Immunoelectrophoresis lab

sophietevans
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From the 04/10/13 Immunology and Disease lab.

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Question Answer
Why was it important to keep replacing the agar in the water bath when making the slides? The molten agar would start to solidify if left out of the water bath, even for a short period of time. If this partially solidified agar were used to produce the slides, the consistency would be uneven and it would set incorrectly, preventing smooth separation of serum proteins during electrophoresis.
What was done to make sure that the agar stuck to the slides even when the slides were upside down in the electrophoresis tank? A drop of agar was quickly spread over the slide and allowed to set for 1 minute, creating a 'sticky' surface onto which the thick layer of 3mL of agar was poured. It provided adhesion for the main body of agar.
Other than not separating proteins accurately, what effect could partially solidified agar have on the results? The agar would not be clear so the results (while likely being inaccurate) would be difficult to interpret.
How was the bromophenol blue dye added to the agar slide? It was mixed with the whole human serum (the control antigen) and the patient serum so when 2µl was added, the dye was already present.
What is the function of the bromophenol blue dye? How did it do this? The dye allowed the progress of the electrophoresis to be visualised and tracked. The molecules of the dye are smaller in size/weight than that of the proteins in the sera, so they travelled furthest/fastest towards the electrode. Once the band of blue dye had almost reached the edge of the slide, it was assumed that the serum proteins were adequately separated.
Which electrode in the electrophoresis tank did the serum proteins and bromophenol blue dye travel towards? Why? The proteins and dye were negatively charged (anions) and so travelled towards the positively charged anode.
How was the number of milli-amps (current) applied to the slides calculated? 8 milli-amps per slide (e.g. 8 milli-amps x 8 slides = 64 milli-amps).
With regard to Ag-Ab interactions, what was first spread throughout the agar by electrophoresis? The antigens - the serum proteins from the control and the patient sample.
With regard to Ab-Ag interactions, what creates the strongest bond between the two? The antibody's binding site and the antigen's epitope must have complementary shapes.
Given that the charge of the proteins determines the direction of travel, what determines the distance travelled? The size of the proteins determines the distance travelled, because the agar contains only small pores so the smallest proteins travel with ease and therefore travel furthest, whereas larger proteins travel more slowly as the pores are navigated. However, mostly it was their CHARGE.
At what point in the assay did the slides cease to be identical? When the different antibodies were added to the troughs in order to visualise different antigens potentially present in the sera.
Given that certain antibodies are used to look for certain antigens, what quality can the technique be said to have? Specificity.
Why is a dye not necessary to visualise the Ab-Ag interactions? The antibody diffuses through the agar and when it comes across a complementary antigen, it binds with it to form a macromolecular complex. These are too large to remain in solution, and so they precipitate out, forming a white/opaque precipitin line if sufficient precipitation occurs (e.g. at the equivalence point).
What is a precipitin? An antibody capable of aggregating soluble antigens.
Why does visible precipitation of antibodies and antigens only occur in the region of equivalence? The antibody has been added in excess to allow it to diffuse. The antigen travelled through the agar to a distance dictated by its size. Only when there is an equivalent ratio of antibody:antigen can a precipitin line be formed. Otherwise a large proportion of antigen (if in excess) remains soluble, or a large number of antibodies do not form macromolecular complexes and precipitate out of solution (again, if in excess).
What is the purpose of immunoelectrophoresis? Therefore, which word can be used to describe the technique? IEP allows a clinician to check whether or not appropriate antigens or antibodies are present. Therefore it is a QUALITATIVE technique.
In what way is immunoelectrophoresis a sort-of quantitative technique? If a precipitin line is particularly thick or bright, there is a large amount of the antibody/antigen being sought present. However, this is not quantitative as it does not produce a number.
Why is IEP a limited technique? IEP is not particularly sensitive. It detects relatively high antibody concentrations (>several hundred µg/ml) and so its utility is limited to the detection of quantitative abnormalities only when the departure from normal is striking e.g. in immunodeficiency states and immunoproliferative disorders.
How might IEP be useful in monitoring therapy? For example, it could be used to track the production of antibodies in a person with an infection, or a person undergoing a vaccination schedule.
What is hypergammaglobulinaemia? A high concentration of IgG in the blood.
List some chronic infections that hypergammaglobulinaema can be indicative of. AIDS, multiple myeloma, chronic hepatitis, multiple sclerosis, and some autoimmune diseases.
What type of hypergammaglobulinaemia would be present in multiple myeloma compared with the other conditions listed? Monoclonal, as opposed to polyclonal, hypergammaglobulinaemia - as a result of rapid proliferation starting with a single cell.
Why does albumin travel the furthest through the agar gel? Because it is the most negatively charged.
What does the arc of the precipitin curve on the agar slide represent? The distance travelled by the bulk of the antigen molecules.
In order for cross-linked antibody-antigen complexes to be formed, what must be true of the antibodies? They must be bivalent, i.e. not truncated, and they will then produce 'star' shaped complexes.
What equipment is required to produce the trough and wells on the agar slides? A measurement tray, a guillotine, a pipette tip (for removing agar from the trough) and a sucker (?!) for removing agar from the wells.
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