What is the Difference Between Direct and Indirect ELISA

The main difference between direct and indirect ELISA is that in direct ELISA, the primary antibody is directly conjugated to the detection enzyme whereas, in indirect ELISA, a secondary antibody which is complementary to the primary antibody is conjugated with the detection enzyme. This means direct ELISA uses a single antibody while indirect ELISA uses two antibodies.

Direct and indirect ELISA are two methods of ELISA (enzyme-linked immunosorbant assay) used to detect the presence of a specific antigen or antibody in the medium.

Key Areas Covered

1. What is Direct ELISA
     – Definition, Procedure, Characteristics
2. What is Indirect ELISA
     – Definition, Procedure, Characteristics
3. What are the Similarities Between Direct and Indirect ELISA
     – Outline of Common Features
4. What is the Difference Between Direct and Indirect ELISA
     – Comparison of Key Differences

Key Terms

Direct ELISA, Detection Enzyme, Indirect ELISA, Primary Antibody, Secondary Antibody, Substrate

What is Direct ELISA

Direct ELISA is a method of ELISA that allows the detection of the antigen with an enzyme-linked primary antibody itself. This ELISA method was first developed Perlmann and Engvall. It is considered as the simplest form of ELISA. The steps of direct ELISA are listed below:

1. Coating the surface of the plate with the sample;
2. Incubation of the plate with the enzyme-conjugated, primary antibody;
3. Washing for the removal of unbound antibodies from the plate;
4. Addition of the substrate required by the enzymatic reaction;
5. Detection of the signals produced from the plate.

Here, the two main types of enzymes conjugated with the primary antibody are HRP (horseradish peroxidase) and alkaline phosphatase. The three types of substrates used along with the HRP are OPD (o-phenylenediamine dihydrochloride), TMB (3, 3’, 5, 5’-tetramethylbenzidine), and ABTS (2, 2’-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt). The substrate used along with the alkaline phosphatase is PNPP (p-Nitrophenyl Phosphate, Disodium Salt). The enzymatic reaction on the substrate causes a color change in the medium, which depends on the type of substrate and the enzyme used for the reaction. This facilitates the detection of samples with the target antigen. 

Figure 1: Direct ELISA

Direct ELISA is more suitable for the detection of the high molecular weight antigen amount in a sample. The fewer number of steps in this ELISA method makes it a faster assay. However, due to the cost and the time incorporated with the labeling of the primary antibody with enzymes, the use of direct ELISA is quite rare. On the other hand, it produces adverse effects on the target antigen as well.

What is Indirect ELISA

Indirect ELISA is another method of ELISA which uses two antibodies for the detection: primary and secondary antibody. The steps of the indirect ELISA are as follows:

1. Coating the surface of the plate with the sample;
2. Incubation of the plate with the primary antibody;
3. Washing to remove the unbound antibodies from the plate;
4. Incubation of the plate with the secondary antibody conjugated with the detection enzyme;
5. Washing to remove the unbound antibodies from the plate;
6. Addition of the substrate required by the enzymatic reaction;
7. Detection of the signals produced from the plate.
   

   Figure 2: Indirect ELISA

Indirect ELISA proceeds through two antibody-binding steps. On the other hand, the secondary antibody is conjugated with the detection enzyme. In general, there are plenty of secondary antibodies conjugated with enzymes. Here, the secondary antibody is mainly a polyclonal, anti-species antibody. Due to the availability of enzyme-conjugated antibodies, the indirect ELISA method is more often used in immunological assays in the detection of parasites, bacteria, and virus. On the other hand, several secondary antibodies can be bound to the primary antibody in the plate. Therefore, the sensitivity of the assay is very high.

Similarities Between Direct and Indirect ELISA

• Direct and indirect ELISA are two methods of ELISA in which the antigen of interest is directly attached to the plate.
• Both methods involve in the detection of the presence of a specific protein molecule in a sample. This protein molecule is directly attached to the plate.
• 96-Well plates or 8-well strip tubes are used in both methods. Both these plates and tubes are made up of polystyrene. The 96-well plates provide plenty of spaces for the determination of optimal dilution for the reaction. In the meanwhile, 8-well strip tubes are better for the finely-tuned assays.

Difference Between Direct and Indirect ELISA

Definition

Direct Elisa refers to a type of ELISA in which the primary antibody is conjugated with the detection enzyme while indirect ELISA refers to a type of ELISA in which the secondary antibody is conjugated with the detection enzyme.

Number of Antibodies Used

Direct ELISA uses one antibody, which is the primary antibody while indirect ELISA uses two antibodies, primary and the secondary antibody. This is the main difference between direct and indirect ELISA.

Detection Enzyme is Linked to

Moreover, the detection enzyme is linked to the primary antibody in direct ELISA while the detection enzyme is linked to the secondary antibody in indirect ELISA.

Intensity of Signals

Another difference between direct and indirect ELISA is that the signals produced by direct ELISA are less intensive while the signals produced by indirect ELISA are more intensive due to the signal amplification.

Background Signals

However, the direct ELISA produces more background signals than the indirect ELISA.

Sensitivity

Direct ELISA is less sensitive while indirect ELISA is more sensitive. This is also a difference between direct and indirect ELISA. 

Cross-Reactivity

Yet another difference between direct and indirect ELISA is the cross-reactivity. That is, the cross-reactivity is minimum in direct ELISA while it is significant in indirect ELISA.

Time

Furthermore, direct ELISA is less time-consuming due to fewer steps while indirect ELISA is more time-consuming due to the doubled antibody-binding steps.

Frequency of Use

While direct ELISA is relatively rare, indirect ELISA is more common.

Conclusion

Direct ELISA only uses the primary antibody for the detection of a specific antigen. This means the primary antibody is conjugated with the detection enzyme in direct ELISA. But, indirect ELISA uses a primary antibody to bind with the antigen while the enzyme-conjugated secondary antibody binds to the primary antibody. Direct ELISA is less common due to the less availability of enzyme-conjugated primary antibody. The main difference between direct and indirect ELISA is the number of antibodies used.

Reference:

1. “Types of ELISA.” Bio-Rad, Bio-Rad Laboratories, Available Here

Image Courtesy:

1. “ELISA diagram” By Cavitri – Own work (CC BY 3.0) via Commons Wikimedia  
2. “Indirect ELISA” By Cawang – Own work (CC0) via Commons Wikimedia