Practical 6 elisa

HBC 1019 Biochemistry 1 Trimester 1, 2012/2013

Practical 6: Enzyme-Linked Immunosorbent Assay (ELISA)

Objectives:1. To understand the immunological concepts in biological science.2. To guide student through processes of a laboratory-based scientific investigation.

Introduction:ELISA stands for Enzyme-Linked Immunosorbent Assay is a powerful antibody-based test which is used in the diagnostic of diseases such as HIV/AIDS and to track pathogenic agents in water, food and the air. ELISA is also used to identify genetically modified organisms (GMOs) and to trace food allergens and molecular markers of pregnancy and drug use.

This practical is basically introducing the studies of immunology in science, particularly about antigen-antibody interactions and the unique properties of antibodies that have revolutionized modern medicine, biotechnology and research. This is because antibodies have becoming vital high-tech tools that being used in biotechnology research and disease diagnosis and treatment. The approach of ELISA in real world applications involves in different context such as antigen detection or antibody detection in the fields of human and veterinary medicine, agriculture, environmental, food safety and quality, etc.

Materials, Reagents and Equipments:Sample A (Egg yolk)Sample B (BSA)Primary antibody (rabbit anti-chicken polyclonal antibody, lyophilized)Secondary antibody (goat anti-rabbit antibody conjugated to horseradish peroxidase (HRP), lyophilized) HRP enzyme substrate 3,3’, 5,5’-tetramethyl benzidine (TMB) PBS with 0.05% Tween 20Disposable plastic transfer pipettesBottles and capsMicroplates with 12 well stripsMicrofuge tubesMicropipettes Pipette tipsPaper towels Beakers

Methods:

1. Arrange your tubes and their contents according to the table below:

Item (Label) ContentsSample Test samples PC (+) Positive control NC (-) Negative control PA Primary antibody SA Secondary antibody

of 5


SUB Enzyme substrate Beaker PBS with 0.05% Tween 20

2. Label your 12-well strip. On each strip label the first 3 wells with “+” for positive controls and next 3 wells with “-” for negative controls. Label the remaining wells with the name of your sample (eg; FN or AF).

3. Bind the antigen to the wells:a. Use a pipette to transfer 25µl of the positive control (+) into the three

“+” wells.b. Use a fresh pipette tip to transfer 25 µl of the negative control (-) into

the three “-“ wells.c. Use a fresh pipette tip for each sample and transfer 25 µl of each of your

samples into the appropriately initiated three wells.

4. Wait 5 minutes while all the proteins in the samples bind to the plastic wells.

5. Wash the unbound sample out of the wells:a. Tip the microplate strip upside down onto the paper towels so that the

samples drain out and gently taps the strip a few times upside down on the paper towels. Make sure to avoid splashing samples back into wells.

b. Discard the top paper towel.c. Use a transfer pipette filled with wash buffer from the beaker to fill each

well with wash buffer taking care not to spill over into neighbouring wells. The same transfer pipette will be used for all washing steps.

of 5


d. Tip the microplate strips upside down onto the paper towels so that the wash buffer drains out, then gently tap the strip a few times upside down on the paper towels.

e. Discard the top 2-3 paper towels.

6. Repeat step 5.

7. Use a fresh pipette tip to transfer 25 µl of primary antibody (PA) from the green tube into all 12 wells of the microplate strip.

8. Wait for 5 minutes for the primary antibody to bind.

9. Wash the unbound primary antibody out of the wells by repeating step 5 for two times.

10. Use a fresh pipette tip to transfer 25 µl of secondary antibody (SA) into all 12 wells of the microplate strip.

11. Wait 5 minutes for the secondary antibody to bind.

of 5


12. Wash the unbound secondary antibody out of the wells by repeating wash step 5 for three times.

13. Use a fresh pipet tip to transfer 50 µl of enzyme substrate (SUB) from the brown tube into all 12 wells of the microplate strip.

14. Wait for 5 minutes. Observe and record your results.

Results: Label the figure below with the same labels you labeled before. In each of the wells, put a “+”if the well turned blue and a “-” if there is no color change.

Questions:

a.Why are enzymes used in this immunoassay?

b. Why do you need to assay positive and negative control samples as well as your experimental samples?

c.The samples that you have added to the microplate strip contain many proteins and may or may not contain the disease antigen. What happened to the proteins in the plastic well if the sample contained the antigen? What if it did not contain the antigen?

d. Why did you need to wash the wells after each step?

e.When you added primary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen?

f. When you added secondary antibody to the wells, what happened if your sample contained the antigen? What if it did not contain the antigen?

of 5


g. If the sample gave a negative result for the disease-causing agent, does this mean that you do not have the disease? What reasons could there be for a negative result when you actually do have the disease?

h. Why did you assay your samples in triplicate?

i. What antibody-based tests can you buy at your local pharmacy?

of 5

Practical 6 elisa

Technology

Transcript of Practical 6 elisa