Questions On Positive And Negative Controls In Biology

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ELISA practical answer sheet

Connie Duffy – B00609996

1. In the assay you have used both a positive and negative control. What is the purpose of both these controls?

Positive control: Positive control is used within ELISA to observe what a sample well would look like with a present antigen. For example an endogenous soluble sample can be used which consists of the protein that is being recognised. It basically establishes the reactions when there is supposed to be one, which in turn displays that the experiment is working.

Negative control: Negative control is used to observe what a sample well would look like without a present antigen. It knowingly does not express the protein being recognised. It is used to detect non-specific binding
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Why do you need four replicates of your positive control, negative control and unknown sample?

Four values were taken and then averaged out to produce more accurate results. Lessens the chance of false results, if for example only one reading was taken and it was abnormal, this could go unnoticed, by doing four examples this lessens the risk of abnormal/false results.

5. Why did you add 0.2M H2SO4 at the end of the assay to stop the reaction?

H2SO4 denatures the protein stopping the reaction, it can alter the pH so that the enzyme within the solution does not act on the substrate, and thus the reaction stops.

6. Record the O.D values obtained for your standard curve and your positive control, negative control and unknown samples below.

Strip 1 O.D. values

Label 1 2 3 4 5 6 7 8 9 10 11 12
O.D 0.573 0.358 0.225 0.39 0.302 0.403 0.514 0.262 0.600 0.176 0.164 0.084

Strip 2 O.D values
Label + + + + - - - - U U U U
O.D 1.929 1.947 1.865 1.635 0.162 0.240 0.187 0.092 0.435 0.362 0.58 0.517

7. Attach a copy of your standard curve.

Title: Graph showing results of ELISA test to determine the concentration of antibody in unknown sample measured at 450nm.

8. What is the concentration of antibody in your unknown sample?

From graph: y=-0.0225x +
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What is meant by quantitative ELISA?

In quantitative ELISA, how much the product “colours” is corresponding to the amount of enzyme-linked antibody involved in binding, which relates to the volume of antibody currently present at that time to bind antigen or conversely the volume of antigen that was present to bind the antibody. A concentration of the antigen is determined, it gives more information then whether the result was negative or positive. As mentioned, colour changes or fluorescence signals the amount of antigen within the sample. Quantitative results are usually displayed via a standard curve, usually from a serial dilution.

10. What is meant by an indirect ELISA?

Indirect ELISA involves two steps, it is a method used for the detection of a labelled secondary antibody. The first step involves incubation of the primary antibody with the antigen. The second step involves a labelled secondary (e.g. an enzyme) antibody that detects the primary antibody getting incubated. The volume of antibody to a certain antigen is indicated by the production of colour.

11. What anti-body based tests can you buy in a

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