Last week we briefly told you about the foundation and operation of the different types of ELISA :
- ELISA direct
- Indirect ELISA
- ELISA sandwich
- Competitive ELISA
As a continuation of that entry, today we bring you a compilation of the main differences between types of ELISA.
DIFFERENCES BETWEEN ELISA TYPES
1.- PLATE UPHOLSTERY
In the case of direct, indirect and competitive ELISA, the surface of the wells that make up the plate is covered with the antigen, whereas in the sandwich ELISA, the plate is coated with the capture antibody.
2.- INCUBATION WITH THE SAMPLE
The incubation step with the test sample only applies to the sandwich ELISA and the competitive ELISA; this step does not take place in the rest of the ELISA types.
3.- INCUBATION WITH ANTIBODIES
All types of ELISA require an incubation step with the primary antibody. However, the indirect ELISA, the sandwich ELISA and the competitive ELISA also require incubation with the secondary antibody.
The antibody that will be labeled with the enzyme is another difference between types of ELISA: in the direct ELISA it is the primary antibody that is labeled with the enzyme, while in the indirect ELISA the secondary antibody is labeled.
In the sandwich ELISA both the detection antibody and the secondary antibody can be labeled if used. And in the competitive ELISA, either the primary or the secondary antibody will be labeled according to the procedure followed in each assay.
Also at the level of specificity there are differences between types of ELISA. It could be said that the direct ELISA is the least specific of the four, since when analyzing heterogeneous samples the probability of nonspecific junctions and high background noise is higher.
The ELISA that offers greater specificity is the sandwich ELISA, because by using two different antibodies directed against the same antigen, it is captured and immobilized more selectively, reducing the probability of non-specific binding and minimizing background noise.
In many occasions, the direct ELISA is usually less sensitive since the conjugation of the antibody to the enzyme can compromise its immunoreactivity.
In contrast, the indirect ELISA generally offers greater sensitivity because it is not the primary antibody that is labeled with the enzyme, allowing its immunoreactivity to be kept intact.
By the number of steps involved in the procedure in each case, we could order the different types of ELISA from least to most complex in their execution as follows: direct ELISA <indirect ELISA <sandwich ELISA <competitive ELISA.