ELISA: Enzyme-Linked Immunosorbent AssayIntroduction and AimIt is of importance to investigate the Enzyme-Linked Immunosorbent Assay (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 virus, monkeypox and influenza among other diseases as well as in the detection of the presence of an antigen. The purpose of this experiment is to utilize multi-step assay in testing samples for infection of a specified pathogen, in this case, measles virus, which is a highly dangerous disease caused by a virus known as paramyxovirus. Background and Principle The Enzyme-Linked Immunosorbent Assay (ELISA) 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 Enzyme-Linked Immunosorbent Assay (ELISA) test is ideal for 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-labelled antibodies are used in the Enzyme-Linked Immunosorbent Assay (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). Proteins alongside other molecules that are generated by pathogens initiate a Specific Immune Response. These proteins are known as antigens.
The T and B cells that are found in the human immune system carry out the reproduction and stimulation of antibodies in the human body for the purposes of fighting infections upon being alerted of the presence of antigens. The pathogens are broken down by the phagocytic macrophages that also display the molecules of antigens for recognition by the lymphocytes (3). Antibodies are defined as immunoglobulin responsible for the binding of antigens and labelling them for destruction. This experiment uses the indirect Enzyme-Linked Immunosorbent Assay (ELISA) test. The indirect Enzyme-Linked Immunosorbent Assay (ELISA) test is a test used in finding pathogenic infection through acknowledging the presence of antibodies in the human blood. Methodology 1.
Have all the regents at room temperatures, and then place the 12-well plastic strip in a carrier frame carefully. 2. Use a pipette and transfer 50uL of measles virus to all the wells, and incubate it for 15 minutes (during this time, remember to cover all the wells).3.
In serum diluent, make up a 1 in 100 dilutions of the Cut-Off control serum in two separate tubes, and make the same dilution 1 in 100 of Positive and Negative Control. 4. Tap all the inside of the wells down the sink, then using a dilute solution (PBS-Tween) to rinse the wells 4 times with a soak time of 5 seconds per rinse. Make sure all the wash solution is removed by drying the strip on the paper towel.
5. Pipette the prepared sample following the table below: Note: From well 7-12, patient samples have already been pre-diluted 1 in 100.Well 1: 50uL diluted Negative Control Well 2: 50uL diluted Negative Control Well 3: 50uL diluted Positive Control Well 4: 50uL diluted Positive ControlWell 5: 50uL diluted Cut-Off Control (1st dilution)Well 6: 50uL diluted Cut-Off Control (2nd dilution)Well 7: 50uL patient serum 1Well 8: 50uL patient serum 1Well 9: 50uL patient serum 2Well 10: 50uL patient serum 2Well 11: 50uL patient serum 3Well 12: 50uL patient serum 36.
On the bench, incubate the strip for 20 minutes at room temperature.7. Using HRP-conjugated anti-human IgG (green) provided and adds 50uL of it to each well. Then incubate the plate-like as step 6.
8. Similar to step 4, tap the wells down the sink and using a dilute solution (PBS-tween) to wash the wells 6 times with a soak time of 5 seconds per wash. Dry the strip by strongly taps it on the paper towel to remove all the wash solution (just be careful to not break the plate).9. Add 50 uL of Tetramethylbenzidine (TMB) in dark bottle to all wells, and then incubate the plate for 10 minutes at room temperature, timing from the initial addition.
The change of yellow colour to blue will indicate the reaction has occurred. 10. Add 50 uL of Stop Solution (0.18M H2SO4; Caution-Corrosive) to stop the reaction. The colour of the liquid will change from blue to yellow.11. Take the readings of the absorbance of each of the wells at a wavelength of 450 nm within 30 minutes of stopping the reaction, and 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 is all conjugated to the enzyme to facilitate the reaction. The substrates in the experiment are used in the reaction with the enzyme so as to generate the coloured products that are 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 Enzyme-Linked Immunosorbent Assay (ELISA) assay. Last but not least, the repeated washing steps are to remove or wash away any unbound or excess. 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).Results Absorbance at 450 nmNegative control 0.09Negative control 0.048Positive control 2.296Positive control 2.
296Cut-Off Control 1st 1.194Cut-Off Control 2nd 1.214Patient serum 1 2.
064Patient serum 1 2.22Patient serum 2 0.062Patient serum 2 0.063Patient serum 3 0.
0683Patient serum 3 0.685Table 1: Absorbance value at 450nm Mean absorbance Passed QC?Negative Control 0.069 YesCut-Off Control 1.204 YesPositive Control 2.296 YesTable 2: Quality Control Mean absorbance % Above or below Cut-Off Control value Standard Units InterpretationPatient 1 2.
142 77.91 17.79 Positive- Measles virus detectedPatient 2 0.0625 94.81 0.52 Negative- Measles virus is not detectedPatient 3 0.
684 43.19 5.68 Negative- Measles virus not detectedTable 3: Patients ResultsResults in Standard UnitsPatient 1=patient mean absorbance value*10/cut off control value =2.142*10/77.91=17.
92Patient 2=patient mean absorbance value*10/cut off control value =0.0625*10/94.81=0.52Patient 3=patient mean absorbance value*10/cut off control value =0.684*43.
19/77.91=5.68DiscussionThere are various types of Enzyme-Linked Immunosorbent Assay (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 the quantative data type. Direct Enzyme-Linked Immunosorbent Assay (ELISA) type is recommended for the estimation of the concentration of antigens having high molecular weight. For the case of indirect Enzyme-Linked Immunosorbent Assay (ELISA) that is used in this experiment, it is a two-step process, which includes the utilization of a primary antibody and a labelled secondary antibody.
The secondary antibody is normally a polyclonal anti-species antibody, which the primary antibody is incubated using antigen-coated, wells. This is the method that is to confirm the presence of measles virus in antigen of the patient, and it is also 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). 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 a virus 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 Enzyme-Linked Immunosorbent Assay (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 a 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 deny the presence of a specified antigen in a sample or samples that have been used for the experiment. The data provides options for 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, the 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, Enzyme-Linked Immunosorbent Assay (ELISA) data is used in making a comparison of the relative levels of antigens using a sample of an assay (9). Extended LearningThis experiment used qualitative indirect Enzyme-Linked Immunosorbent Assay (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.
Enzyme-Linked Immunosorbent Assay (ELISA) tests are also applicable in the diagnosis of other diseases including HIV, influenza, Ebola HF, monkeypox, influenza, food allergy, Chagas disease, RMSF, Lyme disease and leishmaniasis. In all these tests, the concentration of antigens in the known samples or called the blank well is used as the baseline for making comparisons that form the basis of analysis. References1.
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