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Method for measuring viral titer of Marek's disease (MD) turkey herpesvirus live vaccine

A technology of turkey herpes virus and chicken Marek's disease, which is applied in the field of determination of immunogen content, can solve the problems of unsatisfactory protection, failure of chicken immunity, inaccurate measurement results, etc., and achieves simple operation and counting, Easy to adhere to the wall and small error between batches

Active Publication Date: 2013-12-04
RINGPU (BAODING) BIOLOGICAL PHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the measurement results are inaccurate, the actual antigen content of the finished vaccine may be lower than the standard, and the protection produced by the immunized chicken body will not meet expectations, which will directly lead to the failure of the immunization of the chicken flock

Method used

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  • Method for measuring viral titer of Marek's disease (MD) turkey herpesvirus live vaccine
  • Method for measuring viral titer of Marek's disease (MD) turkey herpesvirus live vaccine
  • Method for measuring viral titer of Marek's disease (MD) turkey herpesvirus live vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of chicken embryo fibroblasts:

[0037] ⑴ Select well-grown 9-day-old Merial SPF chicken embryos, strictly disinfect the surface of the chicken embryos, use tweezers to knock out the eggshell in the air chamber, carefully clamp the chicken embryos with elbow ophthalmic tweezers, take them out and place them in a sterilized plate ;

[0038] (2) Remove the eyes, brain and internal organs of the chicken embryo, and wash the embryo body three times with 0.01M PBS pH7.2 washing solution;

[0039] (3) Add trypsin solution with a mass concentration of 0.25% preheated at 38°C in proportion to 10ml of each embryo body, and digest under magnetic stirring for 10 minutes each time. After each digestion, the cell solution is filtered through 2 layers of gauze to Add 2% of the total volume of trypsin solution to the container of fetal bovine serum in advance; repeat this step 3 to 4 times;

[0040](4) Centrifuge the cell solution at 2500rpm for 10min, discard ...

Embodiment 2

[0045] Example 2 Inoculation of chicken Marek's disease turkey herpes virus into chicken embryo fibroblast monolayer

[0046] ⑴ Dilute the virus: Select a live chicken Marek’s disease turkey herpes virus vaccine sample (product of Ringpu (Baoding) Biopharmaceutical Co., Ltd., available in the market), use cell maintenance solution (volume percentage 98% M-199 Cell nutrient solution, 2% newborn bovine serum, pH value 7.4) for 1×10 4 and 5×10 4 During the dilution process, use a vortex mixer to mix the virus solution at a speed of 300rpm, and the mixing time is 10-20s to ensure that the virus cell solution is completely mixed;

[0047] (2) Virus inoculation: Discard the cell growth solution in the 24-well cell culture plate, add the diluted virus solution into the wells, and connect 2 dilutions to 5 wells respectively;

[0048] (3) After adding the virus solution, put it in a 38°C incubator for adsorption for 1 hour;

[0049] ⑷ After the adsorption is completed, add 1.5ml of ...

Embodiment 3

[0050] Example 3 Counting of virus plaques and calculation of poison price:

[0051] The plaques were counted on the 4th day after the virus was inserted, and no plaques appeared in the culture wells of the negative control, and the experiment was established. The plaques in the cell wells were counted under a microscope, from left to right, from top to bottom Observe the plaques in each cell well carefully (some plaques may be fused together, and the germinal centers of the plaques need to be distinguished), according to the number of plaques in each culture well, the volume of the virus solution to be inoculated and the dilution factor Calculate the poisonous price of the virus liquid to be determined.

[0052] Poison Price = The arithmetic mean of each hole plaque at the optimum dilution factor × the optimum dilution factor

[0053] Volume of inoculated virus solution per well

[0054] Note: The optimal dilution factor is the dilution factor in the range of 10-30 plaqu...

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Abstract

The invention discloses a method for measuring viral titer of a Marek's disease (MD) turkey herpesvirus vaccine, live. The method comprises the following steps of: (1) preparing chick embryo fibroblast suspension; (2) performing cell culture; (3) diluting a virus solution to be measured; (4) performing virus inoculation and culture; (5) counting plaques; and (6) calculating the viral titer. The method is easy to impellent, uniform in measurement conditions, strong in controllability and small in batch errors, and shortens the period of measuring the viral titer of the MD turkey herpesvirus vaccine, live.

Description

technical field [0001] The invention relates to a technique for measuring the immunogen content of biological products, in particular to measuring the poison price of chicken Marek's disease turkey herpes virus live vaccine through a plaque formation experiment, and belongs to the field of biotechnology. Background technique [0002] Chicken Marek's disease (Marek's Disease, MD) is a highly contagious lymphoproliferative disease of chickens caused by Marek's disease virus (MDV) of the herpesviridae family. The world chicken industry has taken a huge toll. Chicken Marek's disease is the first neoplastic disease that can be prevented by vaccines. Chicken Marek's disease turkey herpes virus live vaccine is currently the most widely used effective means to prevent and treat chicken Marek's disease. [0003] In the early 1950s, Dulbecco, based on the principle of plaques produced by bacteriophage infection of bacteria, established the virus plaque technology with cultured cell m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/02C12R1/93
Inventor 马明杨保收李汉秋郁宏伟刘涛
Owner RINGPU (BAODING) BIOLOGICAL PHARMACEUTICAL CO LTD
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