se

ea

Pl
us
e
ly

on
th
e
id

va
l
n

rs
io

ve
of
th
e
ns

In
st
ru
ct
io
fo
rU
se

Instructions for use

Prolactin rat ELISAAR E-8200R
ed

ov
id

pr

w
ith

th
e

t

ki

Prolactin rat ELISA
1. INTRODUCTION
1.1. Intended Use
The Prolactin rat ELISA is an enzyme immunoassay for the quantitative measurement of prolactin in rat
serum.
1.2 Summary and Explanation

fo
rU

se

pr

ov
id

ed

w
ith

th
e

ki

t

Rat prolactin (rPRL) is a single-chain polypeptide hormone of the rat anterior pituitary with a molecule massof approximately 23,000. Prolactin from different species exhibits significant variations in the amino acidsequence. Rat prolactin differs from human prolactin at about 50 percent of all residues.
The secretion of rPRL from the pituitary is inhibited by hypothalamic prolactin-inhibitory factor (PIF).
Although dopamine was long thought to be this PIF molecule, today it seems that there is a special peptide
with prolactin-inhibiting activities.
The release of prolactin is certainly stimulated by different peptides, particularly thyrotropin releasing
hormone (TRH) and vasoactive intestinal peptide (VIP). There is also evidence that rat posterior pituitary
lobe contains a special prolactin releasing hormone.
The most important role of prolactin is stimulation of mammary gland growth and lactation. During
pregnancy, blood prolactin levels climb, but the increases can differ enormously between rats. High prolactin
levels are observed during lactation. Prolactin has a wide variety of other physiological actions, for example
on the ovary. In the rat, prolactin has a luteotrophic effect which is not seen in many other species.
Furthermore, prolactin is a stress hormone.
In rats, as in humans, prolactin exhibits a sleep-related diurnal variation. Peak values are seen in the lateafternoon and nadir values in the morning.
Because of the variety of its actions, prolactin is one of the preferred hormones to monitor when testing theinfluence of new therapeutic agents and drugs on the endocrine system in the rat.

ns

2. PRINCIPLE

Pl

ea

se

us

e

on

ly

th
e

va
l

id

ve

rs
io

n

of

th
e

In
st
ru
ct
io

The Prolactin rat ELISA kit is a solid phase enzyme immunometric assay (ELISA) in the microplate format,
designed for the quantitative measurement of rat prolactin. The microplate is coated with a first monoclonal
antibody specific for rat prolactin.
Standards and samples are pipetted into the antibody coated microplate. During a 2 hours incubation
endogenous rat prolactin in the sample bind to the antibodies fixed on the inner surface of the wells. Nonreactive sample components are removed by a washing step. Afterwards, a second polyclonal horseradish
peroxidase-labeled antibody, directed against another epitope of the prolactin molecule, is added. During
another 1 hour incubation, a sandwich complex consisting of the two antibodies and the rat prolactin isformed. An excess of enzyme conjugate is washed out.
A chromogenic substrate, TMB (3,3',5,5'-Tetra-Methyl-Benzidine), is added to all wells. During a 30 minutes
incubation, the substrate is converted to a colored end product (blue) by the fixed enzyme. Enzyme reaction
is stopped by dispensing of hydrochloric acid as stop solution (change from blue to yellow). The color
intensity is direct proportional to the concentration of rat prolactin present in the sample. The optical densityof the color solution is measured with a microplate reader at 450 nm. Bi-chromatic measurement with a 600- 690 nm reference filter is recommended.
3. WARNINGS AND PRECAUTIONS
1.

This kit is strictly intended for research use only. Use by staff, who is specially informed and trained inmethods which are carried out by use of immunoassays.

2.

Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.

3.

The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.

4.

Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.

5.

Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.

6.

Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.

7.

Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.

8.

Allow the reagents to reach room temperature (21-26°C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the samples will not be affected.

Version: 4.0-b

Effective: 2018-09-07

2/8

9.

Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.

10. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
11. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
12. Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
13. Do not use reagents beyond expiry date as shown on the kit labels.
14. All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
15. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof different plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may result slightly different.
16. Avoid contact with Stop Solution. It may cause skin irritation and burns.
17. Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
18. For information please refer to Safety Data Sheets. Safety Data Sheets for this product are available
upon request directly from the manufacturer.

th
e

ki

t

4. REAGENTS

AR E- 8231

ov
id

ed

Microtiterplate - Ready to use
12 x 8 (break apart) strips with 96 wells.

prContents:w
ith

4.1 Reagents provided

fo
rU

se

Removable wells coated with a monoclonal anti-rat prolactin antibody.
Rat Prolactin Master Standard - Lyophilized

AR E-8201

in serum/buffer matrix containing highly purified rat prolactin.

Volume:1 x 80 ng

In
st
ru
ct
io

ns

Contents:the

For reconstitution see “Reagent Preparation”.

ofRat Prolactin Standard/Sample Diluent - Ready to use

AR E-8260
rat prolactin free.

Volume:1 x 6 ml

AR E-8240

Enzyme-Conjugate - Ready to use
containing horseradish peroxidase-labeled polyclonal anti rat prolactin antibody in abuffered solution with preservative. Red.
1 x 22 ml

rs
io
ve
id

va
l

th
e

e

us
se

AR E-8270

Rat Prolactin Sample Buffer - Ready to useeaVolume:onlyContents:

nContents:

yellow

Volume:1 x 6 ml

Pl

Contents:

AR E-0055

Substrate Solution - Ready to use

Contents:

3,3’,5,5’-Tetra-Methyl-Benzidine (TMB) in buffered peroxide solution.

Volume:1 x 22 ml

Hazards
identification:
H360D May damage the unborn child.
Wash Solution - 10x concentrated

AR E-0030
Volume:1 x 50 ml
See “Reagent Preparation”.

Version: 4.0-b

Effective: 2018-09-07

3/8

Stop Solution - Ready to use

AR E-0080
Contents:

contains 2 M Hydrochloric Acid

Volume:1 x 7 ml

Hazards
identification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
H335 May cause respiratory irritation.
4.2 Materials required but not provided

t

ki

th
e









Microplate reader capable for endpoint measurements at 450 nm (optional reference filter in the range of
600 - 690 nm)
Vortex mixer
Microplate mixer operating more than 600 rpm
Distilled or deionized water
Graduated cylinder for 500 ml
Plastic containers for storage of the wash solution
Adjustable pipette for up to 1000 µl
Dispenser or repeatable pipet for 25 µl, 50 µl, and 200 µl

w
ith



ed

4.3 Reagent preparation

fo
rU

se

pr

ov
id

Standards:
Reconstitute lyophilized Master Standard with 1 ml dest. water 30 min. before use (end concentration of 80
ng/ml). Make a dilution series with Standard/Sample Diluent to get standards with 80, 40, 20, 10 and 5
ng/ml.

In
st
ru
ct
io

ns

Wash Buffer:
Dilute with 450 ml dist. water to a final volume of 500 ml.
The diluted Wash Solution is stable for 12 weeks at room temperature.

the

4.4 Storage conditions

th
e

va
l

id

ve

rs
io

n

of

When stored at 2°C to 8°C unopened reagents will be stable until expiration date. Do not use reagents
beyond this date. Opened reagents must be stored at 2° - 8 °C. After first opening the reagents are stablefor 30 days if used and stored properly.
Microtiter wells must be stored at 2°C to 8 °C. Take care that the foil bag is sealed tightly. Store Standards
refrigerated, they will be stable at 2°C to 8 °C for 7 days after reconstitution. For longer storage aliquot andfreeze at -20 °C. Protect Substrate Solution from light.

e

on

ly

5. SPECIMEN

Pl

ea

se

us

For determination of rat prolactin serum is the preferred sample matrix. The procedure calls for 25 µl matrix
per well.
Prolactin is one of the most sensitive stress hormones of the rat. Blood collection should therefore be as
stress-free as possible. The samples may be stored refrigerated at 2 - 8 °C for one week, or up to 2 months
frozen at -20 °C. To avoid repeated thawing and freezing the samples should be aliquoted.
Samples expected to contain rat prolactin concentrations higher than the highest Standard (80 ng/ml)
should be diluted with Standard/Sample Diluent before assay. The additional dilution step has to be taken
into account for the calculation of the results.
6. ASSAY PROCEDURE
6.1 General remarks






Do not interchange components of different lots.
All components should be at room temperature (18 – 28 °C) before use.
All components of this test kit, supplied as concentrate should be diluted to their final concentration atleast 30 minutes prior to use. Mix well, but prevent of foam formation.
Use a disposable-tip micropipette to dispense serum samples. Pipet directly to the bottom of the wells.
Change the tip between samples, to avoid carryover contamination.
For internal quality control we suggest to use Rat Control Set coded AR K-8000. For more
information please contact the manufacturer.

Version: 4.0-b

Effective: 2018-09-07

4/8

6.2 Assay Procedure
1 Preparation of Standards
Label four tubes: E (40 ng/ml), D (20 ng/ml), C (10 ng/ml) and B (5 ng/ml). Pipet 0.1 ml of theStandard/Sample Diluent into all tubes. Pipet 0.1 ml of the reconstituted Master Standard into tube E (40ng/ml), and mix thoroughly. Repeat this process successively to complete the 2-fold dilution series. The
reconstituted Standard will serve as the highest Standard F (80 ng/ml). Use the Standard/Sample Diluent as
the zero Standard A (0 ng/ml).
1
a A
b A
c B
d B
e C
f C
g D
h D

2
E
E
F
F
P1
P1
P2
P2

3
4
5
P3 P..
P3 P..
P4
P4
P5
P5
P6
P6

6

7

8

9

10 11 12

Pipet 25 µl of each standard, control and sample into the wells prepared.

3.

Add 50 µl of Sample Buffer to every well.

4.

Rotate for 2 hours at room temperature (18 - 28 °C) on a plate mixer (600 - 900 rpm).

5.

Discard the content of the wells and wash 4 times with 300 µl buffered Wash Solution.

ov
id

ed

w
ith

th
e

ki

t

2.

se

pr

Remove as much wash solution as possible by beating the microplate carefully.
Add 200 µl of Enzyme Conjugate to all wells.

7.

Shake again for 1 hour (600 – 900 rpm).

8.

Discard the content of the wells and wash 4 times with 300 µl buffered Wash Solution.

In
st
ru
ct
io

ns

fo
rU

6.

th
e

Remove as much Wash Solution as possible by beating the microplate carefully.
Add 200 µl of liquid Substrate Solution to all wells.

10.

Incubate without shaking for 30 minutes in the dark.

11.

Add 50 µl of Stop Solution to each well and mix carefully.

12.

Read the optical density at 450 nm. Bi-chromatic measurement with a reference at 600-690 nm is
recommended.

on

ly

th
e

va
l

id

ve

rs
io

n

of

9.

se

us

e

The developed color is stable for at least 30 minutes. Read optical densities during this time.

Plea

6.2 Calculation of Results

For evaluation of rat prolactin a 4-Parameter-Fit with lin-log coordinates for optical density (linear scale) andconcentration (logarithmic scale) is recommended.
Spline approximation with lin-log coordinates and log-log coordinates are also suitable.
6.3 Calculation of results
For evaluation of rat prolactin a 4-Parameter-Fit with lin-log coordinates for optical density (linear scale) andconcentration (logarithmic scale) is recommended.
Spline approximation with lin-log coordinates and log-log coordinates are also suitable.

Version: 4.0-b

Effective: 2018-09-07

5/8

6.3.1 Example of typical Standard Curve
The figure below shows typical results for Prolactin rat ELISA test kits. These data are intended forillustration only and should not be used to calculate results from another run.
Replicate
(OD)

Mean
(OD)

Binding(%)

Rat Prolactin(ng/ml)

0.073

-

0

0.179

6.9

5

0.342

12.7

10

0.796

29.7

20

1.635

61.0

40

2.677

100

80

B
C
D
E

th
e

F

ki

0.079
0.067
0.189
0.169
0.328
0.357
0.766
0.827
1.591
1.679
2.588
2.765

A

t

Standards

0.318
0.329
0.577
0.603
1.733
1.698

1.716

ed
ov
id
pr

12.1

9.8

22.0

16.4

64.1

47.3

In
st
ru
ct
io

ns

X 003

0.590

se

X 002

0.324

fo
rU

X 001

w
ith

Unknown Samples

7. EXPECTED NORMAL VALUES

rs
io

n

of

th
e

In a reference range study rat serum samples were collected in the morning between 8 and 9 a.m. and in
the evening between 5 and 6 p.m. Diurnal variations have not been observed. Analysis by the Prolactin rat
ELISA kit yielded the following results:
Absolute Range (ng/ml)
nd - 17.9
nd - 23.4
nd = nondetectable

th
e

va
l

id

ve

Group
Normal female rats
Normal male rats

n
15
10

ea

se

us

e

on

ly

Because of differences which may exist between laboratories with respect of population, laboratory
technique and selection of reference groups, it is recommended that each laboratory establishes its own
normal and pathological ranges of rat prolactin. The reference ranges should be regarded as guidelines only.

Pl

8. PERFORMANCE CHARACTERISTICS
8.1 Analytical Sensitivity
The lower detection limit for rat prolactin is approximately 0.6 ng/ml.
8.2 Specificity
The antibodies in the Prolactin rat ELISA procedure are highly specific for rat prolactin. Detectablecrossreactivities to other hormones that may be present in serum samples are not known.
The following substances were tested:

Rat TSH
Rat FSH
Rat LH
Rat GH

Version: 4.0-b

added quantity(ng/ml)measured concentration(ng/ml)cross-reactivity
(%)

5 000
10 000
5 000
4 000

1.5
5.1
nd
nd

0.03
0.5
-

Effective: 2018-09-07

6/8

8.3 Reproducibility
Statistics for Coefficients of variation (CV) were calculated for each of three samples from the results of 20
pairs of wells in a single run for Intra-Assay precision and the Inter-Assay precision was calculated from theresults of 14 different runs of three samples:
Intra Assay
SampleMean(ng/ml)x

SD ± s (ng/ml)

CK (%)

1
2
3

6.6
19.6
33.0

0.20
0.77
1.80

3.0
3.9
5.5

SampleMean(ng/ml)x

SD ± s (ng/ml)

CK (%)

1
2
3

6.5
20.5
33.4

0.28
0.71
1.48

4.3
3.5
4.4

w
ith

th
e

ki

t

Inter Assayed

8.4 Recovery

Measured Concentration

ns

Diluted Solution

In
st
ru
ct
io

Sample

fo
rU

se

pr

ov
id

Three spiking solutions were prepared using the Sample Diluent, to represent the 600, 800 and 1,000
ng/ml, respectively. A 50 µl aliquot of each solution (A, B, C) was spiked into 950 µl aliquots of two different
rat serum samples, for a spiking ratio of 1 to 20, leaving the serum matrix of the spiked samples relatively
intact. All samples were then assayed by the Prolactin rat ELISA procedure.
(ng/ml)

(%)

-

26.1

-

-

54.7

56.1

97

n
rs
io
id

ve

-

va
l

A

66.1

100

76.1

105

24.2

-

-

52.6

54.2

103

67.5

64.2

95

81.1

74.2

91

e

on

ly

th
e

B
C

66.6

79.9

of

B
C
2

Recovery(ng/ml)

A

th
e

1

Expected concentration

seus

8.5 Linearity

Plea

In dilution experiments sera with high rat prolactin concentrations were diluted with sample diluent andassayed in the Prolactin rat ELISA kit. The assay showed linearity over the full measuring range.
Sample1

2

Version: 4.0-b

Diluted Factor

8 in 84 in 82 in 81 in 88 in 84 in 82 in 81 in 8

Measured Concentration

Expected concentration

Recovery(ng/ml)(ng/ml)

(%)

20.9
10.6
5.9
3.0

10.5
5.2
2.6

101
113
115

34.7

-

-

16.7

17.4

96

9.0

8.7

103

4.5

4.3

105

Effective: 2018-09-07

7/8

9. LIMITATIONS OF PROCEDURE
Effect of Anticoagulants
To determine whether anticoagulants interfere with the assay, blood was collected from 30 rats into plain
and EDTA vacutainer tubes. All samples were assayed by the Prolactin rat ELISA procedure, with thefollowing results.
(EDTA) = 1.05 (Serum) – 9.3 ng/ml r = 0.978
Means:

3.68 ng/ml (Serum)
3.78 ng/ml (EDTA)

A limited study with citrated and heparinzed plasma show comparable results to EDTA plasma.
"High-Dose Hook"-EffectRat sera containing up to 300 ng/ml Prolactin were measured with the Prolactin rat ELISA assay. A HighDose Hook effect could not be observed.
10. REFERENCES
Leung, F. C., Russel, S. M.; Nicoll, C. S.
Relationship between bioassay and radioimmunoassay estimates of prolactin in rat serum.
Endocrinology 1978; 103: 1619 - 28.
2. Martinat, N., Hall, E., Ravault, J. P., Dubois, M. P.
Purification of rat prolactin: development of an homologous radioimmunological assay and comparison
with the NIAMDD system. Ann Biol Anim Bioch Biophys 1979; 19: 1771 - 48.
3. Beach, J. E., Miles, D. J., Lukes, Y. G., Vigersky, R. A. Microplate solid-phase radioimmunoassay for rat
prolactin. J Lab Clin Med 1985; 105: 294 - 298.
4. Butcher, R. L., Collins, W. E., Fugo, N. W. Plasma concentration of LH, FSH, prolactin, progesterone
and estradiol-17ß throughout the 4-day estrous cycle of the rat. Endocrinology 1974; 94: 1704 - 8.
5. Barbieri, R. L., Todd, R. B., Morishita, H., Ryan, K. J., Fishman, J, Naftolin, F. Response of serum
prolactin to catechol estrogen in the immature rat. Fertil Steril 1980; 34: 391 - 3.
6. Wong, C. C. Endogene und exogene Einflüsse auf die Variabilität der Hormonausschüttung bei derRatte (Endogenous and exogenous influences on the variability of hormone release in the rat). Thesis,
Hannover (Germany): Univ. of Hannover, 1981.
7. Campbell, G. A., Kurcz, M., Marshall, S., Meites, J. Effects of starvation in rats on serum levels of FSH,
LH, TSH, growth hormone and prolactin: response to LH-releasing hormone and thyrotropin-releasing
hormone. Endocrinology 1977; 100: 580 - 7.
8. Haggi, E., Aoki, A. Prolactin content in rat pituitary gland. RIA of prolactin after different extraction
procedures. Acta Endocrinol 1981; 97: 338 - 42.
9. Reichel, J.
Wirkungen gonadaler Steroide auf die adeno-hypophysären Thyreotropin-ReleasingHormon-Rezeptoren, den Prolactinserumspiegel und die hormonelle Hypophysen-Schilddrüsen-Achse
der Ratte (Effects of gonadal steroids on the pituitary TRH-receptors, the serum prolactin concentrations
and the pituitary-thyroid axis in the rat) Thesis. Lübeck (Germany): University of Lübeck, 1990.
10. Moishige, W. K., Pepe, G. J., Rothschild, I. Serum LH, prolactin and progesterone levels during
pregnancy in the rat. Endocrinology 1973; 92: 1527 - 30.

Pl

ea

se

us

e

on

ly

th
e

va
l

id

ve

rs
io

n

of

th
e

In
st
ru
ct
io

ns

fo
rU

se

pr

ov
id

ed

w
ith

th
e

ki

t

1.

Symbols:

Version: 4.0-bStoragetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2018-09-07

Contains sufficient for<n> tests

For research use
only!
8/8