ELISA Methodology

(Name)
(Instructors’ name)
(Course)
(Date)
ELISA Protocol
ELISA (Enzyme-linked immunosorbent assay) is an assay procedure which is created for checking and quantifying substances like the proteins, peptides, hormones, and antibodies. There are many forms of ELISA which make the ELISA protocol to have few variations. The different types of ELISA protocol include; sandwich, direct, elispot, indirect and competitive ELISA protocol. However, the major ELISA protocol and process are similar. All in all, the ELISA protocol involves three majors steps; plate preparation, assay protocol, and analysis of results.
Plate Preparation
This process involves diluting the extracted antibody to the working samples in the plates. Afterward, you should cover a 96-well microplate with 150 µL per plate of the captured body which is diluted. You should then enhance the sealing of the plate and incubate it overnight at 4 ºC. Aspiration follows which involves washing the plate with at least 300 µl wash buffer and replicate the procedure twice through several washes. To achieve good results, it is crucial to complete the extraction of fluid at every step. After the final wash, you should dispose any residual wash by overturning the plate and use a paper towel for blotting purposes. The last step involves blocking the plate by including 300 µL of blocked buffer to each plate.
Assay Process
The assay process involves adding 150 µl of standards or sample in the dilute sample solution on each plate.

Enhance sealing of the plates and incubating for 2 hours at normal temperature and repeat step 2 of plate preparation that is the aspiration or washing. Add 150 µL of the antibody which is concentrated in antibody buffer to every plate then cover and incubate for at least 1 hour at normal temperature. Again, repeat the washing process similar to that of plate preparation. The next step involves the inclusion of 250 µL of substrate fluid to every plate. Afterward, the mixture should be kept in an incubator for not more than 20 minutes (If the substrate mixture is not a requirement, the time of incubation should be standardized. It is important to note that the mixture should not be exposed to direct sunlight. Finally, add 1000 µL of stop fluid to every pot and slowly hold the plate to ensure sufficient mixing of the solution. Calculation of the optical mass of every well follows which involves using a microplate equipment which has been standardized.
Calculation of Results
Determination of the results is the last procedure of ELISA protocol which involves calculation of the average absorbance rate for every category of identical samples, controls, and standards. After the calculation, you should create a graph by drawing the average absorbance for every standard on the y-axis against the concentrate on the x-axis and then drawing a line of best fit through the plotted sections on the plotted graph. The next step involves calculation of the concentration of the estimated values and locating the estimates average absorbance value on the y-axis and drawing a parallel mark to the arc on the graph. At the section of connection, you should draw a perpendicular line to the x-axis and read the concentration. Finally, if the sample solution has been diluted, the concentration read from the graph must be multiplied by the dilution factor. Due to the advancement in technology, the computer-enabled software has been created that can be used to determine the concentration of the sample.