The common operation problems of ELISA competition inhibition method
July 24 06:02:26, 2024
Two-and-a-half ELISA methods were used, of which HBsAg, HBsAb, and HBeAg were detected using the double antigen / antibody sandwich method, while HBcAb and HBeAb were detected using competitive inhibition methods.
The principle of the competition inhibition method: the enzyme-labeled antibody and the sample antibody are in the same system and compete with the solid-phase antigen for binding. In theory, the sample and the enzyme-labeled antibody should be mixed first, and then the reaction should be added. But this is not the case in actual work, all are added separately to the reaction wells. This will result in the combination of joining first, and the latter is not a fair relationship, so it does not meet the principle of proportionality. Especially in the case of long storage time and high temperature, it has a great influence on the results.
The influence of Changhai Hospital of the Second Military Medical University was studied. 94 negative specimens were diluted 1:30 with normal saline. After the specimens were added, the enzyme-labeled antibody was added after placing it according to the table below. The test results are as follows:
Placement time (min)
Number of negative specimens
Critical value-specimen A450nm value
False positive rate (%)
<0.3
0.3 ~ 0.7
> 0.7
0
75
10
3
6
0
2
68
13
7
6
7.4
5
65
15
8
6
10.6
10
56
16
12
10
20.2
20
38
18
17
twenty one
39.3
30
twenty one
12
18
43
57.4
As can be seen from the above table, if the specimen and enzyme-labeled antibody are added at the same time, no false positive phenomenon occurs. The enzyme-labeled antibody was added after 2 minutes. Because the sample was bound for two minutes before the enzyme-labeled antibody, a false positive of 7.4% occurred. After 30 minutes, the false positive rate was as high as 57.4%. Therefore, it is recommended to add the enzyme-labeled antibody immediately after adding ten samples to the competitive inhibition method, which has no effect on the test results.