ELISA PRACTICAL REPORT
The Enzyme-Linked Immunosorbent assay (ELISA) is a test that is conducted to identify the substance under study with the help of antibodies and changing color elements. It is the “wet-lab” oriented analytic biochemistry assay that consume enzyme immunoassay to detect the existence of a substance (antigen) in a liquid sample. It is of great help in medicine and plant pathology as a diagnostic tool and quality control check.
ELISA test involves one antibody according to the need of the test, where the antigen is immobilized on the solid plate. The method is done specifically and nonspecifically. The report involves the description and construction of two different ELISAs.
Theory behind Fibrinogen
It is possible to differentiate between the intact and processed Fibrinogen using Sandwich ELISA. In theory, the sandwich ELISA is an efficient method to detect sample antigen. The method involves the two layers of antibodies i.e. detection and capture antibodies. An antigen under study must have at least two antigenic epitope that are capable to bind an antibody (monoclonal and polyclonal) and hence easily detectable in the system (ELISA encyclopedia ).
The biochemistry of fibrinogen can be manipulated when designing an appropriate ELISA sandwich assay. On the other hand, intact fibrinogen is only present in plasma. Fibrinogen is a protein produced by the liver that helps to stop bleeding by forming blood clots. During normal clotting, Fibrinogen is broken by thrombin that is also an enzyme, into short sections of fibrin. It can be measured in serum or plasma (Brunner, E.; Smith, G.; Marmot, M.; Canner, R.; Beksinska, M.; O’Brien, J.).
Sandwich ELISA is used to distinguish both Fibrinogen because it utilizes two antibodies to detect. The first antibody is the capture, and other is detection. These two antibodies detect the distal portion of intact fibrinogen that in turns useful to distinguish plasma and serum.
Theory of Gliadin
In this method, the single antibody is used and hence the method is also called Elisa. The theory behind the ELISA is that the single antibody is directed against the antigen that has to be immobilized on the surface. Single antibody makes it simple to detect depending upon the protein.
Gliadin is the complex group of protein that coupled with the gluten protein to determine the properties of wheat flour. It also helps the bread to rise in the process of baking. It is important to study the wheat flour quality.
On the other hand, this method is used to detect the childhood coeliac disease and adult coeliac disease and are preferable for screening of coeliac patients.
There are basically two major sections of the experiment, therefore, for each of these sections the outcomes and aims are different. The experiment holds two different section, in each section ELISA is conducted on human fibrinogen and gliadin proteins. These two proteins from different sources are taken. Hence, these two methods (repeated for one another) holds the objectives respectively.
The Chief aim of the experiment is to establish the ELISA Standard Curve from the provided sample that contains a specific concentration of human fibrinogen and gliadin proteins. Fibrinogen is extracted from the human serum whereas, gliadin is taken from bread. Moreover, the study is also conducted to determine the concentration of above-mentioned protein in unfamiliar tester by using standard curves. Lastly, the experiment is conducted to compare the single and double Ab ELISA methods.
The method of both experiments is same. However, protein Ag is changed in plate 1 and plate 2. The experiment started with the adding 100 µl buffer in the A1, A2, B1 and B2 funnels. These funnels are blank. The Same amount of buffer is also added in the A4-A12, B4-B12, C1-C12 and D1-D12. On the other hand, A3 and B3 are the bells having the 100 µl Ag protein (either fibrinogen or Gliadin). An undiluted amount of antigen is added to it. Remaining wells have same series in which 100 µl of stock of protein is added in A4/B4 with a gentle mix of 100 µl coating buffer. Therefore, these wells contain 200 µl liquid and dilution ratio is 1:2.