Module 5 : Immunological Methods

Lecture 30 : ELISA

 

ELISA

Lab Experiment 30.1 : Measurement of Antibody titre by an indirect ELISA.  

Background Information: ELISA is a immunological technique used to measure the level of antibodies or antigen in the body fluid. It has been used in diagnostics to identify the antigen or cross reactive antibody. ELISA can be performed in two different ways to either measure antibody or antigen. These different variants are given in Figure 30.1.

1. In-direct ELISA- This setup is used to measure the level of antibodies in the serum and used to calculate the titre of the antibodies. In the in-direct ELISA setup, a known amount of antigen is coated to the well and it is incubated with the different dilutions of antibodies. The antigen bound antibody is then recognized by the secondary antibody linked to the enzyme. A colorimetric substrate is used to measure the level of antibody.

2. Sandwich ELISA- This setup is used to measure the level of antigen (such as insulin) in the serum. In the direct ELISA setup, a known amount of antibody specific antibody (capture antibody) to capture the antigen. The antigen is then recognized by the secondary antibody linked to the enzyme. A colorimetric substrate is used to measure the level of antigen.

Figure 30.1: Different types of ELISA.

Reagents and Materials:

Biocarbonate buffer- Prepare the 50mM Biocarbonate buffer pH 9.2 in distilled water and filter sterile with 0.2µm filter.

ELISA plate: Flat Bottom 96 well is more suitable for performing ELISA.

Antigen solution: Prepare 5µg/ml antigen solution in biocarbonate buffer pH 9.2.

BSA: Prepare 10mg/ml BSA solution in distilled water and filter sterile with 0.2µm filter.