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A kind of preparation method of hemoglobin antibody

A hemoglobin and antibody technology, which is applied in the field of hemoglobin antibody preparation, can solve the problems of hemoglobin detachment, conformational instability, hemoglobin preparation and purification, and antibody preparation obstacles, etc., and achieve the effect of high antibody titer

Active Publication Date: 2021-10-08
武汉戴安生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the process of preparing hemoglobin antibody, it is necessary to extract and purify hemoglobin. Hemoglobin is a protein molecule with unstable conformation. The loss of glyceric acid (2,3-DPG) makes the heme detach from the globin subunit, and at the same time, the hemoglobin tetramer is split into dimers, which creates great obstacles for the preparation and purification of hemoglobin and the preparation of antibodies

Method used

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  • A kind of preparation method of hemoglobin antibody
  • A kind of preparation method of hemoglobin antibody

Examples

Experimental program
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Embodiment 1

[0025] The separation and purification of embodiment 1 hemoglobin

[0026] Take 20mL of whole blood from healthy people and centrifuge at 4000rpm to remove the upper layer of plasma and white blood cell layer, wash the precipitate with normal saline three times, add 40mL of normal saline to make red blood cell suspension, add 600mL of sodium phosphate buffer solution with a pH of 7.4, and perform hypotonic hemolysis for 1 hour , use a high-speed centrifuge at 4°C, centrifuge at 15000rpm for 40min, take the supernatant, add the supernatant to a 100kd ultrafiltration centrifuge tube, centrifuge at 4000rpm for 30min, collect the liquid in the collection tube, which is the solution containing hemoglobin.

Embodiment 2

[0027] Embodiment 2 prepares the hemoglobin of liposome encapsulation

[0028] Take 4mL of chloroform and 6mL of ether and mix to obtain a mixed solution, weigh 60mg of soybean lecithin and 30mg of cholesterol to obtain a lipid solution, prepare hemoglobin with ultrapure water to a solution with a concentration of 6.67mg / mL, take 3mL of the diluted hemoglobin solution and use Slowly add the syringe dropwise into the lipid solution, ultrasonicate in a water bath for 5 minutes to form a w / o emulsion, let the emulsion stand for half an hour, reconstitute if it is separated, and place the emulsion in a rotary evaporator at 37°C if there is no separation Evaporate under reduced pressure to remove the organic solvent. After reaching the glued state, add 30mL of ultrapure water, continue to distill under reduced pressure until the gel falls off the bottle wall, pass the gel through a 0.45μm microporous membrane to obtain a milky white liposome suspension Liquid, which is liposome-enc...

Embodiment 3

[0029] Example 3 Preparation of hemoglobin polyclonal antibody

[0030] In the experimental group, 2-3 month-old experimental rabbits were taken. After the above-mentioned liposome-encapsulated hemoglobin was fully mixed with an equal volume of Freund's complete adjuvant, 400ug of antigen per experimental rabbit was subcutaneously injected at multiple points, and measured 15 days later. Serum titer, select experimental rabbits with good immune response to boost immunization, take liposome-encapsulated hemoglobin and an equal volume of Freund’s incomplete adjuvant, mix well, and inject 125 μg antigen subcutaneously at multiple points per experimental rabbit to boost immunization The number of times is 3 times, each interval is 15 days, blood is taken from the carotid artery, and rabbit anti-human hemoglobin antiserum is obtained.

[0031] The hemoglobin directly used in the control group was hemoglobin without liposome encapsulation, and the other steps were the same as those i...

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Abstract

The invention relates to a preparation method of hemoglobin antibody, which comprises the following steps: separating and purifying hemoglobin from human whole blood, wrapping hemoglobin with liposome, and then immunizing animals to prepare hemoglobin antibody. The method is simple and easy to operate, and the titer of the prepared antibody is high.

Description

technical field [0001] The invention relates to the field of antibody preparation, in particular to a preparation method of hemoglobin antibody. Background technique [0002] Colorectal cancer (CRC) is a common malignant tumor. Its incidence rate ranks fifth among malignant tumors in my country. It usually develops from benign adenoma. Early detection is important to improve the cure rate and survival time of patients with colorectal cancer. help. The most common early screening method for colorectal cancer is the fecal occult blood test. Compared with the traditional fecal occult blood detection method, the immunochemical method directly detects hemoglobin in feces (fecal immunochemical test, FIT) has many advantages. The immunoassay fecal occult blood test is a new method of colorectal cancer screening that uses antibodies against the globin component of hemoglobin to detect hemoglobin in stool. The FDA has recommended that people over the age of 60 check one side of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K1/22
CPCC07K16/18
Inventor 胡治勇曾凡明冉凯凯
Owner 武汉戴安生物技术有限公司
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