Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA)

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Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA)


To acquire understanding and knowledge to determine the antibody titre of the Rabbit Anti-Ferritin Antibody using the Enzyme Linked Immunosorbent Assay (ELISA)


ELISA is a rapid immunochemical assay that involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules).

The ELISA is a fundamental tool of clinical immunology, and is used as an initial screen for pathogens detection. Based on the principle of antibody-antibody interaction, in this assay you can easy visualisation the results and can be completed without the use of radioactive materials.

The ELISA technique is the first and most basic assay to determine if an individual is positive for a selected pathogen, such as HIV. The applications of immunoassays are extended to other fields such as infectious diseases, autoimmune diseases, cancer, degenerative diseases, haematology and pharmacology. Applications have not been confined to human health care. Many applications have been described in veterinary medicine, agriculture, environmental health and the food industry. The assay is performed in a microtitre plate (plastic) which contains an 8 x 12 matrix of 96 wells.

They also called indirect ELISA or sandwich ELISA. Microtitre plates are coated with antigen. Samples is added and the bound antibody subsequently detected by addition of an enzyme-labelled antibody specific for the bound antibody. This enzyme labelled antibody is referred as the detector antibody. The detector antibody is not always labelled directly and a second enzyme- labelled antiglobulin antibody, directed against the detector antibody, is sometimes used.


You first need to wash the plate with diluting buffer; removing the content of the plate (pre-coated antigen) in the sink first; spill the content vigorously (but do make sure that you don’t splash the content out, should remain in the sink). Then fill the wells with diluted buffer, spill the content in the sink again and repeat this step 3 times. Place the plate upside down on a paper towel tapping it to remove all residues.

Prepare dilutions of one positive and one negative rabbit serum in diluting buffer (1/25).  You need to prepare a series of dilution in duplication into the microtitre directly. The final volume of each well should be 100µl.

In to wells A1 and B1 of the microtitre plate dispense serial dilutions of the Rabbit Anti-Ferritin antibody. Into to wells D1 and E1 dispense dilutions of the control Normal Rabbit Serum. Wells G1 and G2 should be the test controls i.e. no Goat-Anti Rabbit Serum Conjugate and no Serum control. Make sure to change the pipette tip each time when using a different solution (this is to prevent contamination).

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Details are given in the table below:

Table 1

Once you have dispensed 100µl diluting buffer into the all the well as mentioned in the above table, you then transfer 100µl across starting from A1 to A2 all the way to A12, mix with your micropipette as you go along; this is in affect producing a double dilution. You then do the same with rows D and E. You only add primary antibody to well G1 and not G2; compensate by adding 100µl to well G2. After this procedure has been carried out, you then incubate the ...

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