Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody

A detection method, blood type antibody technology, applied in the field of blood type identification, can solve the problems of lack of standardization, standardization, difficulty in guaranteeing reagent quality, short storage period, etc., and achieve the effect of easy automatic operation, saving manpower, and reducing human errors

Inactive Publication Date: 2015-04-08
SOUTH CHINA AGRI UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, red blood cells are used as reagents for anti-typing detection. Due to its easy hemolysis and short storage period, the quality of reagents is difficult to guarantee. As a result, ABO blood type reverse typing detection has not yet achieved standardization and standardization.
[0003] The red blood cell antibody detection methods established in the current technology include test tube method, paper method, micro-column gel detection card method, etc. These conventional methods cannot avoid the use of reagent red blood cells

Method used

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  • Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody
  • Indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of blood group antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0029] S1. Preparation of Dot-ELISA 96-well plate: Soak the nitrocellulose membrane with distilled water, then dry it slightly, and use a puncher to make a circular membrane of appropriate size. Put the dried nitrocellulose membrane into a 96-well plate, and press the nitrocellulose membrane on the bottom of the 96-well plate with a fixing ring (Fig. 1). Then put the 96-well plate into a drying oven to dry for 2 h, and store it dry for future use.

[0030] The structure of the retaining ring is as figure 1 As shown in C; the fixed ring is made of plastic material, and the fixed ring is a cylindrical ring with a height of 2-4 mm and a thickness of 0.04-0.08 mm. The outer diameter of the ring matches the inner diameter of the 96-well plate used. The outer diameters of both ends of the fixed ring are divided into small and large ends. It is not easy to slip off, and the outer diameter of the small end is slightly smaller than the inner diameter of the 96-well plate, so that it ...

Embodiment 2

[0037] Determination of the amount of antigen: Dilute the cell membrane of the corresponding blood type with 0.01 mol / L PBS solution (pH7.4), dilute the antigen by 2 times, spot 2 μL on the membrane of each Dot-ELISA plate hole, and judge the coating concentration according to the result. The specific steps of the indirect Dot-ELISA detection method for blood group antibodies are the same as in Example 1.

[0038] The criterion for judging is: the greater the concentration of the antigen, the more prone to non-specific binding. Negative antibody serum shows colorless or no spots, and positive antibody serum shows the darkest concentration is the best antigen coating concentration.

[0039] The results of this experiment show that the antigen coating concentration of 1-2 μg (2 μL, 500-1000 μg / ml) can achieve high sensitivity without non-specific binding.

Embodiment 3

[0041] Specificity experiment:

[0042] Different concentrations of canine DEA1.1 positive antigen and ABO antigen were used to react with negative and positive antibody antiserum respectively. Different concentrations of DEA1.1 negative antigen reacted with canine negative and positive antibody antiserum respectively. Observe the specificity of the indirect Dot-ELISA method. The specific steps of the indirect Dot-ELISA detection method are the same as in Example 1.

[0043] The result shows as figure 2 , the erythrocyte membrane with the corresponding antigen reacts with the positive antibody antiserum, the result shows dark brown spots, the result is +++, and the others have no spots, indicating that the indirect Dot-ELISA detection method has high specificity.

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Abstract

The invention specifically disclosed the indirect DOT-Elisa detection method and application of a blood-type antibody.The method is to fix the nitrate cellulose membrane on the 96-hole plate, and then directly react with the antibody to the antibody to be tested, and the antigen-antibody complex is formed through the enzyme standard two antibodies.The Dot-Elisa board can use commercial ELISA to detect and analyze instruments, which can achieve the purpose of automated operations, to save manpower, reduce human errors, and high-throughput detection.The spots for the immune adsorption measurement method used in this study are more than 8 times the indirect Coombs experiments. High sensitivity can increase the accuracy of the detection of low concentration and the detection of weak relatives and antibodies, and further improve the blood type detection method and improve blood transfusion safety.

Description

Technical field [0001] The present invention is a blood type appraisal technology field, which involves an indirect DOT-ELISA detection method and application of a blood-type antibody. Background technique [0002] Blood type identification is very important in blood transfusion treatment and occurs around the world every day.The reagent red blood cells include ABO's anti -fixed -type reagent red blood cells, various red blood cell spectrocytes (Panel Cell RBC) and special or rare blood type red blood cells. They are used for blood type identification, red blood cell blood type antibody research and examination of red blood cell antibodies.It is also a rare necessary work reagent.All blood type serum laboratories must be regularly equipped.At present, most of the reagent red blood cells used in the blood type serum laboratory are mainly washed multiple times of all blood meridians of physiological saline, and then made of a certain amount of red blood cell preservation liquid.At ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/80
CPCG01N33/721G01N33/80
Inventor 李守军李华涛
Owner SOUTH CHINA AGRI UNIV
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