Enzyme Linked Immunosorbent Assay: Introduction, Types, And Applications

Discuss about the Enzyme Linked Immunosorbent Assay.

It is of importance to investigate the ELISA and blood typing experiment as it can be conducted to examine the presence or absence of either an antibody or an antigen in a sample of solution. This makes it an important tool in both the determination of the concentration of serum antibody such as measles, monkey pox, and influenza among other diseases as well as in the detection of the presence of an antigen. The ELISA (Enzyme-Linked Immunosorbent Assay) technique is applicable in the diagnosis of the presence of a disease through the use of two different methods: direct and indirect methods (1).

The direct method of conducting an ELISA test is ideal in the detection of antigens while the indirect method is used in the detection of antibodies. The indirect method identifies antibodies through agglutination reactions which take place during the reaction between the antigens and antibodies. Enzyme labeled antibodies is used in the ELISA test for the purposes of detection of an antigen or antibody.

There are numerous ways in which the human body fights pathogens. One of such ways is through the establishment of the Specific Immune Response. Specific Immune Responses are designed in such a way that they are responsive to a specific pathogen that could be making attempts to invade the human body (2). A Specific Immune Response is initiated by proteins alongside other molecules that are generated by pathogens. These proteins are known as antigens. The reproduction and stimulation of antibodies in the human body for the purposes of fighting infections upon being alerted of the presence of antigens is carried out by the T and B cells that are found in the human immune system. The pathogens are broken down by the phagocytic microphages which also display the molecules of antigens for recognition by the lymphocytes (3).

Antibodies are defined as immunoglobulin responsible for the binding of antigens and labeling them for destruction. This experiment uses the indirect ELISA test. The indirect ELISA test is a test used in finding pathogenic infection through acknowledging the presence of antibodies in the e human blood. The purpose of this experiment is to utilize multi-step assay in testing samples for infection of a specified pathogen, in this case measles, which is a highly dangerous disease caused by a virus known as paramyxovirus.

To investigate the hypothesis that there is a decrease in the spectrophotometer reading with an increase in the dilution of the well from the 1^st to the 12^th well

Materials and Methods

Objectives

• To conduct an indirect ELISA test technique for use in the determination of the immune status to measles virus in test sample
• To adopt the knowledge of immunology and incorporate the testing principles by ELISA and the utilization of labeled reagents

Materials and Methods

1. Have all the regents at room temperatures
2. Handle the 12-well plastic strip carefully from the kit and place it in a carrier frame
3. Pipette 50 uL of measles viral antigen (simulated) to all the wells of the strip
4. Cover the strip and incubate it for 15 minutes (during this time, perform steps 5 and 6)
5. In serum diluent, prepare two separate 1 in 100 dilutions of the Cut-Off control serum
6. In serum diluent prepare 1 in 100 dilutions of Positive and Negative Controls

Note: Patient samples have already been pre-diluted 1 in 100

7. Flick the contents of the wells of the strip down the sink and wash the wells 4 times with diluted solution (PBS-Tween), with a soak time of 5 seconds per wash. Firmly tap the strip dry on paper towel to ensure all the wash solution is removed
8. Add the prepared samples to the wells 1-12 as follows:
9. Cover the strip and incubate the plate for 20 minutes on your bench at room temperature
10. Add 50 uL of HRP-conjugate anti-human IgG (green) to each well(4)
11. Cover the strip and incubate the plate for 20 minutes on your bench at room temperature
12. Flick the contents of the wells down the sink and was the wells 6 times using diluted wash solution (PBS-Tween) at a soaking time of 5 seconds per wash. Firmly tap the strip dry on paper towel to ensure that all the wash solution is removed
13. Add 50 uL of TMB (dark bottle) to all wells
14. Cover and incubate the plate for 10 minutes on the bench at room temperature, timing from the initial addition. The color of the liquid will change to blue.
15. Add 50 uL of Stop Solution (0.18M H2SO4; Caution-Corrosive) to stop the reaction. The color of the liquid will change from blue to yellow.
16. Take the readings of the absorbance of each of the wells at a wavelength of 450 nm within 30 minutes of stopping the reaction.
17. Calculate the results.

An antigen is used in the experiment to coat wells of the microtitre plate, the blocking reagents used in unbounding the sites that would then aid in the prevention of positive reaction results. There are also antibodies, anti-(species) and IgG that are used in the experiment which are all conjugated to the enzyme to facilitate the reaction. The substrates in the experiment are used in reaction with the enzyme so as to generate the colored products that is used to indicate a positive reaction. Besides the reagents used in the procedure, there are additional reagents among them stop solutions, wash buffers and stabilizers that are used in facilitating the quality of the ELISA assy. The choice of an individual reagent should be based on the required sensitivity and whether an individual is attempting to detect an analyte or the response of the antibody to it. The use of quality reagents would aid in the achievement of results that can be reproduced and with reliable and good results (4).

There are various types of ELISA assays that are used in the determination and detection of the presence of various substances in an assay experiment. Such types include direct, indirect and sandwich or immunometric assay. In direct assay, the surface of the plate is directly coated with the sample that is to be tested and the measurement is enabled through the use of an enzyme-tagged antibody. Washing which is used in the elimination of the unbound antibodies follows incubation (5). The recommended substrate is added to the medium generating a signal that is directly proportional to the amount of antigen that is present in the sample.

Such a correlation is usable in the extrapolation of the antigen concentration of the unknown sample using a standard curve for the case of quantative data type. Direct ELISA type is recommended for the estimation of the concentration of antigens having high molecular weight. For the case of indirect ELISA, it is a two-step process which includes the utilization of a primary antibody and a labeled secondary antibody. The secondary antibody is normally a polyclonal anti-species antibody which the primary antibody in incubated using antigen –coated wells. This is the method that is commonly used in the diagnosis of viruses, bacteria, parasites or even the quantity of antibody in comparison to a foreign antigen. Sandwich assays utilize two antibodies that are specific to the antigen in capturing the antigens that are in the well for the purposes of detection (6).

Types of ELISA Assays

The mean absorbances of all the three quality controls met the standards i.e. the negative control, cut off control and positive control. The mean absorbance of each of the patients was determined and comparison made against the cut off control to establish the percentage with which it was above or below the cut off control. From the obtained results following the interpretation of the standard units, patient 1 test positive for measles virus hence there was viruses of measles detected in the blood. Patients 2 and 3 on the other hand, showed negative tests. This means there were no measles viruses detected in their antigens or antibodies (7).

The results on an ELISA test can be analyzed and interrelated using three different approaches depending on the nature of the data from the findings of the experiment. These approaches include qualitative, quantitative and semi quantative (8). With regard to quantative data, the interpretation of the results is based on making comparison between the data and a standard curve which allows the determinations of the concentrations of the antigens in the various samples precisely.

Qualitative data analysis is applied in confirm or denying the presence of a specified antigen in a sample or samples that have been used for the experiment. The data provides options of offering a yes or no response. It is applicable in making comparisons to a blank well which is free from any unrelated control antigen or otherwise any other antigen.  Still, semi-quantative analysis is used in cases where the intensity of the signals differs directly with regard to the concentration of the antigen. In this case, ELISA data is used in making comparison on the relative levels of antigens using a sample of an assay (9).

This experiment used qualitative ELISA test analyzing which the aim was to confirm the presence of measles virus in the antigens of three patients named patient 1, patient 2 and patient 3. As can be established from the results, patient 1 confirmed the presence of measles virus in the blood while patent 2 and patient 3 tested negative for measles in their blood (10). The results can then be used in further analysis of the blood samples to establish the cause for the differences in the results.

ELISA tests are also applicable in the diagnosis of other diseases including HIV, influenza, Ebola HF, monkey pox, influenza, food allergy, chagas disease, RMSF, Lyme disease and leishamaniasis. In all these tests, the concentration of antigens in the known samples or called the blank well is used as the base line for making comparisons which forms the basis of analysis.

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