1) In doing an ELISA, blocking is a critical step. Some blockers require long am
ID: 204044 • Letter: 1
Question
1) In doing an ELISA, blocking is a critical step. Some blockers require long amounts of time, while others require much shorter amounts of time. If blocking is not complete, how would that change the absorbance values that you observe? Explain your reasoning on a molecular basis.
2) Could an anti-digoxin antibody bind to molecules other than digoxin? Explain your reasoning on a molecular basis.
3) What would you expect the effect on absorbance to be if you increased the concentration of enzyme-linked antibody? If you increased the concentration of substrate? Explain.
4) pH can be an important factor in development of ELISAs. Explain what types of things in this assay would be affected by changes in pH. Provide a molecular level explanation.
5) In using ELISA to characterize whether someone is HIV-positive or HIV-negative (has antibodies to HIV or not), the researcher must decide the cut-off value for a positive and a negative identification and often runs a sample that they know is “negative” and they know is “positive.” However, patient samples will not exactly match these values. Below is a sample of data. Would you classify each patient as positive, negative, or indeterminate? (Indeterminate means cannot be determined by the data and assay system used.) Explain your reasoning.
Positive Control
Negative Control
Patient A
Patient B
Patient C
Assay Control
1.689
0.153
0.055
0.412
1.999
0.123
Positive Control
Negative Control
Patient A
Patient B
Patient C
Assay Control
1.689
0.153
0.055
0.412
1.999
0.123
Explanation / Answer
1) if the blocking is not done properly then the background noise increases and an absorbance value increases. This is because a blocking agent increases selectivity of the antigen to the provided antibody by reducing stray binding to the antibody.
2) yes. There is a possibility that the epitope of the digitoxin is present in some other drug/protein which will bind to the antidigitoxin.
3) the absorbance will increase of both substrate and enzyme linked antibodies are increased because they will produce more colour which will increase the absorbance.
4) the reasoning can be based on simple facts yhat we are dealing with mostly proteins- antibodies and antigenic peptides. At an un-optimal pH these biomolecules will degrade and loose it's functionality.
5. Patient A negative
Patient B intermediate
Patient C positive
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