It has been suggested that dysfunction of the serotonergic system is involved in the pathogenesis of clinical depression. To indirectly evaluate
central serotonergic functioning, the increase in plasma prolacatin concentration caused by oral administration of the drug fenfluramine can be
measured. Because of the limitation on use of human tissue in undergraduate practicals, the plasma samples that you have been supplied with in
the following experiment are ‘dummies’. However, the protocol and results are taken from a real study.
Neuroendocrine Challenge
• One depressed patient and one control subject were diagnosed by a psychiatrist according to DSM V criteria.
• Both were drug free and were matched for age, sex and menopausal status.
• Subjects fasted from the previous midnight until the end of the experiment.
• At 8.30am on the day of the study, a baseline blood sample was taken.
• At 9.00am fenfluramine (60mg) was given orally, and hourly blood samples taken for the next 5 hours.
• The blood was centrifuged and the resulting plasma was stored frozen at –20oC until assay.
Principle of Prolactin Assay
This prolactin enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microtiter plate
provided has been pre-coated with a monoclonal antibody specific for prolactin. Standards or samples are then added to the microtiter plate
wells and prolactin, if present, will bind to the antibody pre-coated on the wells. In order to quantitate the amount of prolactin present in
the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody, specific for prolactin are added to each
well to “sandwich” the prolactin immobilized on the plate. The microtiter plate undergoes incubation, and then the wells are thoroughly washed
to remove all unbound components. Next, a TMB (3,3′,5,5′ tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and
substrate are allowed to react over a short incubation period. Only those wells that contain prolactin and enzyme-conjugated antibody will
exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is
measured spectrophotometrically at a wavelength of 450 nm.
In order to measure the concentration of prolactin in the sample, calibration standards are also assayed (4 standards; 0, 5, 25, 50 ng
prolactin/ml)). The calibration standards are assayed at the same time as the samples and allow you to produce a standard curve of Optical
Density (O.D.) versus prolactin concentration (ng/mL). The concentration of prolactin in the samples is then determined by comparing the O.D.
of the samples to the standard curve.
Assay Procedure
1. You will assay all standards and samples. Therefore, you will need 4 wells for the standards and 12 wells for the samples ie. a total of
16 wells.
2. Add 50l of standards or samples to the appropriate well.
3. Add 100l of conjugate to each well. COMPLETE MIXING IN THIS STEP IS IMPORTANT. Cover and incubate for 1 hour at 37oC.
4. Prepare the substrate solution by mixing 3.5ml of substrate A and 3.5ml of substrate B. Prepare this no more than 15 minutes before the
end of the 1 hour incubation.
5. At the end of the incubation, wash the microtiter plate. Remove the incubation mixture by aspirating the contents of the plate into a
sink or waste container.. Using a squeezy bottle, fill each well completely with distilled water, then aspirate the contents into a sink or
container. Repeat this procedure 5 times, so that you have done a total of 5 washes. After the final wash, invert the plate and blot dry by
hitting the plate onto some absorbent paper until no more moisture appears. Note: hold the side of the plate firmly when washing to ensure that
all wells remain firmly in the frame.
6. Add 100l substrate solution to each well. Cover and incubate for 15 mins at 37oC.
7. Add 50l of stop solution to each well. Mix well.
8. Read Optical Density (O.D.) at 450nm within 30 minutes using a microtiter plate reader.
Please base your practical report on the following questions:
Calculation of Results
1. Give your values for optical density (O.D.) at 450nm for each standard and sample. Subtract the blank O.D. (produced by the calibration
standard with 0 ng/ml prolactin) from the O.D. of all the other samples. (1 mark)
2. Plot a standard curve of (measured OD-blank OD) against prolactin
concentration for each standard used. (3 marks)
3. Referring to the standard curve you have produced, determine the prolactin concentration of each of the subjects’samples. (2 marks)
4. Produce a graph showing the plasma prolactin concentration against time for both the depressed and the non depressed subject (2 marks).
Questions
1. Explain why the OD in the blanks was subtracted from all the other samples (2 marks)
2. In the ELISA to determine plasma prolactin, explain what the conjugate, substrate solution and stop solution contained (3 marks).
3. Explain the purpose of the two incubation stages, the washing stage, addition of the stop solution and reading of the plates at 450nm (4
marks).
4. Explain why it was important that both subjects had fasted before the fenfluramine was administered, and that all subjects were given
fenfluramine at the same time of day (2 marks)
5. Compare the way in which the plasma prolactin levels in the depressed patient and the control changed following fenfluramine
administration (2 marks)
6. What could be the underlying neurochemical difference(s) that account for any difference observed in plasma prolactin levels between the
depressed patient and non depressed patient following fenfluramine administration? Suggest experimental work that could be undertaken to
investigate your suggestions further (4 marks)
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