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: 2014-01-01
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • 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 fixed 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 hole used. The outer diameters of both ends of the fixed ring are divided into small and large ends, and the outer diameter of the large end is slightly greater than or equal to the inner diameter of the 96-well plate, so that the fixed ring can ...

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 particularly discloses an indirect Dot-ELISA (enzyme-linked immunosorbent assay) method and application of a blood group antibody. The method comprises the following steps of fixing a nitrocellulose membrane on a 96-hole plate, then directly coating the nitrocellulose membrane with a red cell membrane, and reacting the red cell membrane with a to-be-detected antibody, so as to form an antigen-antibody compound through a second enzyme-labeled antibody. A Dot-ELISA plate can be prepared by utilizing a commercialized ELISA analysis instrument, so that the automatic operation can be achieved, and purposes of saving manpower, reducing personal errors and realizing high-throughput test are achieved. The Dot-enzyme-linked immunosorbent assay method for researching has the advantages that the efficiency is higher than 8 times of that of an indirect Coombs experiment; as the sensitivity is high, the detection accuracies of low-concentration and low-affinity antibodies can be increased, and a blood group detection method and the blood transfusion safety are further improved.

Description

technical field [0001] The invention belongs to the technical field of blood type identification, and in particular relates to an indirect Dot-ELISA detection method and application of blood type antibodies. Background technique [0002] Blood typing is very important in transfusion therapy and happens every day all over the world. Reagent red blood cells include reagent red blood cells for ABO anti-typing, various red blood cell lineage cells (Panel cell RBC) and special or rare blood type red blood cells, which are used for blood type identification, red blood cell antibody research and red blood cell antibody inspection. These red blood cell antigens are very valuable , is also a rare essential working reagent. All blood group serology laboratories must be routinely equipped. At present, the reagent red blood cells used in most blood group serology laboratories are mainly made of whole blood washed with normal saline for many times, and then a certain amount of red bl...

Claims

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Application Information

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Patent Type & Authority Applications(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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