Immunology Practical: Enzyme Linked Immunosorbent Assay (ELISA)

Aim

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

Introduction

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.

Method

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).

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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Details are given in the table below:

Table 1

After 30mins of incubation has passed, you remove the microtitre plate out of the incubator and wash the microtitre plate in the same method as before; using diluting buffer; 3 times.

You then add the secondary antibody which is urease conjugated Goat Anti Rabbit Serum into all wells except for G1; the test control well and compensate it by adding 100µl of diluting buffer. Details are given in table below:

Table 2

Once you have added all the solutions into the wells, you then place the plate into the incubator at 37ºC for 30 minutes again covering the plate. After the 30 minutes are over, you now wash the plate again with diluting buffer 3 times, but this time you are also using distilled water using the same methodology as before.

Now you need to add 100µl of urea; the substrate to all the appropriate wells as mentioned in the table below:

Table3

You then incubate the plate for 24 hour at 37ºC. After 24 hours you should be able to visualise your result.

Result

Discussion

The purpose of this experiment is to calculate the antibody titre of an unknown serum sample. The antibody titre is a reciprocal; the last well that gives a positive result. The result tells you how much specific antibody is present. By looking at my results; the endpoint titre is 1/6400.

Antibody Titre

Antibody titre = Endpoint Titre x Dilution

= 1 x 1

= 6.25x10-6 or 1

160000

The microtitre plates have been coated overnight with antigen; in this experiment we used a protein called ferritin (iron protein). The mechanism behind the assay does not concern what type of antigen you are using but the purpose of the assay. This is an assay which can be used routinely in a clinical laboratory.

Antigen (ferritin) is isolated from the spleen of a horse; the plate is coated overnight with the antigen, so that the antigen is absorbed onto the plastic. In this assay we remove the entire antigen from plate, which was non-specifically bound onto the plastic. The actual experiment was to add the primary antibody; in this experiment we used Rabbit Anti-Ferritin (RbαIgG). The ferritin has been injected into the Rabbit and you get Rabbit Immunoglobulin (IgG), which recognise ferritin in the anti-serum that we are trying to measure the antibody titre for. The way we did this is by using ELISA test, which is also refereed to as a Sandwich ELISA because it actual builds up in layers, in a sandwich manner. Once the primary antibody has been added, the plate is then put in for a 30min incubation period, after this process the primary antibody was then washed off; this removes the entire non-specifically bound antibody. We then added secondary antibody, in this experiment we used Goat Anti-Rabbit IgG, and the secondary antibody needs to be recognised by the primary antibody. In this specific assay the secondary antibody recognises the primary antibody as if it was an antigen. The secondary antibody is quit different because it is actually being conjugated to an enzyme called Urease. Secondary antibody determines how much primary antibody has interacted with the antigen.

Substrate for urease enzyme is urea, when the substrate is dispensed; its colour is yellow; if there is a positive reaction (enzymic reaction) colour is converted into a bright blue/purple colour. You can actually visualise the reaction which determine the antibody titre, no machine or anything of the sort is required to detect the occurrence of the reaction.

The reason behind why the secondary antibody recognises the primary antibody is that the primary antibody (RbIgG) has been injected into the Goat, the Goat sees the Rabbit IgG as an antigen and produces its own IgG, and the Goat IgG is which recognises the Rabbit IgG.

For negative result there is no colour change, it remains yellow because no primary antibody has bound to antigen and no secondary antibody will bind. Consequently, no enzyme to act on substrate is present.

Control well are set in row G, refer to Tables 1, 2 and 3. The reason for having control wells is to see if the reagents are working, because reagents have a shelf life they can possible be degraded. The way we see if the reagents are working is by is adding into well G1; primary antibody and in well G2 we miss out the primary antibody and compensate it by adding 100µl diluting buffer. You then incubate the plate (details are all given in the methodology) for 30 minutes, and then you would add the secondary antibody to the G2 well but not G1 and compensate well G1 by adding 100µl diluting buffer. You then incubate for 30 minutes; after that, you then add substrate (urea) to all the wells.

The control wells tell us whether the reagents are working or not; if there is a colour change from yellow – blue/purple then it indicates that the secondary antibody is reacting directly with the antigen, this is not what we want. If the colour changes to blue/purple then there is a problem with the reagent, it is very important that the reagent have not gone off (shelf life has not expired); the reagents being protein might have degraded.

In this experiment the control well were negative i.e. there was no colour change, hence the reagents are working.

In row D & E there was not reaction i.e. there is a negative result with series of dilution with the addition Normal Rabbit Serum. In wells D3, 6, 7, 8 there is slight contamination.

D3 D6 D7 D8

The reason why we know that this is a contamination is because there is no positive reaction in the first wells (D1 or E1; the highly concentrated wells).

Limitations to ELISA: The drawback to the antigen-coated plate method is that it is precise; limiting quantity of antigen must be bound to the microtitre plate. Furthermore any dissociated antigen will also act as an inhibitor.

Reference:

Definition of Enzyme-linked immunosorbent assay (ELISA) [accessed on the 20 Nov 2008 at 13:52]
G.R. Bullock, D. Van Velzen, M.J. Warhol. (1991), Volume 2. Techniques in diagnostic pathology / edited by G.R. Bullock, D. van Velzen and M.J. Warhol. Vol.2, ELISA techniques: new developments and practical applications in a braod field / volume editor, P. Herbrink. London: Academic Press.
Introduction to ELISA Activity. The Biology Project, The University of Arizona, Wednesday, May 3, 2000. http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/main.html. [Accessed on 27th November 2008].
Kemeny, D. M. (1991). A practical guide to ELISA / D.M. Kemeny. Oxford: Pergamon
Lequin RM. Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clin Chem 2005;51:2415-2418
The Wellcome Foundation Ltd, United Medical and Dental School of Guy’s and St Thomas’s Hospitals. (1988). ELISA and other solid phase immunoassays: theoretical and practical aspects / edited by D.M. Kemeny and S.J. Challacombe. ChichesterbWiley.