CN1645104B - Method for determining polymer content in Vi-rEPA combined vaccine of typhoid - Google Patents

Abstract

The invention relates to a method for determining the content of a polymer conjugate in a typhoid Vi-rEPA conjugate vaccine. The method is to use the typhoid Vi-rEPA conjugate vaccine to be tested as a sample 1, and then add absolute ethanol and calcium chloride to the sample 1 , then centrifuge to remove the supernatant and get the precipitate; dissolve the precipitate with hydrochloric acid and add water to obtain the assay sample 2 of the polymer conjugate of the precipitate; then measure the absorbance values of sample 1 and sample 2 respectively and calculate typhoid fever Vi- The content of the polymer conjugate contained in the rEPA conjugate vaccine; the method of the present invention can simply, accurately and quickly calculate the content of the polymer conjugate contained in the conjugate vaccine, and the potency of the conjugate vaccine can be evaluated by using the method .

Description

Method for Determination of High Molecular Conjugate Content in Typhoid Vi-rEPA Conjugate Vaccine

technical field

The invention relates to a method for determining the content of a high molecular conjugate in a typhoid Vi-rEPA conjugate vaccine.

Background technique

Typhoid fever is an important public health issue of common concern in developing countries, especially in school-age children, where the incidence is relatively concentrated, which has attracted people's attention. The global annual incidence is estimated to be 33,000,000 and the death toll is 500,000 ^[1] . In most developing countries, the improvement and change of living conditions and habits is a relatively slow process, and vaccines are undoubtedly the most effective means of controlling typhoid infection. Existing multiple vaccines have been used to prevent the occurrence of the disease, among which the effect of the whole bacterial vaccine has been affirmed, but there are problems such as local redness and swelling, pain, and fever; the effect of oral typhoid live vaccine needs further evaluation.

In 1934, Felix and Pitt discovered the virulence antigen of typhoid bacteria (Virulence antigen, referred to as Vi antigen). It is now known that Vi antigen is a capsular polysaccharide antigen that exists outside the bacterial cell wall. Acid is highly polymerized. Robbins ^[2] et al. considered that Vi antigen is a protective antigen, immunizing mice with 0.5-1.0 μg purified Vi antigen can provide a high degree of protection against Vi ⁺ strain challenge. The typhoid Vi polysaccharide vaccine produced by extracting bacterial capsular polysaccharides by cold phenol method provides a simple and safe means for preventing typhoid fever ^{[3, 4]} , and the vaccine can provide >70% protection in people over 5 years old. However, Vi polysaccharide is a thymus-independent antigen (TI), its immunogenicity is related to age, and there is no booster immunity after repeated vaccination, so the effect of simple Vi polysaccharide vaccine in children under 5 years old is not ideal, so Some restrictions on the use of the vaccine. Chemically conjugating a protein carrier to a polysaccharide renders it a thymus-dependent antigen (TD), which has the property of enhancing the immune response and providing protection in young children.

Szu ^[6] of the NIH in the United States has successively carried out studies on Vi-protein conjugated vaccines, and proved that the antibodies produced by the conjugated vaccines after immunizing mice were higher than those using Vi antigens alone. The Vi-rEPA (recombinant Pseudomonas aeruginosa toxin) conjugate vaccine developed by them had a protection rate of 91.4% among children aged 2 to 5 in Vietnam.

However, no one has yet developed a method for determining the content of polymeric conjugates in typhoid Vi conjugate vaccines, but judging the content of polymeric conjugates in conjugated vaccines is of great significance to the evaluation of the vaccine's potency.

Contents of the invention

The purpose of the present invention is to provide a method for determining the content of high-molecular conjugates in typhoid Vi-rEPA conjugate vaccines. The method can simply, accurately and rapidly determine the content of the polymer conjugate in the vaccine.

The purpose of the present invention can be achieved through the following measures:

A method for assaying the content of polymer conjugates in a typhoid Vi-rEPA conjugate vaccine, comprising the steps of:

a, take the typhoid Vi-rEPA conjugate vaccine to be tested as sample 1, then add absolute ethanol by volume ratio (2-3): 1; 11): 1 Add calcium chloride solution, mix well and place at room temperature for 50-80 minutes; then centrifuge at 6500rpm for 20-40 minutes; discard the supernatant;

b. Add 40-60 μl of hydrochloric acid to dissolve the precipitate in the centrifuged precipitation tube in the above step a, and let it stand for 20-30 minutes; Determination sample 2 of polymer conjugates of precipitates;

c. Use the O-acetyl method to measure the absorbance values A1 and A2 of sample 1 and sample 2 respectively;

d. Calculate the content of the polymer conjugate contained in the typhoid Vi-rEPA conjugate vaccine according to the formula (A2×2.05)/A1×100%.

The typhoid Vi-rEPA conjugate vaccine is formed by covalently combining purified Salmonella typhi Vi polysaccharide and gene-detoxified Pseudomonas aeruginosa exotoxin A.

Detailed ways

The present invention utilizes the characteristics of the conjugated vaccine to denature and settle the combined Vi polysaccharide in the presence of ethanol, while the free Vi polysaccharide remains unchanged and remains in the supernatant after centrifugation. Determine the polysaccharide content in the precipitate, and calculate the proportion of polysaccharide in the precipitate. Concrete embodiment is as follows: the assay method of polymer conjugate content in the typhoid Vi-rEPA conjugated vaccine, comprises the following steps:

a. Take the typhoid Vi-rEPA conjugate vaccine to be tested as sample 1, then add absolute ethanol at a volume ratio of 2.5:1; mix well and then add calcium chloride at a volume ratio of 10:1 to the typhoid Vi-rEPA conjugate vaccine Solution, after mixing, let stand at room temperature for 60 minutes; then centrifuge at 6500rpm for 30 minutes; discard the supernatant;

b. Add 50μl of 1.0mol/L hydrochloric acid to the sedimentation tube after centrifugation in the above step a to dissolve the sediment, and let it stand for 25 minutes; Determination sample 2 of polymer conjugates of precipitates;

c. Use the O-acetyl method to measure the absorbance values A1 and A2 of sample 1 and sample 2 respectively;

d. Calculate the content of the polymer conjugate contained in the typhoid Vi-rEPA conjugate vaccine according to the formula (A2×2.05)/A1×100%.

The typhoid Vi-rEPA conjugate vaccine is covalently combined with purified Salmonella typhi Vi polysaccharide and gene-detoxified Pseudomonas aeruginosa exotoxin A. It is a colorless, transparent or slightly opalescent liquid, containing preservatives, and does not contain foreign matter or clots.

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.