ManualsRANKL ELISA Kit
For the in vitro determination of mouse/rat sRANKL in serum andcell culture supernatant
For research use only

Valid from 24.05.2011+ 8 °CK 1019+ 2 °C96
Immundiagnostik AG, Stubenwald-Allee 8a, D 64625 Bensheim
Tel.: ++49 6251 70190-0
Fax: ++ 49 6251 849430
e.mail: Info@immundiagnostik.com
www.Immundiagnostik.com

Arbeitsanleitung/ManualsRANKL

Inhaltsverzeichnis

1

Table of contents2

1. INTENDED USE

2

2. SUMMARY AND EXPLANATION OF THE TEST2

3. PRINCIPLE OF THE TEST

3

4. MATERIAL SUPPLIED

3

5. MATERIAL REQUIRED BUT NOT SUPPLIED

4

6. PREPARATION AND STORAGE OF REAGENTS

4

7. PRECAUTIONS

5

8. SPECIMEN COLLECTION AND PREPARATION

6

9. ASSAY PROCEDURE

6PROCEDURAL NOTESPLATE PREPARATIONASSAY PROCEDURE

667

10. RESULTS

8

11. LIMITATIONS

8

12. QUALITY CONTROL

8

13. REFERENCES

9

14. GENERAL NOTES ON THE TEST AND TEST PROCEDURE

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Arbeitsanleitung/ManualsRANKL

1. INTENDED USE
The Immundiagnostik assay is a sandwich ELISA intended for thequantitative determination of mouse/rat sRANKL in serum and cell culture
supernatant. It is for research use only.

2. SUMMARY AND EXPLANATION OF THE TESTRANKL, receptor activator of nuclear factor (NF)-B ligand (also:
osteoprotegerin ligand, OPGL), its cellular receptor, receptor activator of NFB (RANK), and the decoy receptor, osteoprotegerin (OPG) have been
identified as the key molecular regulation system for bone remodelling.
RANKL, a member of the tumor necrosis factor (TNF) family, is the main
stimulatory factor for the formation of mature osteoclasts and is essential for
their survival. Therefore, an increase in RANKL expression leads to bone
resorption and bone loss. RANKL is produced by osteoblastic lineage cells
and activated T lymphocytes. It activates its specific receptor RANK which islocated on osteoclasts and dendritic cells.
The effects of RANKL are counteracted by OPG which is secreted by various
tissues and acts as an endogenous soluble receptor antagonist.
Imbalances of the RANKL/OPG system have been related to thepathogenesis of Paget’s disease, benign and malignant bone tumors,
postmenopausal osteoporosis, rheumatoid arthritis, bone metastases andhypercalcemia. It was shown in several studies that in animal models
restoring of the RANKL/OPG balance (e.g. by administering OPG) reduces
the severity of these disorders.
It has been shown that RANKL is produced as a membrane-bound protein
on murine osteoblasts/stromal cells, and cleaved into a soluble form by ametalloprotease. Stimulators of the osteoclastogenesis such as IL-1beta, IL-6,
IL-11, IL-17, and TNF-alpha, increase the expression of RANKL and decrease
OPG expression in osteoblasts/stromal cells. Cytokines inhibiting theosteoclastogenesis such as IL-13, INF-gamma, and TGF-beta1, suppress theexpression of RANKL and stimulated OPG expression.
Indications
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Postmenopausal and senile osteoporosis
Diseases with locally increased bone resorption activity
Paget´s disease
Periodontal disease
Inflammatory diseases
Immunological disorders
Arthritis
Oncology
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Arbeitsanleitung/ManualsRANKL3. PRINCIPLE OF THE TESTThis sandwich-type ELISA is an assay for the direct determination of sRANKLin serum, plasma and urine. In this assay two highly specific antibodies
against sRANKL are used. The capture antibody is attached to the wells of
the microtiter plate, the detection antibody is labeled with biotin.
In a first incubation step the samples and the biotinylated antibody against
sRANKL react with the coated capture antibody on the microtiter plate. A
sandwich-type complex is formed consisting of the binding antibody on theplate, sRANKL and the biotinylated detection antibody. To remove allunspecific bound substances a washing step is carried out.
In a second step streptavidin – peroxidase is added which reacts with thedetection antibody. After another washing step, the solid phase is incubatedwith the substrate, TMB. An acidic stopping solution is subsequently added.
The blue color changes to yellow. The intensity of the yellow color is directly
proportional to the concentration of sRANKL in the sample.
A dose - response curve of the absorbance units at 450 nm versus
concentration is generated. sRANKL in the samples is determined directlyfrom this calibration curve.

4. MATERIAL SUPPLIED
Cat. no.

Label

Kit Components

Quantity

K1019LMTP

PLATE

Microtiter plate, 12 x 8 strips

96

K1019WPWASHBUF ELISA washing buffer concentrate (10x)100 ml

K1019BeP

COATBUF

Coating buffer

30 ml

K1019A

COATAB

Capture antibody (rat anti-mouse
RANKL), lyophilized

1 vial

K1019A2

2.AB

Detection antibody (goat anti-mouse
RANKL, biotinylated), lyophilized

1 vial

K1019ST

STD

Calibrator (4000 pg) lyophilized

2 vials

K1019K

CONJ

Conjugate, (Strepdavidin-HRPlabeled), ready-to-use

1 vial

K1019DLDIL

Reagent Diluent

100 ml

K1019TMBSUB

TMB substrate (Tetramethylbenzidine),
ready-to-use

22 ml

K1019ACSTOPELISA stop solution, ready-to-use

1 x 15 ml

3Arbeitsanleitung/ManualsRANKL5. MATERIAL REQUIRED BUT NOT SUPPLIED
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Distilled or deionized water
Precision pipettes calibrated to deliver 50 -1000 μl and disposable tips.
Multichannel or Multipipette
Vortex mixer
Conventional glass or plastic tubes
1,5 ml reaction vials (Eppendorf)
ELISA reader equipped with 450 nm filter

6. PREPARATION AND STORAGE OF REAGENTS

To run assay more than once, ensure that reagents are stored atconditions stated on the label. Prepare only the appropriate amountnecessary for each assay. The kit can be used up to 4 times within the
expiry date stated on the label.

Reagents with a volume less than 100 μl should be centrifuged before
use to avoid loss of volume.



The WASHBUF (ELISA wash buffer concentrate) should be diluted withaqua bidist. 1:10 before use (100 ml WASHBUF + 900 ml aqua bidist.),mix well. Crystals could occur due to high salt concentration in the stock
solutions. The crystals must be redissolved at room temperature or at
37°C before dilution. The buffer concentrate is stable at 2-8°C until the
expiry date stated on the label. Diluted buffer solution can be stored ina closed flask at 2-8°C for one month.

COATAB (Capture Antibody, 1 vial), 144 μg/ml of rat anti-mouse RANKL
Reconstitute with 35 μl bidistilled water. After reconstitution, store at
2 - 8°C for up to 60 days or aliquot and store at -20°C to -70°C for up to
6 months. Dilute immediately before use to a working concentrationof 0.4 μg/ml in COATBUF (coating buffer).



2.AB (Detection Antibody, 1 vial), 36 μg/ml of biotinylated goat antimouse RANKL. Reconstitute with 70 μl bidistilled water. After
reconstitution, store at 2 – 8°C for up to 60 days or aliquot and store at 20°C to -70°C for up to 6 months. Dilute to a working concentration of
200 ng/ml in DIL (Reagent Diluent).

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Arbeitsanleitung/ManualsRANKL

STD (Standard, 1 vial), 4000 pg/ml of recombinant mouse RANKL
Reconstitute with 0.6 mL bidistilled water. Allow the standard to sit fora minimum of 15 minutes with gentle agitation prior to making
dilutions. Store reconstituted standard at 2 - 8° C max. overnight or
aliquot and store at -70° C for up to 2 months. A seven point standard
curve using 2-fold serial dilutions in DIL (Reagent Diluent), and a highstandard of 4000 pg/ml is recommended.



CONJ (Streptavidin-HRP, 1 vial), 60 μl of streptavidin conjugated tohorseradish-peroxidase. Store at 2 - 8° C. Do not freeze. Dilute to theworking concentration 1:200 in DIL (Reagent Diluent).

7. PRECAUTIONS

To avoid cross-contamination, change pipette tips between addition ofstandards, controls, samples, antibody, conjugate and substrate. Alsouse separate reservoirs for each reagent.

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Avoid foaming when mixing the reagents.



Do not mix stoppers and caps of different reagents.



Do not use reagents beyond expiration date.

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Do not mix or substitute reagents with those from other lots or sources.



To ensure accurate results, proper adhesion of plate sealers during
incubation steps is necessary.



Substrate solution should remain colourless until added to the plate.

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Stop solution should be added to the plate in the same order as thesubstrate solution.

Protect reagents from direct sunlight.

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Do not pipette by mouth.

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Do not eat, drink, smoke or apply cosmetics where reagents are used.

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Avoid all contact with the reagents by using gloves.



Stop solution is composed of sulfuric acid, which is a strong acid. Even
diluted, it still must be handled with care. It can cause acid burns andshould be handled with gloves, eye protection, and appropriate
protective clothing. Any spills should be wiped out immediately withcopious quantities of water.



Human materials used in kit components were tested and found to be
negative for HIV, Hepatitis B and Hepatitis C. However, for safety
reasons, all kit components should be treated as potentially infectious.
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Arbeitsanleitung/ManualsRANKL

8. SPECIMEN COLLECTION AND PREPARATION
Serum, plasma and urine samples
Serum, plasma and urine samples can be used without any dilution. Serummust be centrifuged and aliquoted within 90 min after collection andstored at –20 °C until use.

9. ASSAY PROCEDURE
Procedural notesDo not mix different lot numbers of any kit component.

Guidelines for medical laboratories should be followed.



Incubation time, incubation temperature and pipetting volumes of thecomponents are defined by the producer. Any variation of the test
procedure, which is not coordinated with the producer, may influence
the results of the test. Immundiagnostik AG can therefore not be held
responsible for any damage resulting from wrong use.

The assay should always be performed according the enclosed manual.

Plate Preparation
1. Coat a 96-well microplate with 100 μL per well of the diluted COATAB(capture antibody). Seal the plate and incubate overnight at room
temperature.
2. Aspirate and wash the wells 5 x with 250 μl ELISA wash buffer. Complete
removal of liquid at each step is essential for good performance. After thelast wash, remove any remaining wash buffer by aspirating or by
inverting the plate and blotting it against clean paper towels.
3. Block plates by adding 250 μl of DIL (reagent diluent) to each well.
Incubate at room temperature for 1 hour.
4. Aspirate and wash the wells 5 x with 250 μl ELISA wash buffer. After thelast wash, remove any remaining wash buffer by aspirating or by
inverting the plate and blotting it against clean paper towels. The plates
are now ready for sample addition.

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Arbeitsanleitung/ManualsRANKL

Assay Procedure
1. Add 100 μl of SAMPLE (sample) or STD (standards) in Reagent Diluent,
per well. Cover with an adhesive strip and incubate 2 hours at room
temperature.
2. Aspirate and wash the wells 5 x with 250 μl ELISA wash buffer. After thelast wash, remove any remaining wash buffer by aspirating or by
inverting the plate and blotting it against clean paper towels.
3. Add 100 μl of the 2. AB (detection antibody), diluted in Reagent Diluent,
to each well. Cover with a new adhesive strip and incubate 2 hours atroom temperature.
4. Aspirate and wash the wells 5 x with 250 μl ELISA wash buffer. After thelast wash, remove any remaining wash buffer by aspirating or by
inverting the plate and blotting it against clean paper towels.
5. Add 100 μl of the working dilution of CONJ (streptavidin-HRP) to eachwell. Cover the plate and incubate for 20 minutes at room temperature.
Avoid placing the plate in direct light.
6. Aspirate and wash the wells 5 x with 250 μl ELISA wash buffer. After thelast wash, remove any remaining wash buffer by aspirating or by
inverting the plate and blotting it against clean paper towels.
7. Add 100 μl of SUB (substrate solution) to each well. Incubate for 20
minutes at room temperature. Avoid placing the plate in direct light.
8. Add 50 μl of STOP (stop solution) to each well. Gently tap the plate toensure thorough mixing.
9. Determine absorption immediately with an ELISA reader at 450 nm
against 620 nm (or 690 nm) as a reference. If no reference wavelength isavailable, read only at 450 nm. If the extinction of the highest standard
exceeds the range of the photometer, absorption must be measured
immediately at 405 nm against 620 nm as a reference.

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Arbeitsanleitung/ManualsRANKL

10. RESULTS
The following algorithms can be used alternatively to calculate the results.
We recommend to use the "4-Parameter-algorithm".
1. 4-parameter-algorithm
It is recommended to use a linear ordinate for optical density and alogarithmic abscissa for concentration. When using a logarithmic
abscissa, the zero calibrator must be specified with a value less than 1(e. g. 0.01).
2. Point-to-point-calculation
We recommend a linear ordinate for optical density and a linear
abscissa for concentration.
3. Spline-algorithm
We recommend a linear ordinate for optical density and a logarithmic
abscissa for concentration. When using a logarithmic abscissa, thezero calibrator must be specified with a value less than 1 (e. g. 0.01).
The plausibility of the pairs of values should be examined before theautomatic evaluation of the results. If this option is not available withthe used program, a control of the paired values should be done
manually.

11. LIMITATIONS
Samples with sRANKL levels greater then the highest standard value should
be further diluted and re-assayed.

12. QUALITY CONTROL
Immundiagnostik AG recommends the use of commercial control samples
for internal quality control if available.
Control samples should be analyzed with each run. Results, generated fromthe analysis of control samples, should be evaluated for acceptability using
appropriate statistical methods. The results for the patient samples may not
be valid, if within the same assay one or more values of the quality control
sample are outside the acceptable limits.

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Arbeitsanleitung/ManualsRANKL13. REFERENCES
1.

Lacey D.L, et al., Osteoprotegerin ligand is a cytokine that regulates
osteoclast differentiation and activation. Cell (1998), 93:165-176.
2. Kong Y.Y. et al., OPGL is a key regulator of osteoclastogenesis,
lymphocyte development and lymph-node organogenesis. Nature(1999), 397: 315-323.
3. Hsu H. et al., Tumor necrosis factor receptor family member RANK
mediates osteoclast differentiation and activation induced byosteoprotegerin ligand. Proc Natl Acad Sci (1999), 96:3540-3545.
4. Josien R, et al., TRANCE, a tumor necrosis factor family member,enhances the longevity and adjuvant properties of dendritic cells in
vivo. J Exp Med (2000), 191: 495-502.
5. Fuller K. et al., TRANCE is necessary and sufficient for osteoblastmediated activation of bone resorption in osteoclasts. J Exp Med (1998),
188: 997-1001.
6. Nakashima T , et. al., Protein expression and functional difference of
membrane-bound and soluble receptor activator of NF-kappaB ligand:modulation of the expression by osteotropic factors and cytokines.
Biochem Biophys Res Commun (2000), 275(3):768-75.
7. Kong Y.Y. et al., Activated T cells regulate bone loss and joint
destruction in adjuvant arthritis through osteoprotegerin ligand. Nature(1999), 402: 304-309.
8. Hofbauer L.C. & A.E. Heufelder, Role of receptor activator of nuclear
factor-KB ligand and osteoprotegerin in bone cell biology. J Mol Med(2001), 79: 243-253.
9. Hofbauer L.C. & A.E. Heufelder, The Role of Osteoprotegerin and
Receptor Activator of Nuclear Factor KB Ligand in the Pathogenesis andTreatment of Rheumatoid Arthritis. Arthritis & Rheumatism (2001),
44:253-259.
10. Hofbauer L.C., et al., The role of receptor activator of nuclear factorkappaB ligand and osteoprotegerin in the pathogenesis and treatmentof metabolic bone diseases. J Clin Endocrinol Metab (2000), 85: 23552363.
11. Teitelbaum S.L., Bone resorption by osteoclasts. Science (2000), 289:
1504-1508.
12. Hofbauer L.C. et al., Effects of oral contraceptives on circulating
osteoprtegerin an soluble RANK ligand serum levels in healthy young
women. (2004) Clin Endocrinol 60:214-219
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14. GENERAL NOTES ON THE TEST AND TEST PROCEDURE
 The test components which are made of human serum are tested forAustralia antigen and HIV and found to be negative. However, sinceno test method can offer complete assurance that infectious agentsare absent, these reagents should be handled as recommended forany potentially infectious human serum or blood specimen. The
normal precautions for laboratory working should be observed.
 Kit reagents contain sodium azide or thimerosal as bactericides.
Sodium azide and thimerosal are toxic. Substrates for the enzymaticcolor reactions are toxic and carcinogenic. Avoid contact with skin or
mucous membranes.
 For research use only.
 Reagents should not be used beyond the expiration date shown on
the kit label.
 Do not mix different lot numbers of any kit component.
 Quality control guidelines should be followed.
 Incubation time, incubation temperature and pipetting volumes of
the components are defined by the producer. Any variation of the test
procedure, which is not coordinated with the producer, may influence
the results of the test. Immundiagnostik AG can therefore not be held
responsible for any damage resulting from wrong use.
 Warranty claims and complaints in respect of deficiencies must be
logged within 14 days after receipt of the product. The product should
be send to Immundiagnostik AG along with a written complaint.

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