CN1645104B - Method for determining polymer content in Vi-rEPA combined vaccine of typhoid - Google Patents
Method for determining polymer content in Vi-rEPA combined vaccine of typhoid Download PDFInfo
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- CN1645104B CN1645104B CN 200410073535 CN200410073535A CN1645104B CN 1645104 B CN1645104 B CN 1645104B CN 200410073535 CN200410073535 CN 200410073535 CN 200410073535 A CN200410073535 A CN 200410073535A CN 1645104 B CN1645104 B CN 1645104B
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Abstract
A determining method includes using typhoid Vi-rEPA combined vaccine to be tested as sample one, adding absolute alcohol and calcium chloride in it then removing clear liquid and holding deposit by centrifugalization, dissolving the deposit with hydrochloric acid and adding water into obtain sample two, measuring absorbance for sample one and two separately and utilizing formula to calculate out content of high polymer solder contained in typhoid Vi-rEPA combined vaccine.
Description
Technical field
The present invention relates to polymer conjugate Determination on content method in a kind of Salmonella typhoid Vi-rEPA combined vaccine.
Background technology
Typhoid fever is the common important public health problem of paying close attention to of developing country, and morbidity is more concentrated in the school-ager especially, more causes people's attention.The annual morbidity number in the whole world is 33,000,000 according to estimates, and death toll is 500,000
[1]In most of developing countries, the raising of living condition and custom and change are processes more slowly, the effective means that vaccine controls beyond doubt that typhoid fever infects.Existing multiple vaccine is owing to prevent the generation of this disease, and wherein the effect of whole cell vaccine has obtained certainly, but has as local redness problems such as pain, fever; The effect of oral typhoid fever live vaccine remains further to be estimated.
Felix in 1934 and Pitt have found eberthian Vi antigen (Virulence antigen is called for short Vi antigen), and present known Vi antigen is a kind of outer capsular polysaccharide antigen of bacteria cell wall that is present in, and is polymerized by 0-acetyl, one ammonia hexuronic acid height.Robbins
[2]Deng thinking that Vi antigen is a kind of protective antigens, with the Vi antigen immune mouse of 0.5-1.0 μ g purifying, to Vi
+Bacterial strain is attacked, and the height protective effect can be provided.The Typhoid Vi Polysaccharide Vaccine that cold phenol method is extracted the manufacturing of bacterial capsule polysaccharide provides simple, safe means for the prevention typhoid fever
[3,4], this vaccine can provide in crowd more than 5 years old〉and 70% protection.But the Vi polysaccharide is a kind of non-thymus gland dependent form antigen (TI), its immunogenicity is relevant with the age, and no booster immunization behind the repeated inoculation, so simple Vi polysaccharide vaccine result of use in children below 5 years old is unsatisfactory, thereby part has limited the use of this vaccine.Can make it to become thymus gland dependent form antigen (TD) after chemically being attached to protein carrier on the polysaccharide, thereby have the character that booster immunization is replied and protection is provided in low age children.
The Szu of America NI H
[6]Deng successively having carried out Vi-protein combination vaccine research, proved the antibody that produces behind the combined vaccine immune mouse all than the independent use Vi antigen immune height of tiring.They reach 91.4% by the protection ratio of Vi-rEPA (reorganization pseudomonal toxin) combined vaccine in 2～5 years old children in Vietnam scene of development.
But at present still nobody works out the polymer conjugate Determination on content method in the Salmonella typhoid Vi combined vaccine, and the content of judging the polymer conjugate in the combined vaccine has great significance to the assessment of tiring of this vaccine.
Summary of the invention
The objective of the invention is for polymer conjugate Determination on content method in a kind of Salmonella typhoid Vi-rEPA combined vaccine is provided.This method can be simply, accurately, the content of polymer conjugate in the fast measuring vaccine.
Purpose of the present invention can realize by following measure:
Polymer conjugate Determination on content method comprises the steps: in a kind of Salmonella typhoid Vi-rEPA combined vaccine
A, get Salmonella typhoid Vi to be measured-rEPA combined vaccine as sample 1, (2-3) then by volume: 1 adds absolute ethyl alcohol; Behind the mixing again by with (9-11) of the volume ratio of Salmonella typhoid Vi-rEPA combined vaccine: 1 adds calcium chloride solution, places 50-80 minute in room temperature behind the mixing; Under 6500rpm centrifugal 20-40 minute again; Supernatant is discarded;
Add the dissolving with hydrochloric acid sediment of 40-60 μ l in b, the sediment tube in above-mentioned steps a after centrifugal, and placed 20-30 minute; And then in sediment tube, add and the identical water of sample 1 volume, behind the mixing to make the mensuration sample 2 of sedimentary polymer conjugate;
C, measure light absorption value A1, the A2 of sample 1, sample 2 respectively with O-acetyl group method;
D, calculate the content of polymer conjugate contained in Salmonella typhoid Vi-rEPA combined vaccine according to formula (A2 * 2.05)/A1 * 100%.
Described Salmonella typhoid Vi-rEPA combined vaccine is that the Pseudomonas aeruginosa exotoxin A covalent bond by the salmonella typhi Vi polysaccharide of purifying and gene detoxification forms.
Embodiment
The present invention utilizes the characteristic of combined vaccine in the presence of ethanol, the Vi polysaccharide sex change sedimentation of bonding state, and the Vi polysaccharide unchangeability of free state, and centrifugal back is in supernatant.Measure the polyoses content in the precipitation, calculate the polysaccharide ratio in the precipitation.Concrete embodiment is as follows: polymer conjugate Determination on content method in Salmonella typhoid Vi-rEPA combined vaccine comprises the steps:
A, get Salmonella typhoid Vi to be measured-rEPA combined vaccine, added absolute ethyl alcohol then in 2.5: 1 by volume as sample 1; Again by adding calcium chloride solutions, placed 60 minutes in room temperature behind the mixing behind the mixing with 10: 1 of the volume ratio of Salmonella typhoid Vi-rEPA combined vaccine; Under 6500rpm centrifugal 30 minutes again; Supernatant is discarded;
Add the 1.0mol/L dissolving with hydrochloric acid sediment of 50 μ l in b, the sediment tube in above-mentioned steps a after centrifugal, and placed 25 minutes; And then in sediment tube, add and the identical water of sample 1 volume, behind the mixing to make the mensuration sample 2 of sedimentary polymer conjugate;
C, measure light absorption value A1, the A2 of sample 1, sample 2 respectively with O-acetyl group method;
D, calculate the content of polymer conjugate contained in Salmonella typhoid Vi-rEPA combined vaccine according to formula (A2 * 2.05)/A1 * 100%.
Wherein Salmonella typhoid Vi-rEPA combined vaccine is to be formed by the salmonella typhi Vi polysaccharide of purifying and the Pseudomonas aeruginosa exotoxin A covalent bond of gene detoxification.Liquid for water white transparency or little band opalescence contains antiseptic, does not contain foreign matter or grumeleuse.
Claims (1)
1. polymer conjugate Determination on content method in Salmonella typhoid Vi-rEPA combined vaccine comprises the steps:
A, get Salmonella typhoid Vi to be measured-rEPA combined vaccine as sample 1, (2-3) then by volume: 1 adds absolute ethyl alcohol; Behind the mixing again by with (9-11) of the volume ratio of Salmonella typhoid Vi-rEPA combined vaccine: 1 adds calcium chloride solution, places 50-80 minute in room temperature behind the mixing; Under 6500rpm centrifugal 20-40 minute again; Supernatant is discarded;
Add the dissolving with hydrochloric acid sediment of 40-60 μ l in b, the sediment tube in above-mentioned steps a after centrifugal, and placed 20-30 minute; And then in sediment tube, add and the identical water of sample 1 volume, tie the mensuration sample 2 of full thing to make sedimentary macromolecule behind the mixing;
C, measure light absorption value A1, the A2 of sample 1, sample 2 respectively with O-acetyl group method;
D, calculate the content of polymer conjugate contained in Salmonella typhoid Vi-rEPA combined vaccine according to formula (A2 * 2.05)/A1 * 100%; Described Salmonella typhoid Vi-rEPA combined vaccine is that the Pseudomonas aeruginosa exotoxin A covalent bond by the salmonella typhi Vi polysaccharide of purifying and gene detoxification forms.
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| CN 200410073535 CN1645104B (en) | 2004-12-21 | 2004-12-21 | Method for determining polymer content in Vi-rEPA combined vaccine of typhoid |
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| CN 200410073535 CN1645104B (en) | 2004-12-21 | 2004-12-21 | Method for determining polymer content in Vi-rEPA combined vaccine of typhoid |
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| CN1645104A CN1645104A (en) | 2005-07-27 |
| CN1645104B true CN1645104B (en) | 2011-03-16 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011709A1 (en) * | 1994-10-17 | 1996-04-25 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Synthesis of typhoid fever vaccine from a plant or fruit polysaccharide |
| CN1156826A (en) * | 1996-02-06 | 1997-08-13 | 广州市第八人民医院 | Method for quickly testing flagellar antigen of salmonella typhi |
| US6797275B1 (en) * | 1998-12-04 | 2004-09-28 | The United States Of America As Represented By The Department Of Health And Human Services | Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine |
| CN1553187A (en) * | 2003-06-04 | 2004-12-08 | 中国热带农业科学院热带生物技术研究 | Rapid testing method for ayfactin fungal antibiotic |
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2004
- 2004-12-21 CN CN 200410073535 patent/CN1645104B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996011709A1 (en) * | 1994-10-17 | 1996-04-25 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Synthesis of typhoid fever vaccine from a plant or fruit polysaccharide |
| CN1156826A (en) * | 1996-02-06 | 1997-08-13 | 广州市第八人民医院 | Method for quickly testing flagellar antigen of salmonella typhi |
| US6797275B1 (en) * | 1998-12-04 | 2004-09-28 | The United States Of America As Represented By The Department Of Health And Human Services | Method of immunizing humans against Salmonella typhi using a Vi-rEPA conjugate vaccine |
| CN1553187A (en) * | 2003-06-04 | 2004-12-08 | 中国热带农业科学院热带生物技术研究 | Rapid testing method for ayfactin fungal antibiotic |
Non-Patent Citations (4)
| Title |
|---|
| ZUZANA KOSSACZKA, FENG-YING C.LIN et. al.Safety and immunogenicity of Vi conjugate vaccinesfor:Typhoid fever in adults, teenagers, and 2-to 4-year-oldchildren in vietnam.Infection and Immunity67 11.1999,67(11),5806-5810. |
| ZUZANA KOSSACZKA, FENG-YING C.LIN et. al.Safety and immunogenicity of Vi conjugate vaccinesfor:Typhoid fever in adults, teenagers, and 2-to 4-year-oldchildren in vietnam.Infection and Immunity67 11.1999,67(11),5806-5810. * |
| 何彦林, 周耀东等.分光光度法测定血液制品生产过程中的乙醇含量.兰州医学院学报28 1.2002,28(1),9-11. |
| 何彦林, 周耀东等.分光光度法测定血液制品生产过程中的乙醇含量.兰州医学院学报28 1.2002,28(1),9-11. * |
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