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A rapid antigen detection method for inactivated oil emulsion vaccine against avian influenza finish products

A technology of avian influenza oil emulsion and inactivated vaccine, which is applied in the field of rapid detection of finished product antigen of avian influenza oil emulsion inactivated vaccine, can solve problems such as chloroform is harmful to the environment, affects the accuracy of HA titer, and is not easy to store safely, and achieves The human body and the environment are safe and non-toxic, the human body and the environment are cheap and easy to obtain, and the effect of reducing time and capital costs

Active Publication Date: 2013-08-07
ZHAOQING DAHUANONG BIOLOGIC PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that chloroform is used as a demulsifier to carry out the potency detection of avian influenza oil emulsion inactivated vaccine HA. This method has the following disadvantages: 1. Chloroform can make protein denaturation, damage HA protein antigenicity, and affect HA efficacy. 2. Chloroform is gradually oxidized into highly toxic phosgene when it encounters air under light, which is not easy to store safely; 3. Chloroform acts on the central nervous system, causing damage to the heart, liver, and kidneys. The human body is carcinogenic, teratogenic, and mutagenic; 4. Chloroform is harmful to the environment and can cause pollution to water bodies
It is also reported that organic solvents such as benzyl alcohol, n-pentanol, and n-hexanol are used as demulsifiers for foot-and-mouth vaccines, but these organic solvents also have the disadvantages of being toxic, flammable, and denaturing proteins, which affect the direct determination of HA titer.

Method used

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  • A rapid antigen detection method for inactivated oil emulsion vaccine against avian influenza finish products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 1. Antigen aqueous phase separation of recombinant avian influenza oil emulsion inactivated vaccine (H5N1 subtype, Re-6 strain)

[0027] Step 1) Take 10ml of recombinant avian influenza oil emulsion inactivated vaccine (H5N1 subtype, Re-6 strain), put it into a 50ml centrifuge tube, add 5-15ml of isopropyl myristate, and place it in a vortex shaker for vigorous shaking more than 5 minutes;

[0028] Step 2) Centrifuge at a speed of more than 3500rpm for more than 5 minutes, oil and water can be seen separated;

[0029] Step 3) Separate about 3ml of the aqueous phase layer.

[0030] Two, get the above-mentioned avian influenza oil emulsion inactivated vaccine aqueous phase, carry out HA potency detection to the vaccine aqueous phase according to the hemagglutination test method of the prior art

[0031] 1) Add 0.025 ml of isotonic phosphate buffered saline (PBS buffer) to wells 1 to 12 of the microreaction plate, and change the dropper.

[0032] 2) Pipette 0.025ml of t...

Embodiment 2

[0049] Example 2 Comparison of Antigen Water Phase HA Titer Determination before and after Demulsification of Recombinant Avian Influenza Oil Emulsion Inactivated Vaccine (H5N1 Subtype, Re-6 Strain)

[0050] Step 1) Take the same batch of H5 subtype Re-6 strain recombinant avian influenza vaccine inactivated with the same batch of known HA titers, and wash them with appropriate amounts of pH 7.2 and 0.01mol / L isotonic phosphate buffered saline (PBS buffered saline) respectively. liquid) diluted to HA titers of 9 log2, 8 log2, 7 log2, and 6 log2 respectively, and the numbers were 1A to 4A in turn, and they were respectively inactivated according to the recombinant avian influenza oil emulsion vaccine (H5N1 subtype, Re-6 strain) Four batches of vaccines were prepared according to the formula and process stipulated in the manufacturing and inspection trial regulations, and the numbers were 1#~4#.

[0051] Step 2) Demulsify the 4 batches of vaccines numbered 1# to 4# respectively ac...

Embodiment 3

[0054] Example 3 Comparative analysis of antigenicity of finished product of avian influenza oil emulsion inactivated vaccine

[0055] Step 1) Take commercially available H5 subtype Re-6 strain recombinant avian influenza vaccine, H5 subtype Re-5 strain recombinant avian influenza vaccine, H5 subtype Re-1 strain recombinant avian influenza vaccine and H9 type Re-2 strain recombinant avian influenza vaccine Each bottle of influenza vaccine was demulsified according to the method described in Example 1 to obtain vaccine aqueous phases numbered Re-6, Re-5, Re-1, and Re-2 respectively.

[0056] Step 2) According to the method described in Example 1, the HA titers of the vaccine water phases of Re-6, Re-5, Re-1, and Re-2 were determined to be 9 log2, 8 log2, 8 log2, and 9 log2, respectively.

[0057] Step 3) According to the HA titer test results of the vaccine water phase, adopt the preparation method of the 4HAU vaccine antigen dilution solution in Example 1, and respectively tak...

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Abstract

The present invention discloses a rapid antigen detection method for inactivated oil emulsion vaccine against avian influenza finish products, and the method comprises the following steps of: 1) mixing uniformly isopropyl myristate and the inactivated oil emulsion vaccine against avian influenza by thoroughly shaking, centrifuging, and separating a water phase layer; 2) performing HA titer detection to the water phase of the vaccine obtained in the step 1); 3) according to the detection result of the HA titer detection of the water phase of the vaccine in step 2), taking the water phase of the inactivated oil emulsion vaccine against avian influenza of step 1) to prepare a 4HAU vaccine antigen diluent; and 4) performing hemagglutination inhibition tests by using the 4HAU vaccine antigen diluent prepared in step 3), wherein a HI titer is expressed as the highest dilution serum that completely inhibits the 4HAU antigen. The method of the invention does not destroy the hemagglutination titer and antigenicity of vaccine antigens, can quickly and accurately determine the HI titer of the water phase of the inactivated oil emulsion vaccine against avian influenza finish products and analyze differences in antigenicity, and reagents in use are safety and non-toxic for human and environment, and are cheap and readily available.

Description

[0001] technical field [0002] The invention relates to a vaccine quality detection technology, in particular to a rapid detection method for the finished antigen of an avian influenza oil emulsion inactivated vaccine. [0003] Background technique [0004] At present, the worldwide epidemic of bird flu has dealt a severe blow to the poultry industry, and also poses a major threat to public health. The prevention and control of avian influenza has become the common responsibility of the international community. All countries in the world have attached great importance to it and invested a lot of manpower and material resources in research on the prevention and control of avian influenza. Vaccination against avian influenza has been proven to be the most effective method for the prevention and control of avian influenza outbreaks. But in fact, the results of bird flu immunization monitoring in recent years show that there are still many immunized populations with a low pas...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 陈瑞爱赖汉漳刘玉鹏汤钦
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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