sandwiched between the solid phase and enzyme- 4. Conduct the assay away from bad ambient conditions.

t conditions. E.g.
linked antibodies. ambient air containing high concentration corrosive gas
 After washing, a complex is generated between the such as sodium hypochlorite acid, alkaline, acetaldehyde
solid phase, the prolactin within the sample and and so on, or containing dust.
enzyme-linked antibodies by immunological reactions. 5. Wash the wells completely. Each well must be fully
PROLACTIN ELISA  Substrate solution is then added and catalyzed by this injected with wash solution. The strength of injection,
Enzyme Immunoassay for the Quantitative complex, resulting in a chromogenic reaction. The however, is not supposed to be too intense to avoid
Determination of Prolactin Concentration resulting chromogenic reaction is measured as overflow. In each wash cycle, dry the liquids in each well.
in Human Serum absorbance. Strike the microplate onto absorbent paper to remove
For In-Vitro diagnostic and professional use only  The absorbance is proportional to the amount of PRL in residual water droplets. It is recommended to wash the
the sample. microplate with an automated microplate strip washer.
Store at 2-8 °C 6. Failure to remove adhering solution adequately in the
Materials aspiration or decantation wash step(s) may result in poor
96 Tests Materials provided replication and spurious results.
1. Coated Microplate, 8 x 12 strips, 96 wells, pre-coated with 7. Do not use reagents beyond the labeled expiry date.
Intended use
mouse monoclonal Anti-Prolactin. 8. Do not mix or use components from kits with different
Immunoassay for the in vitro quantitative determination of
2. Calibrators, 6 white cap vials, 1 ml each, ready to use; batch codes.
prolactin in human serum.
Concentrations: 0(A), 5(B), 10(C), 20(D), 50(E) and 100(F) 9. If more than one plate is used, it is recommended to
ng/mL. repeat the calibration curve.
Introduction
3. Enzyme Conjugate, 1 red cap vial, 11 mL of HRP 10. It is important that the time of reaction in each well is
Prolactin is synthesized in the anterior pituitary and is
(horseradish peroxidase) labeled mouse monoclonal Anti- held constant to achieve reproducible results.
secreted in episodes. The hormone is made up of 198 amino
prolactin in Tris-NaCl buffer containing BSA (bovine serum 11. Ensure that the bottom of the plate is clean and dry.
acids and has a molecular weight of approx. 22‐23 kD.
albumin). Contains 0.1% ProClin300® preservative. 12. Ensure that no bubbles are present on the surface of the
Prolactin appears in serum in three different forms. The
4. Substrate, 1 brown cap vial, 11ml, ready to use, liquid before reading the plate.
biologically and immunologically active monomeric (“little”)
(tetramethylbenzidine) TMB. 13. The substrate and stop solution should be added in the
form predominates (approx. 80 %), 5‐20 % is present as the
5. Stop Solution, 1 yellow cap vial, 6.0 ml of 1 mol/L sulfuric same sequence to eliminate any time deviation during
biologically inactive dimeric (“big”) form and 0.5‐5 % is
acid. reaction.
present as the tetrameric (“big‐big”) form having low
6. Wash Solution Concentrate, 1 transparent cap bottle, 25
biological activity. The target organ for prolactin is the
ml (40X concentrated), PBS-Tween wash solution. Storage
mammary gland, the development and differentiation of
7. IFU, 1 copy. 1. Store at 2-8°C.
which is promoted by this hormone.
8. Plate Lid: 1 piece. 2. Place unused wells in the zip-lock aluminum foiled pouch
High concentrations of prolactin have an inhibiting action on
and return to 2-8 °C, under which conditions the wells
steroidogenesis of the ovaries and on hypophyseal
Materials required (but not provided) will remain stable for 2 months, or until the labeled
gonadotropin production and secretion. During pregnancy the
1. Microplate reader with 450nm and 620nm wavelength expiry date, whichever is earlier.
concentration of prolactin rises under the influence of
absorbent capability. 3. Seal and return unused calibrators to 2-8 °C, under which
elevated estrogen and progesterone production. The
2. Microplate washer. conditions the stability will be retained for 1 month, for
stimulating action of prolactin on the mammary gland leads
3. Incubator. longer use, store opened calibrators in aliquots and
post partum to lactation.
4. Plate shaker. freeze at -20 °C. Avoid multiple freeze-thaw cycles.
Hyperprolactinemia (in men and women) is the main cause of
5. Micropipettes and multichannel micropipettes delivering 4. Seal and return all the other unused reagents to 2-8 °C,
fertility disorders. The determination of prolactin is utilized in
50μl with a precision of better than 1.5%. under which conditions the stability will be retained for 2
the diagnosis of anovular cycles, hyperprolactinemic
6. Absorbent paper. months, or until the labeled expiry date, whichever is
amenorrhea and galactorrhea, gynecomastia and azoo-
7. Distilled water earlier.
spermia. Prolactin is also determined when breast cancer and
pituitary tumors are suspected.
Warnings and Precautions Specimen collection and preparation
1. For in vitro diagnostic use only. For professional use only. 1. Collect serum samples in accordance with correct medical
Test Principle
2. All products that contain human serum or plasma have practices.
Sandwich principle. Total duration of assay: 80 minutes.
been found to be non-reactive for HBsAg, HCV and HIVI/II. 2. Cap and store the samples at 18-25 °C for no more than 8
 Sample, Anti-Prolactin coated microwells and enzyme
But all products should be reared as potential biohazards hours. Stable for 7 days at 2-8 °C, and 1 month at -20 °C.
labeled Anti-Prolactin are combined.
in use and for disposal. Recovery within 90-110 % of serum value or slope 0.9-1.1.
 During the incubation, prolactin presents in the sample
3. Mix the sample in the wells thoroughly by shaking and Freeze only once.
is allowed to react simultaneously with the two
eliminate the bubbles. 3. The sample types listed were tested with a selection of
antibodies, resulting in the prolactin molecules being
 sample collection tubes that were commercially available valid if the control values fall within the concentration ranges Cal D 20 1.153
at the time of testing, i.e. not all available tubes of all printed on the labels.
Cal E 50 2.107
manufacturers were tested. Sample collection systems
Cal F 100 3.158
from various manufacturers may contain differing Procedure
materials which could affect the test results in some cases. 1. Use only the number of wells required and format the Control1 5.84 0.23
When processing samples in primary tubes (sample microplates’ wells for each calibrator and sample to be Control2 28.71 1.11
collection systems), follow the instructions of the tube assayed.
Sample 48.96 1.76
manufacturer. Centrifuge samples containing precipitates 2. Add 25 μL of calibrators or samples to each well.
before performing the assay. Do not use heatin activated 3. Add 100 μL of enzyme conjugate to each well. Limitations - interference
samples. Do not use samples and controls stabilized with 4. Shake the microplate gently for 30 seconds to mix. 1. The assay is unaffected by icterus (bilirubin < 513 μmol/L
azide. 5. Cover the plate with a plate lid and incubate at 37 °C for or < 30 mg/dL), hemolysis (Hb < 0.932 mmol/L or < 1.5
4. Ensure the patients’ samples, calibrators, and controls are 60 minutes. g/dL), lipemia (Intralipid < 1500 mg/dL) and biotin (< 164
at ambient temperature (18-25 °C) before measurement. 6. Discard the contents of the micro plate by decantation or nmol/L or < 40 ng/mL).
5. Sediments and suspended solids in samples may interfere aspiration. If decanting, tap and blot the plate dry with 2. Criterion: Recovery within ± 15 % of initial value.
with the test result which should be removed by absorbent paper. 3. Samples should not be taken from patients receiving
centrifugation. Ensure that complete clot formation in 7. Add 350 μL of wash solution, decant (tap and blot) or therapy with high biotin doses (i.e. > 5 mg/day) until at
serum samples has taken place prior to centrifugation. aspirate. Repeat 4 additional times for a total of 5 washes. least 8 hours following the last biotin administration.
Some samples, especially those from patients receiving An automated microplate strip washer can be used. At the 4. No interference was observed from rheumatoid factors up
anticoagulant or thrombolytic therapy, may exhibit end of washing, invert the plate and tap out any residual to a concentration of approx. 1100 IU/mL.
increased clotting time. If the sample is centrifuged before wash solution onto absorbent paper. 5. There is no high-dose hook effect at prolactin
a complete clot forms, the presence of fibrin may cause 8. Add 100 μL of substrate to each well. concentrations up to 220000 μIU/mL (10000 ng/mL).
erroneous results. Be sure that the samples are not 9. Incubate at ambient temperature (18-25℃）in the dark 6. In vitro tests were performed on 16 commonly used
decayed prior to use. for reaction for 20 minutes. Do not shake the plate after pharmaceuticals. No interference with the assay was
6. Avoid grossly hemolytic, lipemic or turbid samples. substrate addition. found. In rare cases, interference due to extremely high
7. Note that interfering levels of fibrin may be present in 10. Add 50 μL of stop solution to each well. titers of antibodies to analyte‐specific antibodies,
samples that do not have obvious or visible particulate 11. Shake for 15-20 seconds to mix the liquid within the wells. streptavidin or ruthenium can occur. These effects are
matter. It is important to ensure that the blue color changes to minimized by suitable test design.
8. If proper sample collection and preparation cannot be yellow completely. 7. When determining prolactin it should be remembered
verified, or if samples have been disrupted due to 12. Read the absorbance of each well at 450 nm (using 620 to that the measured concentration is dependent upon
transportation or sample handling, an additional 630 nm as the reference wavelength to minimize well when the blood sample was taken, since the secretion of
centrifugation step is recommended. Centrifugation imperfections) in a micro plate reader. The results should prolactin occurs in episodes and is also subject to a
conditions should be sufficient to remove particulate be read within 30 minutes of adding the stop solution. 24‐hour cycle.
matter. 8. The release of prolactin is promoted physiologically by
9. Ensure the patients’ samples, calibrators, and controls are Calculation suckling and stress. In addition, elevated serum prolactin
at ambient temperature (18-25 °C) before measurement. 1. Record the absorbance obtained from the printout of the concentrations are caused by a number of
Mix all reagents through gently inverting prior to use. microplate reader. pharmaceuticals (e.g. dibenzodiazepines, phenothiazine),
10. Adjust the incubator to 37 °C. 2. Calculate the mean absorbance of any duplicate TRH and estrogen.
11. Prepare wash solution concentrate before measurement. measurements and use the mean for the following 9. The release of prolactin is inhibited by dopamine, L‐dopa
Stable for 2 months at ambient temperature. calculation. and ergotamine derivatives.
12. Don’t use Substrate if it looks blue. 3. Plot the common logarithm of absorbance against 10. A number of publications report the presence of
13. Don’t use reagents that are contaminated or have bacteria concentration in ng/mL for each calibrator. macroprolactin in the serum of female patients with
growth. 4. Draw the best-fit curve through the plotted points on various endocrinological diseases or during pregnancy.
linear graph paper. Point-to-Point method is suggested to Differing degrees of detection of the serum
Quality control generate a calibration curve. macroprolactins relative to monomeric prolactin (22‐23
Each laboratory should assay controls at levels in the low, The following data is for demonstration only and cannot be kD) by various immunoassays have also been described.
normal, and elevated range for monitoring assay used in place of data generations at the time of assay. This could make the detection of hyperprolactinemia
performance. There controls should be treated as unknowns Sample Value (ng/mL) Absorbance dependent on the immunoassay used.
and values determined in every test procedure performed. 11. In case of implausible high prolactin values a precipitation
Cal A 0 0.015 by polyethylene glycol (PEG) is recommended in order to
The recommended controls requirement for this assay are to
purchase trueness control materials separately and test them Cal B 5 0.183 estimate the amount of the biological active monomeric
together with the samples within the same run. The result is prolactin.
Cal C 10 0.492
12. For diagnostic purposes, the results should always be Precision ATLAS Medical
assessed in conjunction with the patient’s medical history, Precision was determined using ATLAS reagents, pooled Ludwig-Erhard Ring 3
clinical examination and other findings. human sera, and controls in a modified protocol (EP5-A) of 15827 Blankenfelde-Mahlow
the CLSI (Clinical and Laboratory Standards Institute): 2 times Germany
Limits and ranges daily for 20 days (n = 40). The following results were obtained: Tel: +49 - 33708 – 3550 30
Measuring range Intermediate Email: [email protected]
Repeatability*
1.00‐2128 μIU/mL or 0.0470‐100 ng/mL (defined by the lower precision
detection limit and the maximum of the master curve). Values Mean SD SD PPI1434A01
Sample CV % CV % Rev A (02.09.2019)
below the lower detection limit are reported as < 1 μIU/mL or ng/mL ng/mL ng/mL
< 0.0470 ng/mL. Values above the measuring range are Human Temperature
4.73 0.409 8.64 0.411 8.68 Catalogue Number
reported as > 2128 μIU/mL or > 100 ng/mL. Serum 1 limit
Human In Vitro diagnostic
16.84 1.206 7.16 1.295 7.69 Caution
Lower limits of measurement Serum 2 medical device
Lower detection limit Human Contains sufficient Consult
42.74 2.167 5.07 2.334 5.46 for <n> tests and instructions for
Lower detection limit: 1.00 μIU/mL (0.047 ng/mL). Serum 3
The lower detection limit represents the lowest analyte level PC Relative size use (IFU)
that can be distinguished from zero. It is calculated as the Universal 8.76 0.617 7.04 0.686 7.83 Batch code Manufacturer
value lying two standard deviations above that of the lowest 1
standard (master calibrator, standard 1 + 2 SD, repeatability PC Fragile,
Use-by date
study, n = 20). Universal 17.86 1.047 5.86 0.943 5.28 handle with care
2 Do not use if
Dilution Manufacturer fax
*Repeatability = within-run precision package is
Samples with prolactin concentrations above the measuring number
damaged
range can be diluted with Diluent Universal. The Method comparison Manufacturer Date of
recommended dilution is 1:10. The concentration of the A comparison of the ATLAS Prolactin assay (y) with the Roche telephone number Manufacture
diluted sample must be > 50 μIU/mL or > 2.4 ng/mL. Cobas Prolatctin II (x) using clinical samples gave the following
correlations: Number of samples measured: 121 Keep away from
Expected values Keep dry
sunlight
Men: 2.3 - 17.5 ng/mL Linear regression
Women: 2.9 - 25.8 ng/mL y = 1.0421x + 0.048
A study with the ATLAS Prolactin II assay was performed using r = 0.9863
samples from 300 apparently healthy blood donors. The The sample concentrations were between approx. 0 and 121
following results were obtained. ng/mL.
Each laboratory should investigate the transferability of the Analytical specificity
expected values to its own patient population and if The monoclonal antibodies used are highly specific against
necessary determine its own reference ranges. prolactin. No cross reaction with hGH, hCG, hPL, TSH, FSH and
Percentiles LH has been observed.
th 2.5- th 2.5-
50 th 50 th
97.5 97.5 References
N μIU/mL ng/mL 1. Smith CR, Norman MR. Prolactin and growth hormone:
Men 404 189 74-350 8.91 3.5-16.5 molecular heterogeneity and measurement in serum. Ann
Women(not- Clin Biochem 1990;27:542-550.
1169 268 84-605 12.64 4-28.5
pregnant) 2. 2 Runnebaum B, Rabe T. Gynäkologische Endokrinologie
und Fortpflanzungsmedizin Springer Verlag 1994. Band
Specific performance data 1:21,124-126,179-181,613, Band 2:412-417,436. ISBN 3-
Representative performance data are given below. Results 540-57345-3, ISBN 3-540-57347-X.
obtained in individual laboratories may differ.