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Enzyme-Linked Immunosorbent Assay or ELISA as it is more commonly known is an enzyme immunoassay (EIA), which is one of the most widely used applications of monoclonal antibodies.

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Introduction

ELISA Enzyme-Linked Immunosorbent Assay or ELISA as it is more commonly known is an enzyme immunoassay (EIA), which is one of the most widely used applications of monoclonal antibodies. Immunosorbent assays, as the term suggests, involve adsorption of either the antigen or the antibody to a surface, in most cases the bottom of a plastic well. The ELISA is used to detect and amplify an antigen-antibody reaction. It is the most basic test to determine if an individual is positive for a selected pathogen. It screens everything from drugs to the AIDS virus in humans. ELISA techniques have advantages of specificity and sensitivity when compared with more traditional tests. It is generally a cheap and simple method. The ELISA is based on antibody recognition of a particular antigenic epitope. Monoclonal antibodies can be cross-linked to one of nine different enzymes and used in the ELISA. Anti-rabbit immunoglobulin is conjugated with alkaline phosphatase (1:3 by weight) by addition of glutaraldehyde. Glutaraldehyde is a bifunctional cross-linker used to join the enzyme to the antigen or antibody. Maleimide derivatives can link two separate protein molecules together, one through an amide bond and the other through a thioether bond. Based on the principle of antibody-antibody interaction, the ELISA technique allows for easy visualization of results and can be completed without the additional concern of radioactive materials use. ...read more.

Middle

It shows that serum A had variations in optical density with different dilutions, whereas serum B did not. Many of the results for serum B were negative and therefore converted into zero, to plot the graph, as a negative reading indicates no binding between the antibody and antigen. The line of best fit plotted for serum A1 and A2 shows that as optical density increases so does the concentration of the serum (becoming more dilute). The optical density for serum A2 is greater for the more concentrated solutions. Figure C represents the same data as figure A, however the scale of the x-axis is different. Here, instead of plotting the actual dilutions, I have coded each dilution 1-12, so that each point is equally spaced from each other. By doing it this way the graph goes in order of the dilutions in the wells, where 1 represents the most concentrated i.e. 1/64?l and 12 is the most dilute, i.e. 1/131072?l. Figure C is generally a better way to display the results as it is easier to read off data and goes in the correct direction of concentration, and the points are not all un-proportionally placed like they are in B. The end-point can be determined using figure C, whereas in figure B this cannot be done as it does not show where the values become negative or cross the x-axis. ...read more.

Conclusion

There is greater colour intensity here, as there will be more formation of antibody - antigen complexes. As it becomes more dilute, the optical density begins to decrease. This may be due to the fact that the difference in dilution at the start is a lot less than as the dilutions proceed. Figure 3 uses the same data as figure 2 but adds trend lines. I think that this is a better way to present the results as it shows both the actual results as well as the general trend so different interpretations can be made, such as the rate at which the optical density decreases (by calculating gradient). 4 - the errors that could have been introduced as a result of undertaking serial dilutions directly into the coated plate are that air bubbles may have formed in the pipette resulting in inaccurate measurements for proceeding dilutions. The tips should be changed for each proceeding dilution rather than for each serum as this could lead to contamination. Also, the serial dilutions could have been prepared in test tubes prior to the wells to enable proper mixing of the serum with conjugate, substrate etc. The first incubation, was left without being covered which would also cause error. *The conclusion describes some aspects of the above answers in more detail. ...read more.

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