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Method for detecting antibody-producing cells by using in-vitro quantitative hemolysis spectrophotometry

A spectrophotometric method and a technology for detecting antibodies, which is applied in the biological field, can solve the problems of large individual differences in organisms, unstable detection results, and high detection costs, and achieve the effects of low cost, stable results, and easy operation

Inactive Publication Date: 2018-09-07
派生特(福州)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The methods for detecting antibody-producing cells in the prior art are all detected on solid animals (mice). The detection cost is high, the cycle is long, and the individual organisms vary greatly, and the detection results are relatively unstable.

Method used

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  • Method for detecting antibody-producing cells by using in-vitro quantitative hemolysis spectrophotometry
  • Method for detecting antibody-producing cells by using in-vitro quantitative hemolysis spectrophotometry
  • Method for detecting antibody-producing cells by using in-vitro quantitative hemolysis spectrophotometry

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preparation example Construction

[0021] Preparation of the 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 appropriate amount of Hank's solution, centrifuge at 1500r / min for 10min, 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 2000r / min for 10min, discard the supernatant, and the pelleted cells are the accumulated SRBC cells.

[0022] Preparation of 5% SRBC suspension: take 20 μl of accumulated SRBC, add physiological saline to 400 μl, that is, 5% SRBC suspension.

[0023] Preparation of 10% SRBC suspension: take 20 μl of accumulated SRBC, add physiological saline to 200 μl, that is, 10% SRBC suspension.

[0024] 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 the complement an...

Embodiment 1

[0034] The method for detecting antibody-producing cells by quantitative hemolysis spectrophotometry in vitro, comprising the following steps:

[0035] 1) Preparation of porcine spleen lymphocyte suspension:

[0036] 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 2 times;

[0037] 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 fluid, centrifuge at 2000r / min for 25min, absorb lymphocytes and place in In another centrifuge tube, add Hank's solution to the centrifuge tube to wash the lymphocytes, then centrifuge...

Embodiment 2

[0045] The method for detecting antibody-producing cells by quantitative hemolysis spectrophotometry in vitro, comprising the following steps:

[0046] 1) Preparation of porcine spleen lymphocyte suspension:

[0047] 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;

[0048] 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, then centrifug...

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Abstract

The invention discloses a method for detecting antibody-producing cells by using an in-vitro quantitative hemolysis spectrophotometry. The method comprises the following steps: (1) taking a cell culture plate, and adding porcine spleen lymphocyte suspension and physiological saline into each well of a blank group; adding the porcine spleen lymphocyte suspension, the physiological saline and sheepred blood cell (SRBC) suspension into each well of a test group, and culturing for 72-84h; (2) adding the sensitized SRBC suspension into each well, continuously culturing for 1-2h, centrifuging, taking 500 volume parts of supernatant from each well and placing into another cell culture plate, using a microplate reader to determine the absorbance of the supernatant in each well at the position of415 nm wavelength, and recording a result as OD415nm; (3) expressing experimental results by means of an antibody production cell positive rate A, wherein A=[(test group OD415nm mean value / blank groupOD415nm mean value)-1]*100%. When being demonstrated on cells, the method can eliminate individual differences and unify all test groups to the same level, thus enabling the test results to be more convincing. The method has the advantages of simpleness and convenience in operation, short period, low cost, relatively stable results, and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting antibody-producing cells using in vitro quantitative hemolysis spectrophotometry Background technique [0002] Quantitative Hemolysis Spectrophotometry (Quantitative Hemolysis Spectrophotometry, QHS), also known as B cell-mediated quantitative hemolysis spectrophotometry of red blood cells, can measure the hemoglobin released by lysing red blood cells with antibodies secreted by B cells, expressed in terms of optical density (OD ), which reflects the humoral immune function of the body, and is one of the common methods for detecting antibody-producing cells. [0003] The methods for detecting antibody-producing cells in the prior art are all detected on solid animals (mice), and the detection cost is high, the cycle is long, and the individual organisms vary greatly, and the detection results are relatively unstable. Therefore, it is of great significance to e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 叶盛聪吕小婷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