DELFIA Sm-Labelling kit 1244-303

13901952-4 (en)
DELFIA® Sm-Labelling kit 1244-303
This DELFIA® Sm-Labelling kit is intended for labelling of proteins with samarium (Sm3+),
for use in dissociation-enhanced time-resolved fluoroimmunoassays. Sm3+ can be used
together with europium (Eu3+) in dual label assays.
Each DELFIA Sm-Labelling kit contains:
0.2 mg Labelling reagent
Sm-Standard and Enhancement Solution for measuring Sm3+
Stabilizer, purified BSA for increasing the stability of labelled proteins
An uncoated microtitration plate, DELFIA Assay Buffer and Wash Concentrate for
testing of labelling results.
Samarium forms a highly fluorescent chelate with ligands present in the DELFIA
Enhancement Solution. The long fluorescence life-time enables the use of the chelate in
time-resolved fluorometry. The time-resolved principle is applied in fluoroimmunoassays to
eliminate background interferences (1,2,3).
The Labelling reagent is the Sm-chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamineN1,N2,N3,N3-tetraacetic acid (DTTA) (Figure 1). The DTTA group forms a stable complex
with Sm3+ and the isothiocyanate-group reacts with a free amino group on the protein to
form a stable, covalent thiourea bond (2,4) (Figure 2). The high water solubility and the
stability of the chelate, in addition to the mild coupling conditions of the isothiocyanate
reaction, enable easy labelling of e.g. antibodies with up to 10-20 Sm3+/IgG.
Figure 1: Chemical structure of the Sm-Labelling reagent, N1-(p-isothiocyanatobenzyl)diethylene-triamine-N1,N2,N3,N3-tetraacetic acid chelated with Sm3+.
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Figure 2: The conjugation reaction between the aromatic isothiocyanate group of the
labelling reagent and an amino group of a protein.
In order to use a simple gel filtration for fractionation of labelled proteins and separation of
proteins from free Sm-chelates, the molecular weight of the protein needs to be at least
5000. For smaller peptides or other amine-containing compounds to be labelled, separate
purification systems need to be specifically developed.
The thermodynamic stability of the chelate allows long-term storage of labelled proteins
and the kinetic stability allows use of the labelled reagents in assays in contact with e.g.
serum samples.
The labelled protein as such is practically non-fluorescent. Consecutively to the immunoreactions and appropriate washing steps, however, Sm3+ is efficiently released from the
chelate within a few minutes by the low pH of the Enhancement Solution. Free Sm3+
rapidly forms a new highly fluorescent chelate with the components of the Enhancement
Solution (2,4). The fluorescence is then measured with the time-resolved fluorometer.
The same Enhancement Solution used for the measurement of Eu3+ is also optimal for
Sm3+, and this enables a simultaneous determination of the two lanthanides. The different
main emission wavelengths of the lanthanides (613 nm for Eu3+ and 643 nm for Sm3+) and
their different emission life-times (730 µs for Eu3+ and 50 µs for Sm3+) serve to minimize
the spillover between the respective signals, when measured with separate filters and
time-windows (3, 5).
If the Eu-concentration exceeds 0.1 nmol/L the minor peak at 650 nm of the Eu-emission
can cause signal spillover into the Sm-channel (643 nm). This Eu-interference, due to its
longer decay-time, can be automatically corrected for in the DELFIA Research
Fluorometer. This signal overlapping can be calculated also manually by measuring
standards with the relevant instrument programs (correction-factors obtained are specific
for delay- and window-times used).
The expiry date of the complete package is stated on the outer label. Store at +2 - +8°C.
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Shelf life and storage
Sm-Labelling Reagent
1 vial, 0.2 mg
< +8°C until expiry date stated
on the vial label.
0.2 mg (300 nmol) of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3tetraacetic acid chelated with Sm3+, lyophilized.
1 vial, 0.5 mL
+2 - +8°C until expiry date
stated on the vial label.
1 µmol/L of Sm3+ in 0.1 mol/L acetic acid.
1 vial, 0.5 mL
+2 - +8°C until expiry date
stated on the bottle label.
Bovine serum albumin (BSA), 7.5 %, in Tris-HCl buffered salt solution (pH 7.8),
containing < 0.1 % sodium azide as preservative. BSA is highly purified from heavy metal
Enhancement Solution
1 bottle, 50 mL
+2 - +8°C until expiry date
stated on the bottle label.
(+20 - +25°C: see the bottle
label). Avoid direct sunlight.
Ready for use Enhancement Solution with Triton X-1001, acetic acid and chelators.
Assay Buffer
1 bottle, 50 mL
+2 - +8°C until expiry date
stated on the bottle label.
Ready for use Tris-HCl buffered (pH 7.8) salt solution with bovine serum albumin, bovine
gammaglobulin, Tween 40, diethylenetriaminepentaacetic acid (DTPA), an inert red dye,
and < 0.1 % sodium azide as preservative.
Wash Concentrate
1 bottle, 40 mL
+2 - +8°C until expiry date
stated on the bottle label.
A 25-fold concentration of Tris-HCl buffered (pH 7.8) salt solution with Tween 20.
Contains Germall II2 as preservative.
Triton is a registered trademark of Rohm and Haas Co.
Germall is a registered trademark of Sutton Laboratories Inc.
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Microtitration Plate
1 plate
One uncoated plate of microtitration strips, 8 x 12 wells.
1. Labelling buffer: Carbonate buffer, 50 mmol/L (pH 9.0 - 9.3), containing 0.9 % NaCl.
pH of the buffer can be in the range of 8.5 to 9.8. A high pH, however, may damage
sensitive proteins, e.g. some monoclonal antibodies cannot withstand overnight
incubation at a high pH and room temperature.
2. Elution buffer: 50 mmol/L Tris-HCl (pH 7.8), containing 0.9% NaCl and < 0.1 % NaN3.
3. Column decontamination buffer: 10 mmol/L potassium hydrogen phthalate (pH 4.0),
containing 0.001 % diethylenetriaminepentaacetic acid (DTPA), and 0.1 % Germall II
or < 0.1 % NaN3 as preservative.
4. Precision pipettes: range 10 µL - 500 µL.
5. Chromatographic system: Gel filtration columns for changing buffers for proteins
prior to labelling and for purification and fractionation of labelled proteins.
PD-10 columns or NAP™-103 columns (Pharmacia Biotechnology) are recommended
for removing interfering substances (e.g. NaN3 and primary amines) from the protein
solution prior to labelling. PD-10 columns can also be used for crude separation of
labelled proteins from unreacted chelates. The use of such short columns may,
however, impose a risk of unsatisfactory separation of the protein from free chelates
and from possible aggregates.
Alternatively, Sephadex G-25 or -50 (Pharmacia Biotechnology) with suitable column
sizes (e.g. 1.5 x 30 cm) can be used for separation of labelled proteins from free
Sepharose 6B (Pharmacia Biotechnology) (e.g. 1.5 x 40 cm) is recommended for
fractionation and purification of labelled antibodies from unreacted chelates and
aggregated proteins. By combining Sepharose 6B with Sephadex G-50 (e.g. 10 cm of
Sephadex on the top of the Sepharose column) the resolution between free Euchelates and proteins can be improved.
Fraction collector, peristaltic pump, UV-detector and tubings.
6. Spectrophotometer for measurement of protein concentrations.
7. Automatic shaker.
NAP-10 is a trademark of Pharmacia Biotechnology.
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8. 1420 VICTOR multilabel counter or alternatively 1234 DELFIA Research
Fluorometer with Multi-Calc® 4 software. Additionally laboratory computer and
This DELFIA Sm-Labelling kit is intended for research use only.
The handling of concentrated Sm3+ solutions constitutes a contamination risk which may
cause elevated backgrounds in time-resolved fluoroimmunoassays. Keep the labelling
reagents away from the place where the assay is performed. Also ensure that accessories
used for the labelling procedure are kept separate from those needed for the assay. Avoid
contaminating Sm-reagents with Eu3+. Use separate accessories and columns for Sm3+
and Eu3+.
Reagents contain sodium azide (NaN3) as a preservative. Sodium azide may react with
lead or copper plumbing to form highly explosive metal azides. On disposal, flush with a
large volume of water to prevent azide build-up.
Disposal of all waste should be in accordance with local regulations.
Conditions for labelling
The labelling depends upon the nature and concentration of the protein to be labelled, the
temperature and pH of the reaction and the intended final labelling yield. The proteins to
be labelled must be in a buffer that does not contain any amines or sodium azide
(carbonate buffer, pH 9.0 - 9.3, is recommended). If there is any doubt about the protein
stability at higher temperature, labelling at +4°C is recommended.
When labelling antibodies for IFMA, generally about 5 - 15 Sm3+/IgG is an optimal yield
giving high sensitivity with low background. For many assays even a lower labelling yield
gives acceptable results. Labelling of antibodies with over 20 Sm3+/IgG may occasionally
cause aggregation and an elevated background, especially after storage.
The labelling yield needs to be optimized separately for each particular protein and the
assay requirements. Especially monoclonal antibodies may behave individually. Table 1
gives examples of labelling yields obtained with different proteins, when labelled according
to the protocol described below. The reactivity of different proteins in labelling depends on
the number of available amino-groups on the protein surfaces and the isoelectric point. For
example avidin is a protein with a high isoelectric point, and accordingly it reacts easily
with the reagent. The incorporation of negatively charged chelates on avidin decreases its
non-specific binding properties, and thus a relatively high chelate density is optimal (8 - 15
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Sm3+/Protein). In contrast, streptavidin has a lower isoelectric point resulting in lower
incorporation, but it has also a lower labelling yield optimum (4 - 10 Sm3+/Protein).
Monoclonal Anti-AFP
Monoclonal Anti-TSH
Monoclonal Anti-CEA
Monoclonal Anti-FSH
Bovine gammaglobulin
Table 1. Examples of labelling yields for immunoglobulins and avidins.
Labelling was performed according to recommended conditions with 0.5 mg of proteins.
The kinetics of the conjugation reaction depend upon the pH and temperature used
(Figure 3). When higher labelling yields are desired, the labelling reaction can be extended
to 2-3 days at room temperature, or higher temperatures (up to +37°C) may be used.
Similarly, higher incorporation rates are achieved at higher pH’s (up to 9.8). On the other
hand, if a lower labelling yield is desired a lower pH, lower temperature or shorter reaction
time is recommended (Figure 3). The labelling yield is not dependent on the protein
amount up to 1 mg, thereafter increasing protein amounts give a slightly decreasing
labelling yield (Figure 4).
Figure 3a
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Figure 3b
Figure 35: Effect of temperature on labelling kinetics at different pHs. One mg of IgG was
labelled in 500 µL buffer at indicated temperatures at pH 8.5 (Figure 3a) or pH 9.8 (Figure
Figure 46: The labelling yields obtained when various amounts of bovine gammaglobulin
are labelled with the 0.2 mg Labelling reagent. Labelling was performed according to the
kit insert.
Study performed at Wallac Oy, Turku, Finland.
as above
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A thorough understanding of this kit insert is necessary for succesful use of the SmLabelling kit. The procedure described in this insert is intended for the labelling of an
'average IgG' to a final labelling yield of 5 - 15 Sm3+/IgG. It must be taken into
consideration that individual antibodies (e.g. monoclonal antibodies) may behave
differently with respect to reactivity (labelling yield), and dependence on pH,
temperature and reaction times. When working with monoclonal antibodies with
unknown characteristics, it may be worth labelling them initially at a low pH (e.g. at
8.3) to a relatively low Sm3+/IgG level (2-5 Sm3+/IgG).
For the labelling, do not use buffers which contain free amines or bacteriostatic
agents (e.g. NaN3 interferes with the reaction). Buffers containing even trace
amounts of primary amines (e.g. Tris or glycine) or secondary amines (HEPES,
MOPS, BICINE etc.) cannot be used.
Do not store labelled proteins in Assay Buffer (prod. nos. 1244-106 and 1244111) or phosphate buffer. If during storage of labelled antibodies the background
level of the assay tends to increase due to aggregation formation, the labelled
antibodies should be filtered through a 0.2 µm membrane.
Free Sm3+ and Eu3+ contaminate the gel filtration column material. Therefore
separate columns should be used for purification of Sm- and Eu-labelled proteins.
Columns should be decontaminated between purifications by washing the column
with decontamination buffer (use a volume of approximately 1/3 of the volume of the
column). Re-equilibrate the column carefully with elution buffer before adding
sample, since the decontamination buffer contains DTPA.
To avoid Eu3+ and Sm3+ contaminations which can result in a high fluorescence
background in assays, high standard pipetting and washing techniques are required.
Avoid contaminating pipettes with Eu3+ or Sm3+ reagents or labelled proteins.
Pre-treatment of proteins to be labelled (buffer exchange):
It is recommended that the buffer be changed to the labelling buffer prior to labelling
in order to avoid possible interferences and additional pH adjustment. PD-10 or
NAP™-10 columns (Pharmacia Biotechnology) are recommended. When small
amounts or diluted solutions of proteins are to be labelled, concentration with an
appropriate method may be necessary, or buffer exchange may, alternatively, be
done e.g. by dialysis.
1.1. Equilibrate the PD-10 column with 25 mL of labelling buffer.
1.2. Add the protein solution to the column and rinse with labelling buffer (do not exceed a
total volume of 2.5 mL).
1.3. Collect the protein fraction after the 2.5 mL void volume. Because the labelling
procedure is for 500 µL volume of pre-treated protein, it may be worth collecting the
protein in as small a volume as possible. The maximum volume of protein for buffer
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exchange (2.5 mL for PD-10) produces 3.5 mL of eluted protein solution. (Respective
volumes for NAP™-10 columns are 1 and 1.5 mL). Smaller protein volumes can be
eluted with smaller buffer volumes provided that the protein elution is monitored e.g.
by absorption measurement.
2.1. Open the Sm-Labelling reagent vial carefully.
2.2. Add 500 µL of the protein in the labelling buffer (max. ~1 mg) to the reagent vial.
2.3. Mix gently to dissolve the reagent and incubate overnight at room temperature (1418h, +20 - +25°C).
3.1. Equilibrate a chromatography column with 3 x void volume of elution buffer. If the
column has been used earlier with Eu-labelled proteins, it is recommended to wash it
with decontamination buffer prior to the equilibration. Add the reaction mixture to the
re-equilibrated column and rinse the labelling vial with a small volume of elution
buffer and elute with the same buffer.
3.2. Monitor the eluate by UV-absorbance at 280 nm, collect fractions of 1 - 2 mL.
3.3. Measure Sm3+ concentrations in the fractions by measuring their fluorescence after
appropriate dilution with Enhancement Solution (1:1000 - 1:10 000, e.g. by serial
dilution of 10 µL with 990 µL Enhancement Solution twice). Use a clean (rinsed with
Enhancement Solution) plastic pipette tip to dispense 200 µL of diluted samples into
the microtiterstrip wells. Mix gently using a plateshaker for about 5 minutes before
measuring with the time-resolved fluorometer.
3.4. Pool fractions containing the labelled proteins (Figure 5). Avoid pooling aggregated
Figure 5: The elution profile of labelled IgG from a column of Sephadex G-50 and
Sepharose 6B. The fractions recommended to be pooled (monomeric IgG) are indicated
with arrows.
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Characterization of the labelled proteins:
4.1. Sm3+ Content: The exact concentration of Sm3+ after dilution with Enhancement
Solution (1:10 00 - 1:10 000), is calculated by measuring the fluorescence in
microtiterstrip wells (200 µL/well; in duplicates) and comparing to the fluorescence of
10 nmol/L Sm-standard (stock standard diluted 1:100 in Enhancement Solution).
4.2. Protein Content: Protein concentration in the pooled fractions can be measured with
appropriate methods, e.g. Lowry’s method, or it can be calculated from the protein
absorbance at 280 nm after subtracting the absorbance of the formed aromatic
thiourea-bonds (0.008/µmol/L).
4.3. Eu-Contamination: The original Eu-contamination level of Sm-Labelling reagent is
below 0.02%. A relatively small additional amount of Eu-contamination can cause a
signal in the Eu-channel in dual label assays. Possible contamination of the labelled
protein can be checked by measuring the fluorescence in both Sm- and Eu-channels.
The spillover can be calculated from these signals and is used for correction of the
4.4 Calculations: For IgG the following equations can be used. They are valid when the
labelling yield is < 20 Sm3+/IgG. 1.34 is used for absorptivity value (for 1 mg/mL) of
IgG, and 160 000 for MW.
Sm3+ (µmol/L) =
Sm-counts x dilution factor
100 x counts of 10 nmol/L Sm3+
Protein (mg/mL) =
Abs(280) - 0.008 x Sm3+(µmol/L)
Protein (µmol/L) =
Protein (mg/mL) x 1 000 000
160 000 (g/L)
Yield (Sm3+/IgG) =
Sm3+ (µmol/L)
Protein (µmol/L)
Recovery (%) =
100 x Protein (mg/mL) x volume of pooled fractions (mL)
Protein added (mg)
5.1. To increase the stability of labelled proteins, the Stabilizer (purified BSA) can be used
as a carrier protein at a final concentration of 0.1 %. Store labelled proteins at +4°C.
Repeated freezing and thawing should be avoided.
In cases where bovine albumin cannot be used as a carrier protein, the labelled
proteins could be stored as such, if the protein concentration is over 50 µg/mL. If
other proteins are used as carriers, these need to be purified from any heavy
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metal contaminations prior to addition. The carrier used also needs to be free from
chelating agents.
Sm-labelled immunoreagents can be applied in different types of immunoassays based on
solid-phase separation (e.g. competitive or non-competitive assays). The design of an
immunoassay depends on the analyte, the antibodies, the possibility of using a sandwichtype assay or the need to employ a competitive assay-design, the required sensitivity and
dynamic range etc.
As a general rule, about 25 - 100 ng of labelled antibodies per well is enough for noncompetitive sandwich-type assays, but the actual optimal level depends on the purity and
affinity of the antibodies and the desired signal levels. For competitive assays no general
rules can be given and the assay always has to be separately optimized.
DELFIA Assay Buffer (prod. nos. 1244-106 and 1244-111) is optimal for most assays. In
some assays, additional components might be needed to overcome cross-reactivity
Sm-labelled immunoreagents are suitable for use in dual-label assays together with Eu3+
as the second label because the same DELFIA Enhancement Solution is optimal for their
measurement. Eu3+ gives higher fluorescence (QEu = 70 %), and is recommended for use
in assays requiring higher sensitivity. Additionally, assays should be optimized to give
relatively high signal levels of Sm3+ in order to compensate for the lower fluorescence
intensity of Sm3+-chelates (QSm = 2 %).
The minor spillover from Eu-emission to the Sm-channel can be corrected, where
necessary, automatically or manually. If the Sm-standard dilution gives a signal in the
normal Eu-counting channel, it implies Eu-contamination in reagents. Calculated
theoretical signal spillover is about 0.01 %, which corresponds to a Eu-contamination level
of 2 ppm in the Sm-reagent. Generally Eu-contamination in Sm-Labelling reagents is about
100 ppm, but may be increased during labelling and purification. In double-label assays
this contamination spillover may be subtracted by using a correction factor measured for
each particular labelled protein (see 4.3).
The performance data presented here is obtained using the labelling procedure indicated
and antibody solutions without interfering compounds. In the indicated conditions the
labelling reagent is able to react with available free aminogroups of proteins. Change of
buffers or variations in protein characteristics can cause alterations in the labelling
The reagent is covered by patents on both the chemical structure (6) and use in DELFIAtype assays (7). Purchase of this reagent gives the purchaser the right to use this material
in his own research. Further distribution of this reagent or products resulting from its use is
expressly prohibited. Purchase of this product implies agreement with these conditions of
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1. Soini, E. and Kojola H. (1983): Time-resolved fluorometer for lanthanide chelates a new generation of non-isotopic immunoassays. Clin. Chem. 29, 65-68.
2. Hemmilä, I., Dakubu, S., Mukkala, V.-M., Siitari, H. and Lövgren, T. (1984): Europium
as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335-343.
3. Hemmilä, I. (1988): Lanthanides as probes for
immunoassays. Scand. J. Clin. Lab. Invest. 48, 389-400.
6. Mukkala, V.-M., Mikola, H. and Hemmilä, I. (1989): The synthesis and use of activated
N-benzyl derivatives of diethylenetriaminetetraacetic acid: Alternative reagents for
labelling of antibodies with metal ions. Anal. Biochem. 176, 319-325.
4. Saarma, M., Järvekülg, L., Hemmilä, I., Siitari, H. and Sinijärv, R. (1989):
Simultaneous quantification of two plant viruses by double-label time-resolved
immunofluorometric assay. J. Virol. Methods 23, 47-54.
5. Mikola, H., Mukkala, V.-M. and Hemmilä, I. (1987): Eur. Patent No. 139,675.
6. Mikola, H., Mukkala, V.-M. and Hemmilä, I. (1989): US Patent No. 4,808,541.
Last revision May 2000
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Tel. +358-2-2678 111
Fax +358-2-2678 357