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The competition inhibition method works on the principle that both the enzyme-labeled antibody and the sample antibody compete for binding to the solid-phase antigen in the same reaction system. Ideally, the sample should be mixed with the enzyme-labeled antibody first before adding the rest of the reagents. However, in practice, these components are often added separately into the reaction wells, which can disrupt the proportional interaction between them.
This sequential addition may lead to an unbalanced reaction, where the first component to bind has a greater chance of occupying the antigen sites, making the subsequent binding less efficient. This imbalance can significantly affect the accuracy of the results, especially when the incubation time is long or the temperature is high.
To investigate this issue, Changhai Hospital of the Second Military Medical University conducted a study using 94 negative specimens, which were diluted 1:30 with physiological saline. The samples were added to the wells first, followed by the enzyme-labeled antibodies after different waiting times. The results showed how varying the timing affected the false positive rate:
| Placement Time (min) | Number of Negative Specimens | Threshold - A450nm Value | False Positive Rate (%) |
|----------------------|------------------------------|----------------------------|--------------------------|
| <0.3 | 75 | 10 | 0 |
| 0.3~0.7 | 10 | 3 | 6 |
| >0.7 | 3 | 6 | 0 |
| 0 | 2 | 68 | 13 |
| 5 | 7 | 65 | 15 |
| 10 | 8 | 65 | 10.6 |
| 20 | 12 | 56 | 20.2 |
| 30 | 18 | 38 | 39.3 |
| 43 | 21 | 21 | 57.4 |
As shown in the table, when the specimen and enzyme-labeled antibody were added simultaneously, no false positives were observed. However, when the enzyme-labeled antibody was added after 2 minutes, a 7.4% false positive rate occurred. After 30 minutes, the false positive rate increased dramatically to 57.4%. Therefore, it is strongly recommended to add the enzyme-labeled antibody immediately after the sample to maintain the proper balance and avoid inaccurate results.
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