the well and exogenously added biotinylated monoclonal anti-PRL 8. Timer. 1.

8. Timer. 1. Format the microplate wells for each serum reference

antibody. 9. Quality control materials calibrator, control and patient specimen to be assayed in
duplicate. Replace any unused microwell strips back into
Upon mixing monoclonal biotinylated antibody, the enzyme- 5.0 PRECAUTIONS the aluminum bag, seal and store at 2-8°C
labeled antibody and a serum containing the native antigen, 2. Pipette 0.025 ml (25µl) of the appropriate serum reference
reaction results between the native antigen and the antibodies, For In Vitro Diagnostic Use calibrator, control or specimen into the assigned well.
without competition or steric hindrance, to form a soluble Not for Internal or External Use in Humans or Animals 3. Add 0.100 ml (100µl) of PRL Tracer Reagent to all wells.
sandwich complex. The interaction is illustrated by the following 4. Swirl the plate gently for 20-30 seconds to mix and cover.
equation: All products that contain human serum have been found to be 5. Incubate 45 minutes at room temperature.
ka non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV 6. Discard the contents of the microplate by decantation or
Enz
Antibodies by FDA licensed reagents. Since no known test can aspiration. If decanting, blot the plate dry with absorbent
Ab(p) + Ag PRL + BtnAb( m) Enz
Ab(p) - Ag PRL - BtnAb( m) offer complete assurance that infectious agents are absent, all paper.
k-a human serum products should be handled as potentially 7. Add 0.350ml (350µl) of wash buffer (see Reagent Preparation
Btn
Ab( m) = Biotinylated Monoclonal Antibody (Excess Quantity) hazardous and capable of transmitting disease. Good laboratory Section), decant (tap and blot) or aspirate. Repeat four (4)
Ag PRL = Native Antigen (Variable Quantity) procedures for handling blood products can be found in the additional times for a total of five (5) washes. An automatic or
Enz
Ab(p) = Enzyme labeled Antibody (Excess Quantity) Center for Disease Control / National Institute of Health, manual plate washer can be used. Follow the
Enz
Ab(p) - Ag PRL - Btn Ab( m) = Antigen-Antibodies Sandwich Complex "Biosafety in Microbiological and Biomedical Laboratories," 2nd manufacturer’s instruction for proper usage. If a squeeze
ka = Rate Constant of Association Edition, 1988, HHS Publication No. (CDC) 88-8395. bottle is employed, fill each well by depressing the
k-a = Rate Constant of Dissociation container (avoiding air bubbles) to dispense the wash.
Safe Disposal of kit components must be according to local Decant the wash and repeat four (4) additional times.
Simultaneously, the complex is deposited to the well through the regulatory and statutory requirements.
high affinity reaction of streptavidin and biotinylated antibody. This 8. Add 0.100 ml (100µl) of working signal reagent to all wells (see
interaction is illustrated below: Reagent Preparation Section). Always add reagents in the
6.0 SPECIMEN COLLECTION AND PREPARATION same order to minimize reaction time differences between
Prolactin Hormone (PRL) Test System
Enz
Ab( m) - Ag PRL - BtnAb( m) + Streptavidin C.W. ⇒ immobilized complex
wells
Streptavidin C.W. = Streptavidin immobilized on well The specimens shall be blood, serum in type and the usual
Product Code: 775-300 DO NOT SHAKE THE PLATE AFTER SIGNAL ADDITION
Immobilized complex = sandwich complex bound to the well precautions in the collection of venipuncture samples should be 9. Incubate at room temperature in the dark for five (5) min.
observed. For accurate comparison to established normal values, 10. Read the relative light units in each well, for minimum 0.5 – 1.0
1.0 INTRODUCTION After a suitable time, the antibody-bound fraction is separated a fasting morning serum sample should be obtained. The blood
from unbound antigen by decantation or aspiration. The enzyme seconds, using a microplate luminometer. The results should
should be collected in a plain redtop venipuncture tube without be read within thirty (30) minutes of adding the signal
Intended Use: The Quantitative Determination of Prolactin activity, determined by reaction with a substrate that generates additives or anti-coagulants. Allow the blood to clot. Centrifuge the
Hormone Concentration in Human Serum by a Microplate light, in the antibody-bound fraction is directly proportional to the solution.
specimen to separate the serum from the cells.
Enzyme Immunoassay, Chemiluminescense native antigen concentration. By utilizing several different serum
references of known antigen values, a dose response curve can 10.0 CALCULATION OF RESULTS
In patients receiving therapy with high biotin doses (i.e.
2.0 SUMMARY AND EXPLANATION OF THE TEST be generated from which the antigen concentration of an unknown >5mg/day), no sample should be taken until at least 8 hours
can be ascertained. A dose response curve is used to ascertain the concentration of
after the last biotin administration, preferably overnight to
Prolactin hormone (PRL), secreted from the lactotrophs of the ensure fasting sample. prolactin (PRL) concentration in unknown specimens.
anterior pituitary, is a protein consisting of a single polypeptide 4.0 REAGENTS 1. Record the RLUs (Relative Light Unit) obtained from the
chain containing approximately 200 amino acids. The primary Samples may be refrigerated at 2-8°C for a maximum period of printout of the microplate luminometer as outlined in Example
biological action of the hormone is on the mammary gland where Materials Provided: five (5) days. If the specimen(s) cannot be assayed within this 1.
it is involved in the growth of the gland and in the induction and A. PRL Calibrators – 1 ml/vial – Icons A-F time, the sample(s) may be stored at temperatures of -20°C for up 2. Plot the RLUs for each duplicate serum referenceversus the
maintenance of milk production. There is evidence to suggest that Six (6) vials of references for PRL antigen in serum at levels of to 30 days. Avoid use of contaminated devices. Avoid repetitive corresponding PRL concentration in ng/ml on linear graph
prolactin may be involved in steroidogenesis in the gonad, acting 0(A), 5(B), 10(C), 25(D), 50(E) and 100(F) ng/ml. Store at freezing and thawing. When assayed in duplicate, 0.100ml (100µl) paper.
synergistically with luteinizing hormone (LH). High levels of 2-8°C. A preservative has been added. of the specimen is required. 3. Draw the best-fit curve through the plotted points.
prolactin appear to inhibit steroidogenesis as well as inhibiting LH Note: The calibrators, human serum based, were calibrated 4. To determine the concentration of PRL for an unknown, locate
and follicle stimulating hormone (FSH) synthesis at the pituitary using a reference preparation, which was assayed against the 7.0 QUALITY CONTROL the average RLUs of the unknown on the vertical axis of the
gland.1,2 WHO 3rd, IS (84/500). graph, find the intersecting point on the curve, and read the
Each laboratory should assay controls at levels in the low, concentration (in ng/ml) from the horizontal axis of the graph
The clinical usefulness of the measurement of prolactin hormone B. PRL Tracer Reagent – 13 ml/vial – Icon E medium and high range for monitoring assay performance. These (the duplicates of the unknown may be averaged as indicated).
(PRL) in ascertaining the diagnosis of hyperprolactinemia and for One (1) vial containing enzyme labeled atibody, biotinylated controls should be treated as unknowns and values determined in In the following example, the average RLUs (33555) of the
the subsequent monitoring the effectiveness of the treatment has monoclonal mouse IgG in buffer, dye, and preservative. Store every test procedure performed. Quality control charts should be unknown intersects the calibration curve at (13.9ngml) PRL
been well established.3,4 at 2-8°C. maintained to follow the performance of the supplied reagents. concentration (See Figure 1).
C. Light Reaction Wells – 96 wells – Icon ⇓ Pertinent statistical methods should be employed to ascertain
In this method, PRL calibrator, patient specimen or control is first One 96-well white microplate coated with streptavidin and trends. Significant deviation from established performance can Note: Computer data reduction software designed for
added to a streptavidin coated well. Biotinylated monoclonal and packaged in an aluminum bag with a drying agent. Store at indicate unnoticed change in experimental conditions or chemiluminescence assays may also be used for the data
enzyme labeled antibodies (directed against distinct and different 2-8°C. degradation of kit reagents. Fresh reagents should be used to reduction. If such software is utilized, the validation of the
epitopes of PRL) are added and the reactants mixed. Reaction software should be ascertained.
D. Wash Solution Concentrate – 20 ml/vial – Icon determine the reason for the variations.
between the various PRL antibodies and native PRL forms a
sandwich complex that binds with the streptavidin coated to the One (1) vial containing a surfactant in buffered saline. A EXAMPLE 1
preservative has been added. Store at 2-8°C. 8.0 REAGENT PREPARATION
well. Sample Well Mean Value
E. Signal Reagent A – 7ml/vial – Icon CA RLU (A)
1. Wash Buffer I.D. Number RLU (B) (ng/ml)
After the completion of the required incubation period, the One (1) vial containing luminol in buffer. Store at 2-8°C.
Dilute contents of Wash Concentrate to 1000ml with distilled or
enzyme-prolactin hormone antibody bound conjugate is separated F. Signal Reagent B – 7ml/vial – Icon C B A1 113
deionized water in a suitable storage container. Store diluted Cal A 139 0
from the unbound enzyme-prolactin hormone conjugate by One (1) vial containing hydrogen peroxide (H2 O2) in buffer. B1 164
buffer at 2-30°C for up to 60 days.
aspiration or decantation. The activity of the enzyme present on Store at 2-8°C. C1 12028
G. Product Instructions 2. Working Signal Reagent Solution - Store at 2 - 30°C. Cal B 12485 5
the surface of the well is quantitated by reaction with a suitable
Determine the amount of reagent needed and prepare by D1 12941
substrate to produce light.
Note 1: Do not use reagents beyond the kit expiration date. mixing equal portions of Signal Reagent A and Signal Reagent E1 24693
B in a clean container. For example, add 1 ml of A and 1ml of Cal C 24603 10
The employment of several serum references of known prolactin Note 2: Avoid extended exposure to heat and light. Opened F1 24513
hormone levels permits the construction of a dose response curve reagents are stable for sixty (60) days when stored at B per two (2) eight well strips (A slight excess of solution is G1 53221
made). Discard the unused portion if not used within 36 Cal D 53344 25
of activity and concentration. From comparison to the dose 2-8°C. Kit and component stability are identified on the H1 53468
response curve, an unknown specimen's activity can be correlated label. hours after mixing. If complete utilization of the reagents is
A2 76850
with prolactin hormone concentration. Note 3: Above reagents are for a single 96-well microplate anticipated, within the above time constraint, pour the contents Cal E 77335 50
of Signal Reagent B into Signal Reagent A and label B2 77820
4.1 Required But Not Provided: accordingly. C2 98568
3.0 PRINCIPLE Cal F 100000 100
1. Pipette capable of delivering 0.025ml (25µl) and 0.050ml D2 101432
(50µl) volumes with a precision of better than 1.5%. Note: Do not use reagents that are contaminated or have E2 14555
Immunoenzymometric assay (TYPE 3): Ctrl 1 14690 5.9
2. Dispenser(s) for repetitive deliveries of 0.100ml (100µl) and bacteria growth. F2 14825
The essential reagents required for an immunoenzymometric
assay include high affinity and specificity antibodies (enzyme 0.350ml (350µl) volumes with a precision of better than 1.5%. G2 42680
3. Microplate washers or a squeeze bottle (optional). 9.0 TEST PROCEDURE Ctrl 2 42186 18.3
labelled and immobilized), with different and distinct epitope H2 41692
recognition, in excess, and native antigen. In this procedure, the 4. Microplate luminometer. A3 32955
5. Absorbent Paper for blotting the microplate wells. Before proceeding with the assay, bring all reagents, serum Patient 33555 13.9
immobilization takes place during the assay at the surface of a B3 34155
6. Plastic wrap or microplate cover for incubation steps. reference calibrators and controls to room temperature (20-27°C).
microplate well through the interaction of streptavidin coated on
7. Vacuum aspirator (optional) for wash steps. **Test Procedure should be performed by a skilled individual
or trained professional**
* The data presented in Example 1 and Figure 1 is for illustration 3. The reagents for the test system have been formulated to (95% certainty) statistic to calculate the minimum dose. It was 16. Cavaco, B., Prazeres, S., Santos, M.A., Sobrinho, L.G. and
only and should not be used in lieu of a dose response curve eliminate maximal interference; however, potential interaction determined to be 0.11 ng/ml. Leite, V., ’Hyperprolactinemia due to big big prolactin is
prepared with each assay. In addition, the RLUs of the calibrators between rare serum specimens and test reagents can cause differentlyt detected by commercially available immunoassays’,
have been normalized to 100,000 RLUs for the F calibrator erroneous results. Heterophilic antibodies often cause these 14.3 Accuracy J Endocrino Invest , 22, 203-208 (1999).
(greatest light output). This conversion minimizes differences interactions and have been known to be problems for all kinds The PRL AccuLite® CLIA test system was compared with a
caused by efficiency of the various instruments that can be used of immunoassays (Boscato LM, Stuart MC. ‘Heterophilic reference Elisa method. Biological specimens from normal and Revision: 5 Date: 2019-Jul-16 DCO: 1353
to measure light output. antibodies: a problem for all immunoassays’ Clin. Chem. abnormal populations were assayed. The total number of such MP775 Product Code: 775-300
1988:3427-33). For diagnostic purposes, the results from this specimens was 85. The least square regression equation and the
assay should be in combination with clinical examination, correlation coefficient were computed for the PRL AccuLite® CLIA Size 96(A) 192(B)
Figure 1
patient history and all other clinical findings. test system comparison with the reference method. The data
120000 4. For valid test results, adequate controls and other parameters obtained is displayed in Table 4. A) 1ml set 1ml set
100000 must be within the listed ranges and assay requirements.
B) 1 (13ml) 2 (13ml)

Reagent (fill)
5. If test kits are altered, such as by mixing parts of different kits, TABLE 4
80000 which could produce false test results, or if results are Mean Least Square Correlation C) 1 plate 2 plates
Method
incorrectly interpreted, Monobind shall have no liability. (x) Regression Analysis Coefficient
RLU

60000
6. If computer controlled data reduction is used to interpret the Monobind 18.5 y = -1.63+1.01(x) 0.978 D) 1 (20ml) 1 (20ml)
40000 results of the test, it is imperative that the predicted values for Reference 19.4
E) 1 (7ml) 2 (7ml)
20000 Patient the calibrators fall within 10% of the assigned concentrations.
7. Patients receiving preparations of mouse monoclonal Only slight amounts of bias between this procedure and the F) 1 (7ml) 2 (7ml)
0 antibodies for diagnosis or therapy may contain human anti- reference method are indicated by the closeness of the mean
0 20 40 60 80 100 120
mouse antibodies (HAMA) and may show either falsely values. The least square regression equation and correlation
elevated or depressed values when assayed. coefficient indicates excellent method agreement.
PRL Values in ng/ml
8. Pregnancy, lactation, and the administration of oral
contraceptives can cause an increase in the level of prolactin. 14.4 Specificity
11.0 Q.C. PARAMETERS The cross-reactivity PRL AccuLite® CLIA test system to selected
9. Drugs such as morphine, reserpine and the psychotropic drugs
substances was evaluated by adding the interfering substance to
In order for the assay results to be considered valid the increase prolactin secretion.5,6,7
a serum matrix at various concentrations. The cross-reactivity was
following criteria should be met: 10. Since prolactin hormone concentration is dependent upon
calculated by deriving a ratio between dose of interfering
1. The Dose Response Curve should be within established diverse factors other than pituitary homeostasis, the
substance to dose of prolactin hormone needed to produce the
parameters. determination alone is not sufficient to assess clinical status.
same light intensity.
2. Four out of six quality control pools should be within the
established ranges. 13.0 EXPECTED RANGES OF VALUES
Substance Cross Concentration
A study of an apparent normal adult population was undertaken to Reactivity
12.0 RISK ANALYSIS Prolactin Hormone (PRL) 1.0000 --
determine expected values for the PRL AccuLite® CLIA test
system. The expected values (95% confidence intervals) are Luteinizing Hormone (LH) < 0.0001 1000ng/ml
The MSDS and Risk Analysis Form for this product is available on Follitropin (FSH) < 0.0001 1000ng/ml
request from Monobind Inc. presented in Table 1.
TABLE I Chorionic gonadotropin (CG) < 0.0001 1000ng/ml
Expected Values for the PRL AccuLite® CLIA (in ng/ml) Thyrotropin (TSH) < 0.0001 1000ng/ml
12.1 Assay Performance Growth Hormone (GH) < 0.0001 1000ng/ml
1. It is important that the time of reaction in each well is held Women
constant to achieve reproducible results. Adult (Number = 70) 1.2-19.5
Postmenopausal (Number = 10) 1.5-18.5 15.0 REFERENCES
2. Pipetting of samples should not extend beyond ten (10)
minutes to avoid assay drift. Men
1. Maddox, P.R., Jones, D.L., Mansel, R.E., Acta Endocrinol,
3. Highly lipemic, hemolyzed or grossly contaminated Adult (Number = 50) 1.8-17.0
125, 621 (1991).
specimen(s) should not be used. 2. Gonzales, E.R., JAMA 242, 401 (1979).
4. If more than one (1) plate is used, it is recommended to repeat It is important to keep in mind that establishment of a range of
values which can be expected to be found by a given method for a 3. Tolis, G., Hosp. Pract. 15, 85 (1980).
the dose response curve. 4. Balagura, S., Frantz, A.G., Houseplan, E.M., J Neurosurg 51,
5. The addition of signal reagent initiates a kinetic reaction, population of "normal"-persons is dependent upon a multiplicity of
factors: the specificity of the method, the population tested and 42 (1979).
therefore the signal reagent(s) should be added in the same 5. Friesen, H., Hwang, P., Ann Rev Med, 24, 251 (1973).
sequence to eliminate any time-deviation during reaction. the precision of the method in the hands of the analyst. For these
reasons each laboratory should depend upon the range of 6. Frantz, A. G., N Eng J Med, 298, 201 (1978).
6. Failure to remove adhering solution adequately in the 7. Parkes, D.N., J. Med. 301, 873. (1979)
aspiration or decantation wash step(s) may result in poor expected values established by the Manufacturer only until an
8. Kao, P.C., Jiang, N.C. and Abboud, C.F., ‘Radioimmunoassay
replication and spurious results. in-house range can be determined by the analysts using the
method with a population indigenous to the area in which the of human homologous prolactin in serum with commercially
7. Use components from the same lot. No intermixing of reagents available reagents’ Clin Chem. 23, 1563-1568. (1977)
from different batches. laboratory is located.
9. Haus, E., Lakatua, D.J., Halberg, F., Halberg, E., Cornelissen,
8. Patient specimens with abnormally high prolactin levels can G., Sackett, L.L et.al: ’Chronobiological studies of plasma
cause a hook effect, that is, paradoxical low results. If this is 14.0 PERFORMANCE CHARACTERISTICS
prolactin in women in Kyushu, Japan and Minnesota, USA.’
suspected, dilute the specimen 1/100 with ‘0’ calibrator; J.Clin.Endocrinol Metab, 51, 632-640. (1980)
reassay (multiply the result by 100). However, values as high 14.1 Precision
The within and between assay precision of the PRL AccuLite® 10. Christensen, J.M., Poulsen, O.M. and Anglov, T, ”Method,
as 3000ng/ml have been found to absorb greater than the evaluation, quality control, and extreme quality assurance
value of the highest calibrator CLIA test system were determined by analyses on three different
levels of control sera. The number, mean value, standard systems of analytical procedures”; in: Seiler HG, Seigel, H
9. Accurate and precise pipetting, as well as following the exact Eds. Handbook on Metals in Clinical and Analytical Chemistry.
time and temperature requirements prescribed are essential. deviation (σ) and coefficient of variation for each of these control
New York: Marcel Dekker 45-61 (1994).
Any deviation from Monobind’s IFU may yield inaccurate sera are presented in Table 2 and Table 3.
11. Phillips, G.B: ’Relationship between serum levels of
results. dehydroepiandrosterone sulfate, androstenedione and sex
10. All applicable national standards, regulations and laws, TABLE 2
Within Assay Precision (Values in ng/ml) hormone in men and women.’ Eur.J.Endocrinol 134, 201-206.
including, but not limited to, good laboratory procedures, must (1996)
be strictly followed to ensure compliance and proper device Sample N X σ C.V.
12. Touitou Y., Carayon, A., Reinberg, A., Bogdan, A., Beck, H.,
usage. Level 1 20 5.4 0.23 4.3%
’Differences in seasonal rhythmicity of plasma prolactin in
11. It is important to calibrate all the equipment e.g. Pipettes, Level 2 20 18.4 0.67 3.6%
elderly human subjects; Detection in women but not in men’, J
Readers, Washers and/or the automated instruments used Level 3 20 40.8 2.78 6.8%
Endocrinol, 96, 65-71 (1983).
with this device, and to perform routine preventative 13. Costonga, J., Janson PCW, Hermans, J., Van Wersch, JWJ
maintenance. TABLE 3
and Bombacher PJ.: ’Short term and long term intra-individual
12. Risk Analysis- as required by CE Mark IVD Directive 98/79/EC Between Assay Precision* (Values in ng/ml)
variations and critical differences of clinical laboratory
- for this and other devices, made by Monobind, can be Sample N X σ C.V.
parameters’, J.Clin.Chem.& Clin.Biochem, 985, 23, 7-16.
requested via email from Monobind@monobind.com. Level 1 20 5.8 0.57 9.8% 14. John R., McDowell, IFW, Scanton. MF and Ellis, AR.:
Level 2 20 19.8 1.73 8.8% ’Macroprolactin reactivities in prolactin assays: an issue for
12.2 Interpretation Level 3 20 43.8 2.97 6.8% clinical laboratories and equipment manufacturers”, Clin
1. Measurements and interpretation of results must be *As measured in ten experiments in duplicate.
performed by a skilled individual or trained professional. Chem, 46, 884-885 (2000).
15. Lindstedt, G., ‘Endogenous antibodies against prolactin – a
2. Laboratory results alone are only one aspect for determining 14.2 Sensitivity
patient care and should not be the sole basis for therapy, “new” cause of hyperprolactinemia”, Eur. J. Endo 130, 439-42
The sensitivity (detection limit) was ascertained by determining
(1994).
particularly if the results conflict with other determinants. the variability of the 0ng/ml serum calibrator and using the 2σ