Method for measuring potency of chicken bursin by using in vitro quantitative hemolysis spectrophotometry

Inactive Publication Date: 2018-09-04
派生特(福州)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, these detection methods have limitations, and due to individual

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  • Method for measuring potency of chicken bursin by using in vitro quantitative hemolysis spectrophotometry

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preparation example Construction

[0026] Preparation of overstocked sheep red blood cell solution (SRBC): Take 5.0ml of sheep venous blood, add it to 5.0ml of Arbor's solution, wash the cells with an appropriate amount of Hank's solution, centrifuge at 1000-2000r / min for 10-20min, discard the supernatant, and wash three times ( The operation is the same as before). Add an appropriate amount of Hank’s solution to resuspend the pelleted cells, centrifuge at 1500-2500r / min for 10-20min, discard the supernatant, and the pelleted cells are the accumulated SRBC cells.

[0027] Preparation of 5% SRBC suspension: take 20 μl of accumulated SRBC, add physiological saline to 400 μl, that is, 5% SRBC suspension.

[0028] Preparation of 10% SRBC suspension: take 20 μl of accumulated SRBC, add physiological saline to 200 μl, that is, 10% SRBC suspension.

[0029] Preparation of 0.18-0.22% sensitized SRBC suspension: take 1ml of normal saline to dissolve the freeze-dried complement, and place it at 4°C for later use; mix th...

Embodiment 1

[0044] Utilize in vitro quantitative hemolysis spectrophotometry to measure the method for chicken bursalin effectiveness, comprising the following steps:

[0045] 1) Preparation of porcine spleen lymphocyte suspension:

[0046] A. Take fresh pig spleen, sterilize it in 75% alcohol, wash it several times with PBS, remove the superficial tissue, cut it into pieces, add Hank's solution to grind it, filter it through a 100-mesh sieve, wash the cells with Hank's solution, 1500r / min Centrifuge for 4 minutes, discard the supernatant, collect the precipitated cells and continue to wash 3 times;

[0047] B. Resuspend the precipitated cells collected after the last wash with an appropriate amount of Hank's solution to obtain a cell suspension, add the cell suspension to a centrifuge tube containing a cell separation medium, centrifuge at 2000r / min for 20min, absorb lymphocytes and place in In another centrifuge tube, add Hank's solution to the centrifuge tube to wash the lymphocytes, ...

Embodiment 2

[0058] Utilize in vitro quantitative hemolysis spectrophotometry to measure the method for chicken bursalin effectiveness, comprising the following steps:

[0059] 1) Preparation of porcine spleen lymphocyte suspension:

[0060] A. Take fresh pig spleen, sterilize it in 75% alcohol, wash it several times with PBS, remove the superficial tissue, cut it into pieces, add Hank's solution to grind it, filter it through a 100-mesh sieve, wash the cells with Hank's solution, 1000r / min Centrifuge for 5 minutes, discard the supernatant, collect the precipitated cells and continue to wash 3 times;

[0061] B. Resuspend the precipitated cells collected after the last wash with an appropriate amount of Hank's solution to obtain a cell suspension, add the cell suspension to a centrifuge tube containing cell separation medium, centrifuge at 1500r / min for 30min, absorb lymphocytes and place in In another centrifuge tube, add Hank's solution to the centrifuge tube to wash the lymphocytes, th...

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Abstract

The invention discloses a method for measuring the potency of chicken bursin by using the in vitro quantitative hemolysis spectrophotometry. The method comprises the following steps: firstly, preparing porcine splenic lymphocyte suspension under a germfree condition, adding SRBC (Sheep Red Blood Cell) and a complete medium, placing the medium at 37 DEG C for cultivation or sensitization, adding the SRBC and an addiment after certain time, placing the medium at 37 DEG C for incubation, taking supernate, putting the supernate into a microplate reader to measure an OD value, and calculating the activity of the bursin through a formula. The method disclosed by the invention has the advantages of sensitivity, stability, objectiveness and the like, and is easy and convenient to operate, short inperiod and low in cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for determining the efficacy of chicken bursalin by using in vitro quantitative hemolysis spectrophotometry Background technique [0002] Bursalin is an active substance isolated from the bursa, the central lymphoid organ of humoral immunity in poultry. Studies have shown that chicken bursalin can effectively promote the differentiation and proliferation of poultry and mammalian B lymphocyte precursors, enable the body to quickly generate an immune response, promote the secretion of antibodies, and improve the body's humoral immunity. At present, there are few test methods for detecting the efficacy of chicken bursalin, and most of the detection methods use chicken bursalin and vaccines in combination, and the efficacy of chicken bursalin is reflected by measuring the antibody titer of the vaccine. Therefore, these detection methods have limitations, and are prone to test i...

Claims

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Application Information

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IPC IPC(8): G01N21/31G01N1/28
CPCG01N1/28G01N21/3103
Inventor 吕小婷叶盛聪邱灵姗陈盛勇刘楚楚徐磊刘友霖
Owner 派生特(福州)生物科技有限公司
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