, the present invention can be accurately, fast, simple determination goes out the content of free polysaccharide in bond, saves amount of samples.
Brief description of the drawings:
The HPLC collection of illustrative plates of sample A in Fig. 1 embodiment 1, retention time is 14.612min
The HPLC collection of illustrative plates of sample B in Fig. 2 embodiment 1, retention time is 14.627min
Embodiment:
The method of measuring polyoses content can be chemical method, as sulfuric acid anthrone (pneumonia capsular polysaccharide content mensuration), Ribose concentration are measured (haemophilus influenzae capsular polysaccharide content mensuration), phosphorus detection (A group meningitis cocci capsular polysaccharide content mensuration), sialic acid content is measured methods such as (C group meningitis cocci capsular polysaccharide content mensuration), also can be immunological method, as enzyme linked immunosorbent assay (ELISA), or immunoturbidimetry.
Free measurement of the polysaccharide content in experimental example 9V-CRM197 bond
Get 1ml 9V-CRM197, wherein 9V concentration is 110ug/ml.Under ice-water bath condition, add 100ul 1% deoxycholic acid sodium solution, fully mix static 30min.Then add 100% trichloroacetic acid solution 5ul, mix latter 4 DEG C, 12000rpm, 15min is centrifugal.Getting supernatant is working sample A, and getting bond stoste to be measured is testing sample B, utilizes respectively polyoses content a and the b in working sample A and sample B of sulfuric acid anthrone method, and utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
It is 4.5% that dissociation amylase content=a/b * 100% calculates dissociation amylase content in 9V-CRM197 bond stoste.
The prerequisite of measuring dissociation amylase content is to meet the following conditions:
1 albumen is precipitated completely;
According to formula:
Protein recovery=c/d * 100%, the protein recovery calculating in 9V-CRM197 bond supernatant is 0.
2 free polysaccharides are not precipitated;
Getting 1ml 9V-CRM197 is sample A, and wherein 9V concentration is 110ug/ml; 1ml concentration is that the 9V aqueous solution of 45ug/ml is sample B; The potpourri of 1ml 9V-CRM197 and free 9V is sample C, and wherein in 9V-CRM197 bond, the concentration of 9V is 110ug/ml, and the concentration of free 9V is 50ug/ml.Under ice-water bath condition, add respectively 100ul 1% deoxycholic acid sodium solution, fully mix static 30min.Then add 100% trichloroacetic acid solution 5ul, mix latter 4 DEG C, 12000rpm, 15min is centrifugal, gets respectively supernatant.Utilize sulfuric acid anthrone method to measure polyoses content.
Sample A, the polysaccharide concentration in its stoste and supernatant is respectively a, b; Sample B, the polysaccharide concentration in its stoste and supernatant is respectively c, d; Sample C, the polysaccharide concentration in its stoste and supernatant is respectively e, f.
The results are shown in Table 1.
The table 1 free polysaccharide recovery
Table 1 shows, 9V solution, i.e. and sample B, after NaTDC and trichloroacetic acid processing, the free sugar recovery in its supernatant is 98%, illustrates that free 9V polysaccharide can be not precipitated, is still present in supernatant.
Sample C, the polyoses content in stoste c approximates polysaccharide concentration sum in stoste a and stoste c; The polysaccharide concentration of supernatant f is about polysaccharide concentration sum in supernatant b and supernatant d, illustrate and protein bound glycocalix precipitation, and free polysaccharide is all present in supernatant, not precipitated.
3 polysaccharide can not be degraded
If glycocalix is degraded into micromolecular segment, can from bond, come off, dissociate out.Thereby the measurement result of free sugar is bigger than normal compared with actual value.If glycocalix degraded, its mean molecular weight can reduce, and shows as the variation of retention time on HPLC.
Get 1ml 9V polysaccharide solution (sample A), concentration is 1mg/ml.Under condition of ice bath, add 100ul 1% deoxycholic acid sodium solution, static 30min, adds 5ul 100% trichloroacetic acid solution, 4 DEG C of standing 24h(sample B).Utilize the mean molecular weight of HPLC analytic sample A and B.
HPLC condition is as follows: chromatographic column is: TSK5000 pwxl (7.5 mm × 30 cm); Mobile phase is: 0.15-0.2M NaCl solution; Flow velocity: 0.5ml/min; Loading volume: 20ul; Signal detector: RID differential detecting device.
Result shows that the retention time of A is 14.612min, sees Fig. 1; The retention time of sample B is 14.627min, sees Fig. 2.The retention time of A and B is consistent, without significant change.So, utilize this invention to carry out the mensuration of dissociation amylase content, 9V polysaccharide can not be degraded.
HPLC method is described
Chromatographic column is: TSK5000 pwxl (7.5 mm × 30 cm)
Mobile phase is: 0.15-0.2M NaCl solution
Flow velocity: 0.5ml/min
Loading volume: 20ul
Signal detector: RID differential detecting device
Sample concentration: 1mg/ml
Utilize the method for experimental example, detected the dissociation amylase content in certain several streptococcus pneumoniae polysaccharides protein conjugates and haemophilus influenzae bond PRP-crm197.The results are shown in following table 2.
The content of free polysaccharide in the various PS-CRM197 bond of table 2
Embodiment 1
Get 1ml 9V-CRM197, wherein 9V concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 0.3%, and addition is 5% of bond stoste volume, fully mixes static 20min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.1% of bond stoste volume, mixes latter 4 DEG C, 8000rpm, centrifugal 15;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize sulfuric acid anthrone method respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculating 9V-CRM197 bond stoste, dissociation amylase content is 4.5%.
Protein recovery=c/d * 100%, the protein recovery calculating in 9V-CRM197 bond supernatant is 0.
Embodiment 2
Get 1ml 9V-CRM197, wherein 9V concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 3%, and addition is 15% of bond stoste volume, fully mixes static 40min; Then add 100% trichloroacetic acid solution 5ul, addition is 1% of bond stoste volume, mixes latter 8 DEG C, 12000rpm, centrifugal 20min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize sulfuric acid anthrone method respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculating 9V-CRM197 bond stoste, dissociation amylase content is 6.5%.
Protein recovery=c/d * 100%, the protein recovery calculating in 9V-CRM197 bond supernatant is 0.。
Embodiment 3
Get 1ml PRP-CRM197, wherein PRP concentration is 110ug/ml.Under ice-water bath condition, add 100ul 1% deoxycholic acid sodium solution, fully mix static 30min.Then add 100% trichloroacetic acid solution 5ul, mix latter 4 DEG C, 12000rpm, 15min is centrifugal.Getting supernatant is working sample A, and getting bond stoste to be measured is testing sample B, utilizes respectively PRP content a and the b in working sample A and sample B of Ribose concentration determination method, and utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculating PRP-CRM197 bond stoste, dissociation amylase content is 6.1%.
Protein recovery=c/d * 100%, the protein recovery calculating in PRP-CRM197 bond supernatant is 0.
Embodiment 4
Get 1mlA group meningitis cocci polysaccharide-CRM197, wherein polysaccharide concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 2%, and addition is 10% of bond stoste volume, fully mixes static 25min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.4% of bond stoste volume, mixes latter 5 DEG C, 10000rpm, centrifugal 18min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize phosphorus detection method respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculations incorporated thing stoste, dissociation amylase content is 7.1%.
Protein recovery=c/d * 100%, the protein recovery in calculations incorporated thing supernatant is 0.
Get 1ml 7F-CRM197, wherein 7F concentration is 110ug/ml, under ice-water bath condition, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 2%, and addition is 10% of bond stoste volume, fully mixes static 25min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.4% of bond stoste volume, mixes latter 5 DEG C, 10000rpm, centrifugal 18min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize enzyme linked immunosorbent assay respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculations incorporated thing stoste, dissociation amylase content is 0.1%.
Protein recovery=c/d * 100%, the protein recovery in calculations incorporated thing supernatant is 0.
Embodiment 6
Get 1ml 14-CRM197, wherein polysaccharide concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 2%, and addition is 10% of bond stoste volume, fully mixes static 25min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.4% of bond stoste volume, mixes latter 5 DEG C, 10000rpm, centrifugal 18min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize immunoturbidimetry respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculations incorporated thing stoste, dissociation amylase content is 2.3%.
Protein recovery=c/d * 100%, the protein recovery in calculations incorporated thing supernatant is 0.
Embodiment 7
Get 1ml 4-CRM197, wherein polysaccharide concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 2%, and addition is 10% of bond stoste volume, fully mixes static 25min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.4% of bond stoste volume, mixes latter 5 DEG C, 10000rpm, centrifugal 18min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize immunoturbidimetry respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculations incorporated thing stoste, dissociation amylase content is 2.3%.
Protein recovery=c/d * 100%, the protein recovery in calculations incorporated thing supernatant is 0.
Embodiment 8
Get 1ml A group meningitis cocci polysaccharide-CRM197, wherein meningococcal polysacharide concentration is 110ug/ml, under ice-water bath condition, adds the deoxycholic acid sodium solution of 100ul 2%, and addition is 10% of bond stoste volume, fully mixes static 25min; Then add 100% trichloroacetic acid solution 5ul, addition is 0.4% of bond stoste volume, mixes latter 5 DEG C, 10000rpm, centrifugal 18min;
The supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize immunoturbidimetry respectively the polyoses content in working sample A and sample B be a and b; Utilizing BCA method to measure respectively protein content in A and B is c and d, according to formula:
In GL-PP bond, in dissociation amylase content=a/b × 100% calculations incorporated thing stoste, dissociation amylase content is 2.3%.
Protein recovery=c/d * 100%, the protein recovery calculating in 9V-CRM197 bond supernatant is 0.
Claims (10)
1. a free measurement of the polysaccharide content method in GL-PP bond, is characterized in that step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, add deoxycholic acid sodium solution, fully mix, static, add trichloroacetic acid solution, mix rear low-temperature centrifugation;
2) supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize chemical method or immunological method respectively the polyoses content in working sample A and sample B be a and b;
3) in GL-PP bond, dissociation amylase content is a/b × 100%.
2. assay method as claimed in claim 1, is characterized in that step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, add 0.3%～3% deoxycholic acid sodium solution, addition is 5%～15% of bond stoste volume, fully mixes static 20min～40min; Then add 100% trichloroacetic acid solution, addition is the 0.1%-1% of bond stoste volume, mixes latter 4～8 DEG C, 8000rpm～12000rpm, centrifugal 15～20min;
2) supernatant of getting after centrifugal is working sample A, and getting bond stoste to be measured is testing sample B, utilize chemical method or immunological method respectively the polyoses content in working sample A and sample B be a and b;
3) in GL-PP bond, dissociation amylase content is a/b × 100%.
3. assay method as claimed in claim 1, is characterized in that step is as follows:
1) get GL-PP bond stoste to be measured, under ice-water bath condition, 1% deoxycholic acid sodium solution, addition is 10% of bond stoste volume, fully mixes static 20min～40min; Then add 100% trichloroacetic acid solution, addition is 0.5% of bond stoste volume, mixes latter 4 DEG C, 12000rpm, centrifugal 15min;
2) getting supernatant is working sample A, and getting bond stoste to be measured is testing sample B, and utilizing the polyoses content in chemical method or immunological method difference working sample A and sample B is a and b;
3) in GL-PP bond, dissociation amylase content is a/b × 100%.
4. assay method as claimed in claim 1, is characterized in that, described GL-PP bond is haemophilus influenzae GL-PP bond, streptococcus pneumoniae polysaccharides protein conjugates, or meningococcal polysacharide protein conjugates.
5. assay method as claimed in claim 1, is characterized in that, the chemical method described in step 2 comprises sulfuric acid anthrone method, ribose determination method, phosphorus detection method or Application of Sialic Acid Measurement method.
6. assay method as claimed in claim 1, is characterized in that, the immunological method described in step 2 is enzyme linked immunosorbent assay or immune turbidimetry.
7. assay method as claimed in claim 1, is characterized in that, the protein content c in the supernatant sample A obtaining according to the method for step 1, and protein content d in GL-PP bond stoste B, and the result of utilizing Lowry method or BCA method to measure is; Wherein c/d<0.01.
8. the assay method of stating as claim 4, is characterized in that, the GL-PP bond that described streptococcus pneumoniae polysaccharides protein conjugates is following any one streptococcus pneumonia: 1 type, 2 types, 3 types, 4 types, 5 types, 6A type, 6B type, 7F type, 8 types, 9V type, 9N type, 10A type, 12F type, 14 types, 15B type, 17F type, 18C type, 19A type, 19F type, 20 types, 22F type, 23F, 33F type.
9. assay method as claimed in claim 1, is characterized in that, wherein, in described GL-PP bond stoste, polysaccharide concentration is 50-150ug/ml.
10. assay method as claimed in claim 1, is characterized in that, wherein, in described GL-PP bond stoste, polysaccharide concentration is 100ug/ml.
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