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Enzyme immunoassay or enzyme-linked immunosorbent assays is a particularly resourceful test system that is able to be modified to detect either antibody or antigen in a patient's serum, as well as being able to detect the various antibody isotypes, that is, immunoglobulin G (IgG) or immunoglobulin M (IgM). This method originated in 1971 in both the Netherlands and Sweden where it was used in the quantitative detection of protein antigen, rabbit IgG by Engvall and Perlmann (1971) and conjugation of human chorionic gonadotrophin (HCG) to the enzyme horse radish peroxidise (HRP) by Van Weemen and Schuurs (1971). Development of ELISA followed worries about the safety of the current technique being used at the time; radioimmunoassay, which used radioactively labelled antigens or antibodies that provided the signal as to whether that component was present in the sample (Yalow and Berson, 1960). The EIA method is both safer and simpler than radioimmunoassay and uses an enzyme attached to an antibody which reacts with its substrate to produce colour as a detection system for specific antibody/antigen pairs.
EIA is a significant technique used in microbiology for detection of antibodies or antigen in human sera, which proves a powerful diagnostic tool. The immune response produces antibodies when an infection resides in the human body. Thus, if antibodies are detected in EIA, it indicates infection in that individual. Furthermore, if acute serum (day zero) and convalescent serum (a few weeks later) are both taken then it is possible to determine whether the antibody is present due to a prior infection or recent infection. If the concentration of antibodies is increased in the convalescent serum as compared with the acute serum, then it is likely that the infection is recent. Whereas if the concentration remains the same, it is likely that antibody presence is due to prior infection. The way that this is done is by diluting the patient's serum and determining the point at which there is no longer enough antibodies present to produce a colour change. The greatest dilution that still records a positive result is called the antibody titre (e.g. an antibody titre of 64 means that the patient's serum shows a positive result at any dilution down to 1/64 - 1 part serum to 63 parts diluent).
In this exercise human sera is analysed using a 'sandwich' technique of EIA to detect for antibodies against the Leptospira spp antigen. An outline of the method involved and the importance of each step in this type of EIA is demonstrated with the use of a flow chart as follows.
Figure 1. Diagrammatical flow chart demonstrating the key steps involved in EIA.
In this exercise, ten wells of a microtitre tray are coated with Leptospira spp antigen and allowed to incubate overnight at 4áµ’C. Electrostatic forces between the tray and the antigen cause it to bind to the tray walls and then the excess is washed off twice with PBS-Tween and blotted dry on benchcote. Tween is a non-ionic surfactant which is added to the PBS washing solution in order to act as a mild detergent by preventing non specific binding between two proteins. Washing the trays between steps is very important in order to avoid false positive results.
The wells are then loaded with 1% bovine serum albumin (BSA) in PBS-Tween and incubated for 1 hour at 37áµ’C. The purpose of the BSA is to block the surfaces of the well where antigen did not bind so that other proteins (e.g. antibody) do not bind to the plastic. If this step is missed then false positives will be obtained as antibody will bind non-specifically.
The tray was then washed 6 times with PBS-Tween and blotted dry. 50Î¼l of PBS-Tween was added to wells 1 and 2, positive serum added to wells 3 and 4, negative serum added to wells 5 and 6, a patient's serum (test serum 1) added to wells 7 and 8, and, another patient's serum (test serum 2) added to wells 9 and 10. Following incubation for 30 minutes at 37áµ’C, the tray was washed 6 times as before and blotted dry.
50Î¼l of PBS-Tween was then added to wells 1 and 2, and a second antibody system, goat anti-human immunoglobulin conjugated with horseradish peroxidase (HRP) was added to wells 3 to 10 and incubated again for 20 minutes at 37áµ’C. PBS-Tween was used to wash the tray 3 times, and then distilled water was used to do the same.
Peroxidase substrate reagent (hydrogen peroxide + ortho-phenylenediamine/OPD) was added to all wells and incubated at 37áµ’C for a further 20 minutes. The reaction that occurs is as follows:
The HRP has enzymatic activity which allows the breakdown of hydrogen peroxide (H2O2) into oxygen (O2) and water (H2O). If the secondary antibody did not bind to the human antibody in step 4 then there will be no HRP in the well to cause the reaction, and the components will be left colourless. However, if there is HRP present, then the well will appear brown due to the oxygen reacting with OPD to create a brown colouring as below:
Next, one drop of 4M sulphuric acid (H2SO4) is added to stop the reaction. If the well appears brown it indicates that the individual whose serum it is has been exposed to Leptospira spp and has antibodies against this antigen. If the well remains colourless, then no antibodies are present against Leptospira spp.
The aims of the exercise were to perform and explain the principles behind an EIA test, including describing the purpose of the controls and the value of the system as a diagnostic tool (BMS2052, Class Notes, 2012). Also, the exercise aimed to allow analysis and interpretation of the results to reach a meaningful conclusion in a way that would provide a better understanding of the practical applications that such a technique offers.
Materials and Methods
For materials and procedure see BMS2052, Practical Class Notes, 2012. The exercise was carried out in accordance with these notes; however, the expression 'blot dry' was construed as an action in which the components were physically displaced by knocking the upside down microtitre tray onto the benchcote.
An EIA/ELISA was performed in this exercise involving the Leptospira spp. PBS-Tween was added to wells 1 and 2, a positive control serum to wells 3 and 4, a negative control serum to wells 5 and 6, test serum 1 (a patient's serum) to wells 7 and 8 and finally test serum 2 (a patient's serum) was added to wells 9 and 10. A secondary antibody conjugated with peroxidase was added to wells 3-10 and after being washed with PBS-Tween and distilled water the peroxidase substrate reagent was also added and allowed to react. Sulphuric acid was used to stop to reaction and finally the wells were observed for any colour change and the results recorded and presented in table 1.
Table 1. Summary of the various EIA components added to each of the wells and the resultant colour changes observed in response to an enzyme-substrate reaction where homologous antibody was present.
Component added to the well:
1 & 2
3 & 4
5 & 6
7 & 8
(test serum 1)
9 & 10
(test serum 2)
Leptospira spp antigen
1% bovine serum albumin (BSA) in PBS-Tween
+ve control serum
-ve control serum
Test serum 1
Test serum 2
Results (+ = brown and - = colourless)
= component was added to the well
= component was not added to well
It was observed that wells containing only PBS-Tween (1 & 2), wells containing a negative control sample (5 & 6), and wells containing test serum 2 did not present with a colour change, as was expected. Conversely, wells containing a positive control sample (3 & 4) and wells containing test serum 1 (8 & 9) did demonstrate a distinct colour change, with these wells presenting brown.
Diff between indirect & direct ELISA
Purpose of each of the wells?
What is leptospira? (treatment, characteristics)
Question 5 of questions