Question

Exercise : Enzyme-Linked Immunosorbent Assay (ELISA)

At the conclusion of this exercise you should be able to:

• explain the principles of the enzyme-linked immunosorbent assay (ELISA).
• determine the qualitative results of a clinical scenario using ELISA.
• understand the purpose of the reagents used in ELISA.

Background

Immunology is the study of the immune response. This involves the interaction between antigens, antibodies, and cells. Immune responses are generated by the body to recognize substances considered to be “foreign”. Microorganisms can generate different immune responses when they infect a host. This leads to the formation of antibody in a host. This exercise will explore some of the basic principles of immunology using an indirect ELISA to identify the presence of antigen in a sample of serum.

Scenario

A serum sample was obtained from a patient who was affected by a severe gastrointestinal infection, which was presumed to be caused by a bacterial infection. In order to investigate the causative agent, an ELISA using antibodies of known intestinal pathogens was performed to identify the microorganism that caused this infection.

Materials

1. A sample of serum was obtained from the child.
2. Antigens obtained from three known gastrointestinal pathogens:

Shigella sonnei, Escherichia coli, and Salmonella typhimurium

3. Microtitre polystyrene plate.
4. Coating buffer (phosphate buffer solution, pH 9.6; PBS).
5. Blocking buffer (PBS/Tween)-Tween is a detergent added to buffer to prevent non-specific binding of molecules.
6. Secondary antibody (anti-human immunoglobulin). This antibody is attached to an enzyme that will react with a substrate that is added in the last step of the process.
7. Enzyme substrate: this is used in immunoassays. For this assay, the substrate used is horseradish peroxidase, which causes a yellow reaction once it attaches to the secondary antibody.

Procedure

1. Add 50 µl of each of the three antigens diluted in coating buffer to rows A-C and columns 1-12 of a microtiter plate.

Row A: Shigella sonnei

Row B: Escherichia coli

Row C: Salmonella typhimurium

2. After incubating for 1 hour at room temperature remove liquid by tapping onto paper towel.
3. Add 300 µl blocking solution to each well and leave for 5minutes.
4. Tap out residual solution.
5. Add 50 µl of PBS/Tween to rows A-C and columns 1-12.
6. Add 50 µl of the patient’s serum to column 2 and rows 1-3.
7. Proceed to make two-fold dilutions in each row by removing 50 µl from column 2 and transferring to column 3. Continue until the last column is reached (column 12).
8. Incubate the plate at room temperature for 1 hour.
9. Empty plate by tapping out fluid on a paper towel.
10. Fill each well with wash solution (PBS/Tween), invert and remove residual liquid. Repeat 3 times.
11. Add 50 µl of secondary antibody to each well and incubate 1 hour at room temperature.
12. Remove residual liquid as described above and complete 3 washes as above.
13. Add 50 µl of substrate to each well and note the change in colour in each well.

Results

The results of this experiment are shown below.

	1	2	3	4	5	6	7	8	9	10	11	12
A
B
C

Questions

1. Which organisms was the causative agent of infection for this patient? Explain you answer in comparison to the other samples provided.

2. What is the purpose of using a negative control? Explain what it means of column 1 wells appear yellow.

3. What are the fundamental differences between direct and indirect ELISA methods?

4. Explain the purpose of diluting the serum sample provided.

5. Describe the purpose of each of the components in the ELISA.

Answer 1

Ans 1. Escherichia coli is the causative agent of infection.

Explanation: ELISA(Enzyme-Linked Immunosorbent Assay) is a serological test used for the qualitative and quantitative analysis of serum sample obtained from the patient. It is based on antigen-antibody reaction and generally used for the quantitative analysis. It is performed generally in a microtiter plate made up of polystyrene with 96 wells on it. Indirect ELISA is used to detect the presence of antibodies in the serum sample.

During the course of ELISA following steps are followed:

1. The antigen is added to the wells of a microtiter plate and incubated. The antigen is bound to the well. After this wash the plate so that unbound antigen is washed away.

2. Now the blocking agent is added to the wells of a microtiter plate.

3. Now the serum sample containing primary antibody is added to the wells of microtiter plate and incubate. If serum sample contains the antibody for the bound antigen it binds to the antigen. After this wash the plate so that unbound antibodies washed away and the antigen antibody complexes will remain attached to the well surface.

4. Now secondary antibody(specific for primary antibody) linked with enzyme is added to the wells of microtiter plate and incubate. If the well surface contains attached antigen-antibody(primary) complexes then secondary antibody binds to the complex. After this wash the plate so that unbound secondary antibody washed away and the antigen-antibody(primary and secondary) will remain attached to the well surface.

5. Now substrate is added to the wells of microtiter plate and incubate. If the wells containing antigen-antibody(primary and secondary) complex the substrate will bind to the enzyme-linked with secondary antibody. Enzyme-Substrate reaction takes place and enzyme converts substrate into coloured product. ELISA assay uses a substrate with a chromophore group(colour-producing).

6. By using the microtiter plate reader we can observe the colour change.

Other samples did not contain antibody for the bound antigen hence the complex of antigen and primary antibody could not be formed and during the washing process, all the unbound antibodies washed away. The secondary antibodies also washed away during washing and no enzyme-substrate reaction takes place because there is no enzyme in well for the substrate.

Ans 2. A negative control is used to verify the accuracy of. result. It is a tool to verify that the produced result is accurate and there was not any none specific binding occurs during a process that can generate a false-positive result.

Ans3. Indirect ELISA no secondary antibody is used. On the other hand, during indirect ELISA secondary antibody linked with enzyme is used. During direct ELISA the primary antibody is linked with the enzyme.

Ans4. By diluting the titer of antibody can be measured effectively.

Ans5. The colour production in negative control shows nonthe -specific binding between antibody and antigen.