1. Summary and background of the test:
Human Placental Lactogen (hPL) is a dimer of two polypeptide chains of
equivalent weight (19.000) with lactogenic, luteotropic and growth activities.
HPL, which is produced by trophoblastic cells of the normal placenta or by
trophoblastic tumor tissue, has an amino acid composition quite similar to that
of hGH, and to a lesser extent to that of prolactin. HPL becomes detectable in
serum from about 6th week of pregnancy : later on hPL levels in
serum increase progressively throughout pregnancy to reach a plateau of 2-10
µg/ml by the 34th week reflecting directly the growth of the
placental tissue. Because of its short plasma half life (±
20 minutes), hPL becomes undetectable in the serum 4 hours after delivery.
2 Principle of the test :
The Bio-Line hPL-Irma is a competition between a labelled and an unlabelled
antigen for a limited number of binding sites on a specific antibody.In the
present kit, a tracer amount of 125I-hPL is incubated in tubes coated
with anti-hPL monoclonal antibodies ; the incubation medium also known
amounts of unlabelled hPL (refernce curve) or a given volume of the unknown
samples. After one hour incubation (at room tamperature), the content of each
tube is aspirated, the tubes are washed with 2 ml of washing silution and
aspirated again. A standard curve is constructed and the hPL concentrations of
the samples are determined by dose interpolation from this curve.
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. hPL human serum with sodium merthiolate based standards & control:
8 lyophilised vials to reconstitute with each 0.5
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 µl, 100 µl, 500 µl and 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 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.
Avoid successive freezing and thawing.
Serum, heparinized or EDTA plasma provide similar results.
Y (serum) = 1.07x (hep. plasma) + 0.11 r = 0.99 n = 22
Y (serum) = 0.96x (EDTA plasma) + 0.03 r = 0.99 n = 22
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 100 µl of tracer into each tube.
4. Shake the rack containing the tubes gently by hand.
5. Incubate for 1 hour at room temperature (alternative incubations :
15 minutes or 2 hours at RT).
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. Count the tubes in a gamma counter for 60 seconds.
8. hPL Coated Tubes Flow chart
