The paper 'Enzyme-Linked Immuno Sorbent Assay' is a great example of translational researchEnzyme-linked Immunosorbent assay is also known as ELISA or EIA. ELISA is mainly dependent on an enzyme conjugated to an antibody. This enzyme on reaction with a chromogenic substrate gives a coloured product. Various kinds of enzymes are used in ELISA and several variants of the assay have been developed (Stryer et al, 2002). HIV is a global problem today and several current enzyme immunoassays help in early detection of antibodies and one of the assays is ELISA (Fearson, 2005).
In this experiment, we used Indirect ELISA to determine whether a person has been exposed to the Human Immunodeficiency virus or not. In the assay, the antigen is immobilized in wells. Next, the patient’ s serum is added in the wells. If the sera contain antibodies specific for the antigen it binds to the antigen. Next, a secondary antibody conjugated with an enzyme (horseradish peroxidase) is added to the well. This binds to the primary antibodies. Detection depends on the addition of a chromogenic substrate which gives a colour if the assay is positive.
If a person is exposed to the HIV, either directly or through vaccination, his body will produce antibodies. The production of antibodies can be detected through ELISA and a positive test confirms exposure to the pathogen. METHODS AND MATERIALS To conduct the experiment we used 12 wells in total consisting of 3 columns and 4 rows. The columns were marked A, B and C, while the rows were marked 1, 2, 3 and 4. We worked with two different substrates i. e. Substrate1 and substrate2. The table below shows the substances that were added in each of the wells: A B C 1 Patient Sera Secondary antibody Substrate 1 (S1) Patient sera Secondary antibody Substrate 1 (S1) Primary antibody Secondary antibody Substrate 1 (S1) 2 Antigen Patient’ s antisera Secondary antibody S1 Antigen Patient’ s antisera Secondary antibody S1 Antigen Primary antibody Secondary antibody S1 3 Antigen Secondary antibody Substrate 2 (S2) Antigen Secondary antibody Substrate 2 (S2) Antigen Secondary antibody Substrate 2 (S2) 4 Antigen Patient’ s antisera Secondary antibody Substrate 2 (S2 Antigen Patient’ s antisera Secondary antibody Substrate 2 (S2 Antigen Primary antibody Secondary antibody Substrate 2 (S2 S1 contains hydrogen peroxide and amino salicyclate which is naturally colourless but a brown colour indicates a positive result. S2 contains hydrogen peroxide and azino-di-ethylbenzthiazaline sulfonate (ABTS) which turns from colourless to green indicating Positive result. RESULTS The experiment was carried out after proper incubation at room temperature colours developed as follows: A B C 1 Colourless Colourless Colourless 2 Reddish Brown Reddish-brown Reddish-brown 3 Seafoam Green Seafoam Green Seafoam Green 4 Green Green Green We noticed that no colour developed in Row 1.
Reddish-brown colour development was seen in Row2. Seafoam green was detected in Row 3 while Green was observed in Row 4. DISCUSSION The development of colour was analyzed in each of the wells. Row 1 and 2 were conducted using substrate 1 and Row 3 and 4 were conducted using substrate 2. In Row1, no colour developed since no antigen was added at all.
Row 1 served as Negative Control for antigen requirement showing that without the antigen the test cannot be positive since the antibodies in the sera would not anything to bind with and hence would get washed away. Row 2 was reddish-brown and it gave a positive result since all of the necessary components for ELISA i. e. antigen, patient’ s sera, secondary antibody and substrate was added. The green colouration in ROW2 A and B indicates that the patient’ s sera contained an antibody against the antigen Row 2 C serves as the positive control. In Row 3, we obtained seafoam green which was negative.
This happed since in Row 3no patient’ s antibody nor any primary antibody was present. This entire row served as Negative Control for the presence of antibody for ELISA. In row 4, we observed green colour indicating a positive test. This occurred since in Row 4 all columns had all ELISA components i. e. antigen, primary antibody or patient’ s antibodies, secondary antibody and substrate 2. Row 4 column C served as Positive Control. Therefore, analyzing the overall results we may say that the patients tested POSITIVE for exposure to HIV.
The experiment was therefore successful because the antibody was present in the patient’ s sera. When this sera was put in an antigen-coated well, the antibody formed a bond with the antigen. When the secondary antibody was added, it attached to the antibody of the patient’ s sera. Now, since the secondary antibody was conjugated to an enzyme, it gave a colour when the substrate was added. Thereby confirming a positive test. The experiment also established the importance of both positive and negative controls.
Controls help in eliminating errors. Positive controls determine that the experimental design is working while the main purpose of negative controls is to establish the role of certain components of the experimental design (Lipsitch et al, 2010). In our experiment, we designed negative controls by leaving out essential ingredients to verify that without the presence of the ingredient a Negative Result will be produced. CONCLUSION ELISA is a handy tool for the detection of antibodies in the sera. In this case, the patient tested positive for HIV exposure. In the clinical context, ELISA can even be used on paired serum and saliva samples for detection of HIV (Holmstrom et al, 1990).
ELISA, being a relatively easier and cost-effective assay to perform can help in early detection of several diseases including prenatally acquired HIV infection (Liberatore et al, 1996).
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