1. Summary and background of the test:
Prolactin (PRL) is e polypeptide hormone (molecula weight 20,000) secreted by
the pituitary gland which plays a key role in the development of the mammary
gland, the production and secretion of milk and the control of male and female
gonadal functions.Prolactin secretion is under hypothalamic control exerted
directly by dopamine, several prolactin releasing factors (PRF) and perhaps VIP
(vasoactive intestinal polypeptide) or a closely related peptide. TRH also acts
directly at the pituitary level to stimulate prolactin release but its
physiological role in the control of prolactin secretion has not been
established yet. Several factors, involving serotoninergic or noradrenergic
pathways are also invoved in the control of prolactin secretion. The plasma
concentration of prolactin increases in various physiological situations sush as
stress, pregnancy and lactation. Physiological levels fluctuate according to a
nycthemeral rhythm, a significant rise being observed at night. Drugs with
anti-dopamine activity (psychotropic agents) and ovulatory supressants, increase
prolactin secretion.
2. Principle of the test :
The Bio-Line Prolactin-Irma is an immunoradiometnc assay based on coated-tube
separation in which several monoclonal antibodies directed against distinct
epitopes of Prolactin have been used.. Mabs1, the capture antibodies, are
attached to the lower and inner surface of the plastic tube. Standards or
samples added to the tubes will at first show low affinity for Mabs1. Addition
of Mab2, the signal antibody labelled with 125I, will complete the
system and trigger the immunological reaction. After washing, the remaining
radioactivity bound to the tube reflects the antigen concentration. The use of
several distinct Mabs avoids hyperspecificity.
3. Precautions:
1. Radioactive material: Radioactive material may be received, acquired,
possessed and used only by physicians, clinical laboratories, or hospitals for
"In-Vitro" clinical or laboratory tests not involving internal or
external administration of the material, or the radiation therefrom, to human
beings or animals.
Compliance with these basic rules of radiation safety should provide
adequate protection:
1. Do not eat, drink, smoke, or apply cosmetics in areas where radioactive
material is used.
2. Do not pipet by mouth reagents containing radioactive materials.
3. Wear protective clothing; i.e., lab coats and disposable gloves, in
order to avoid direct contact with radioactive reagents.
4. Work with radioactive materials should be performed in a designed area.
5. Radioactive materials should be stored in an acceptable location.
6. A log should be kept for receipt and disposal of radioactive materials.
7. Radioactive spills or accidents should be taken care of immediately
according to established procedures.
8. Disposal of radioactive materials must comply with
prevailing regulations and guidelines of the agencies holding
jurisdiction over
the laboratory.
2. Sodium azide: Sodium Azide, used as a bacteriostatic agent, is toxic in
acid medium. In addition, it may form potentially explosive lead or copper
azides. To avoid dangerous deposits, waste solutions should be flushed away with
large volumes of water.
3. Hepatitis and Acquired Immune Deficiency Syndrome (HTLV-III): All Bio-Line
reagents included in this kit have been tested and found to be non reactive for
hepatitis B surface antigen. They have also been screened and determined to be
non-reactive for HTLV-III antibody. However, human serum products should be
handled as if potentially capable of transmitting hepatitis, Acquired Immune
Deficiency Syndrome, or other infectious agents.
4. Materials provided:
Kit contains sufficient reagents for 100 determinations.
1. Human serum with sodium merthiolate and azide based standards &
control: 8 lyophilised vials to reconstitute with 0.5 ml exept the
standard with 2.0 ml.
See exact values on vial labels.
2. 125I-Monoclonal-anti-Prolactin tracer: 2 vials (red
solution) containing 10.5 ml. Activity < 4.75 µCi or 175 kBq.
3. Coated tubes: 2 x 50 tubes, coated with Anti-Prolactin monoclonal
antibodies.
4. Wash solution concentrate: 1 vial of 10 ml of concentrate, to be
diluted into 700 ml distilled water and stored at 4°
C.
5. Materials required but not provided:
The following material is required but not provided in the kit:
1. Distilled water
2. Pipettes for delivery of: 25, 200 and 500 µl, 2 ml (the use of accurate
pipettes with disposable plastic tips is recommended)
3. 5 ml automatic syringe (Cornwall type) for washing
4. Aspiration system (optional)
5. Gamma counter, set for 125I counting and Vortex mixer and
magnetic stirrer.
6. Specimen collection and preparation:
Serum or plasma :
Serum and plasma must be kept at 2 – 8°C.
If the test is not run within 24 hours, storage at –20°C is recommended
and will not result in loss of immuno-reactivity for at least 6 months.
Serum or EDTA plasma provide similar results.
Y(serum) = 1.09x (hep. plasma) + 0.05 r = 0.95 n = 30
Y(serum) = 0.96x (EDTA plasma) + 0.14 r = 0.98 n = 30
Do not use haemolysed samples.
7. Assay procedure:
Do not use the kit or components beyond expiration date. Do not mix materials
from differents kit lots. Bring all the
reagents to room temperature prior to
use. Thoroughly mix all reagents and samples by gentle agitation or swirling.
Use a clean disposable pipette tip for addition of each different reagent
and sample in order to avoid cross-contamination.
High precision pipettes or automated pipetting equipment will improve the
precision. Respect the incubation times.
Prepare a standard curve for each run, do not use data from previous runs.
1. Label coated tubes in duplicate for each standard, sample, control. For
determination of total counts, label 2 normal tubes.
2. Vortex mix briefly standards, samples, controls and dispense 25 µl of
each into the respective tubes.
3. Dispense 200 µl of tracer into each tube, including the ones for Total
count.
4. Shake the rack containing the tubes gently by hand.
5. Incubate for 2 hours at room temperature.
6. Aspirate (or decant) the content of each tube (except total counts). Be
sure that the plastic tip of the aspirator reaches the bottom of the coated
tube in order to remove all the liquid.
7. Wash the tubes with 2 ml Wash Solution (except total counts). Avoid
foaming during the addition of the Wash Solution.
8. Aspirate (or decant) the content of each tube (except total counts).
9. After the washing let the tubes standing upright for 2 minutes and
aspirate the remaining drop of liquid.
10. Count the tubes in a gamma counter for 60 seconds.
8. Prolactin Coated Tubes Flow chart
