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Symmetric Dimethylarginine (SDMA), Elisa Kit
Quantitative determination of endogenous symmetric dimethyl arginine (SDMA) in serum or EDTA plasma of dogs and cats.
Article number:
EA203/96
Content:
Units
Selling unit:
Kit
This ELISA kit contains reagents for the quantitative determination of derivatized SDMA in plasma and serum of dogs or cats.
Specifications
Sample: plasma or serum from dog or cat
Content: 96 Wells (8*12)
Assay range: Dogs: 0.30 – 0.65 µmol/l (6.0 – 13 µg/dl), Cats: 0.30 – 0.75 µmol/l (6.0 – 15 µg/dl).
Sensitivity: 0,03 µmol/l (0,6 µg/dl)
Test type: competitive enzyme immunoassay
Storage at 2–8 °C
sdmavet-e_15.docx 2025-01-10
Instructions for Use
SDMA vet ELISA
Enzymimmunoassay for the
Quantitative Determination of
Endogenous Symmetric Dimethylarginine (SDMA)
in Serum or Plasma of Dogs and Cats
For Veterinary Diagnostic Only
REF
EA203/96
12 x 8
2 – 8 °C
DLD Gesellschaft für Diagnostika und medizinische Geräte mbH
Adlerhorst 15
22459 Hamburg
Telefon: 040/ 555 87 10
Fax: 040/ 555 87 111
Internet: http://www.dld-diagnostika.de
E-Mail: contact@dld-diagnostika.de
SDMA vet ELISA
Page 3
Table of Contents
1 Introduction and Principle of the Test .......................................................... 5
2 Precautions .................................................................................................. 6
3 Storage and Stability .................................................................................... 6
4 Contents of the Kit ....................................................................................... 7
5 Sample Collection ......................................................................................... 9
6 Preparation of Reagents and Samples .......................................................... 9
7 Test Procedure ELISA .................................................................................. 11
8 Calculation of the Results ........................................................................... 12
9 Assay Characteristics .................................................................................. 13
10 Literature ................................................................................................... 19
11 Changes to declare ..................................................................................... 19
Pipetting Scheme - Sample Preparation ........................................................... 20
Pipetting Scheme - ELISA .................................................................................. 20
Symbols
CONT
Content
Expiry Date
Lot Number
Store at
Manufactured by
Sufficient for ... determinations
Catalogue Number
Consult Instructions for Use
Hazard Pictograms
Warning
Danger
SDMA vet ELISA
Page 5
1 Introduction and Principle of the Test
Symmetric dimethylarginine (SDMA) is a methylated arginine amino acid.
SDMA is derived from intranuclear methylation of L-arginine residues and is
released into the cytoplasm after proteolysis. SDMA is excreted by the
kidneys.
Several studies have found that 1 in 3 cats and 1 in 10 dogs are likely to
develop a kidney disease during their lifetime.
SDMA is an early biomarker of kidney function. It correlates well with
glomerular filtration rate (GFR). On average, SDMA increases in chronic kidney
disease (CKD) with 30 to 40% loss of kidney function. Creatinine, however,
does not increase until 75% of kidney function is lost. SDMA will enable
veterinarians to diagnose chronic kidney disease (CKD) much earlier than
Creatinine or Cystatin C tests.
SDMA is specific for kidney function. It is not impacted by other diseases such
as liver disease, cardiovascular disease, inflammatory disease and endocrine
diseases. Another exciting feature of SDMA is that it is not impacted by
muscle mass either, which simplifies diagnosing and monitoring CKD in thin
geriatric animals, especially cats and animals with other diseases that cause
muscle wasting.
The competitive SDMA-ELISA uses the microtiter plate format. SDMA is bound
to the solid phase of the microtiter plate. SDMA in the samples is acylated and
competes with solid phase bound SDMA for a fixed number of rabbit anti-
SDMA antiserum binding sites. When the system is in equilibrium, free
antigen and free antigen-antiserum complexes are removed by washing. The
antibody bound to the solid phase SDMA is detected by anti-rabbit /
peroxidase. The substrate TMB / peroxidase reaction is monitored at 450 nm.
The amount of antibody bound to the solid phase SDMA is inversely
proportional to the SDMA concentration of the sample.
SDMA vet ELISA
Page 6
2 Precautions
For veterinary diagnostic only. For professional use only.
Before carrying out the test, the valid instructions for use, as included in
this kit, should be read completely and the content understood.
Material of animal origin used in the preparation of the kit has been
obtained from animals certified as healthy but these materials should be
handled as potentially infectious.
Individual components of different lots and test kits should not be
interchanged. The expiry dates and storage conditions stated on the
packaging and the labels of the individual components must be
observed.
When handling the reagents, controls and samples, the current
laboratory safety guidelines and good laboratory practice should be
observed.
Wear protective clothing, disposable gloves, and safety goggles while
performing the test.
Some of the components of this test kit contain hazardous substances.
These components bear the appropriate hazard symbol on their label.
Further information can be found in 4. Contents of the kit and on the
relevant safety data sheets.
Avoid any actions that could result in ingestion, inhalation or injection of
the reagents. Never pipette by mouth.
Avoid contact with individual reagents.
Dispose of waste according to state and local environmental protection
regulations.
Broken glass can cause injury. Be cautious with glass vials.
3 Storage and Stability
The kit is shipped at ambient temperature. Upon arrival, store the kit at
2 - 8 °C to keep it stable until its expiry date. Once opened the kit is stable
until its expiry date. The shelf life of the ready-to-use reagents is indicated on
the respective bottle label. For stability of prepared reagents refer to 6.
Reagents must equilibrate to room temperature before use and be
refrigerated immediately after use.
SDMA vet ELISA
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4 Contents of the Kit
MT-Strips
STRIPS
12 strips
8 wells each, break apart, precoated with SDMA
Standards (1 - 6)
CAL 1 - 6
6 vials
each 4 ml, ready for use
Standard
1
2
3
4
5
6
µmol/l
0
0.2
0.4
0.7
1.2
3
ng/ml
0
40
81
141
242
606
Control 1 & 2
CON 1 & 2
2 vials
each 4 ml ready for use, Range: see QC certificate
Acylation Reagent
ACYL-REAG
3 vials
lyoph., dissolve contents in 3 ml Solvent before use
Acylation Buffer
ACYL-BUFF
1 vial
3.5 ml, ready for use, blue coloured
Warning
Solvent
SOLVENT
1 vial
10 ml ready for use,
contains DMSO,
Please note that Solvent reacts with many plastic
materials including plastic trays; Solvent does not react
with normal pipette tips and with glass devices
Warning
Danger
Antiserum
AS
1 vial
7 ml, ready for use, Rabbit-anti-N-acyl-
SDMA, yellow coloured
Warning
Enzyme Conjugate
CONJ
1 vial
13 ml, ready for use,
Goat-anti-rabbit-IgG-peroxidase
Warning
SDMA vet ELISA
Page 8
Wash Buffer
WASH
1 vial
20 ml, conc. (50x), Dilute with dist. Water to 1000 ml total volume
Substrate
SUB
1 vial
13 ml TMB Solution, ready for use
Stop Solution
STOP
1 vial
13 ml, ready for use, contains 0.3M sulphuric acid, not corrosive
Reaction Plate
ACYL-PLATE
1 piece
For acylation
Equalizing Reagent
EQUA-REAG
1 vial
lyoph., dissolve contents with 21 ml dist. water,
dissolve carefully to minimize foam formation
Foil
FOIL
2 pieces
Ready for use
Additional materials and equipment required but not provided:
Pipettes (20, 50, 100 and 200 µl)
Multipette
Orbital shaker
Microplate washing device
Microplate photometer (450 nm)
Vortex mixer, roll mixer
Paper towels, pipette tips, timer
SDMA vet ELISA
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5 Sample Collection
Serum and Plasma
The test can be performed with serum as well as with EDTA plasma.
Hemolytic and lipemic samples should not be used.
The samples can be stored up to 6 hours at 2 – 8 °C. For a longer storage (up
to 18 months) the samples must be kept frozen at -20 °C
Repeated freezing and thawing should be avoided.
6 Preparation of Reagents and Samples
Microtiter strips
Before opening the packet of strip wells STRIPS , allow it to stand at room
temperature for at least 10 minutes. After opening, place any unused wells in
the original foil packet with the desiccant provided. Reseal carefully and store
at 2 – 8 °C.
Wash Buffer
Dilute the contents of WASH with dist. water to a total volume of 1000 ml,
mix shortly. The diluted wash buffer must be stored at 2 – 8 C and is stable
for 4 weeks. Should the kit be used in several runs, then prepare only the
required amount of wash buffer for each run.
Equalizing Reagent
Dissolve the contents of EQUA-REAG with 21 ml dist. water, mix shortly and
leave on a roll mixer for 20 minutes. Avoid excess formation of foam. The
reconstituted Equalizing Reagent should be stored frozen at -20 °C and is
stable until expiry date.
Acylation Reagent
Dissolve the contents of one bottle ACYL-REAG with 3 ml Solvent SOLVENT
and shake for 10 minutes on an orbital shaker. After use the reagent has to be
discarded. The Acylation Reagent has always to be prepared immediately
before use and is stable for minimum 3 hours. The two other bottles allow a
SDMA vet ELISA
Page 10
second and third run of the test. If the whole kit is to be used in one run, it is
recommended to pool the dissolved contents of the two vials of Acylation
Reagent.
Please note that Solvent reacts with many plastic materials including plastic
trays which are used as reservoir for multichannel pipettes. Solvent does not
react with normal pipette tips and with glass devices. It is recommended to
use a multipette, fill it directly from the vial and add the Acylation Reagent to
the wells.
All other reagents are ready for use.
Preparation of Samples (Acylation)
Bring all reagents to room temperature and mix them carefully, avoid
development of foam. The wells of the reaction plate ACYL-PLATE for the
acylation can be used only once. Please mark the respective wells before use
to avoid repeated use.
1. Pipette each 20 µl standard 1 - 6 CAL 1 – 6 , each 20 µl control 1 & 2
CON 1 & 2 and each 20 µl patient sample into the respective wells of the
Reaction Plate ACYL-PLATE (duplicates are recommended).
2. Pipette 20 µl Acylation Buffer ACYL-BUFF into each well.
3. Pipette 200 µl reconstituted Equalizing Reagent EQUA-REAG (see 6.3) into
each well.
4. Mix the reaction plate for 10 seconds on an orbital shaker.
5. Pipette 50 µl of freshly prepared Acylation Reagent ACYL-REAG (see 6.4)
into each well, mix immediately.
It is recommended to use a multipette, fill it directly from the vial and add
the Acylation Reagent to each well.
Colour changes to violet.
6. Incubate for 20 minutes at room temperature (approx. 20 °C) on an
orbital shaker. Do not cover wells or plate, leave the plate open on the
shaker.
Take each 20 µl of the acylated samples for the SDMA-ELISA.
SDMA vet ELISA
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7 Test Procedure ELISA
Bring all reagents to room temperature and mix them carefully, avoid
development of foam.
Sample Incubation
Transfer each 20 µl acylated Standards 1 to 6, 20 µl acylated controls and
20 µl acylated samples from the Reaction Plate into the respective wells of the
coated microtiter strips STRIPS .
Pipette 50 µl Antiserum AS into each well.
Cover the plate with adhesive foil FOIL and incubate Microtiter Strips for
90 minutes at room temperature (20 – 25 °C) on an orbital shaker.
Washing
Discard or aspirate the contents of the wells and wash thoroughly with each
300 µl diluted Wash Buffer WASH (see 6.2). Repeat the washing procedure
4 times. Remove residual liquid by tapping the inverted plate on clean
absorbent paper.
Conjugate Incubation
Pipette 100 µl enzyme conjugate CONJ into each well.
Incubate for 30 minutes at room temperature on an orbital shaker.
Washing
Repeat step 7.2.
Substrate Incubation
Pipette 100 µl Substrate SUB into each well and incubate for 25 ± 5 minutes
at room temperature on an orbital shaker.
Stopping
Pipette 100 µl Stop Solution STOP into each well and mix on an orbital shaker
for approx. 30 seconds.
Reading
Within 15 minutes, read the optical density at 450 nm (reference wavelength
between 570 and 650 nm) in a microplate photometer.
SDMA vet ELISA
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8 Calculation of the Results
Standard
1
2
3
4
5
6
µmol/l
0
0.2
0.4
0.7
1.2
3
ng/ml
0
40
81
141
242
606
Conversion factor: 1 µmol/l = 202 ng/ml = 20.2 µg/dl
The concentration of the standards (x-axis, logarithmic) are plotted against
their corresponding optical density (y-axis, linear). Cubic spline, 4 parameter
or similar iteration procedures are recommended for evaluation of the
standard curve.
The concentration of the controls and samples can be read directly from this
standard curve by using their average optical density.
Typical standard curve:
SDMA conc. (µmol / l)
0,1 1 10
0,21
0,41
0,61
0,81
1,01
1,21
1,41
SDMA ELISA
y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2
Std (Standards: Concentration vs MeanValue) 1,799 0,772 1,157 -0,104 1
Quality Control: Test results are valid only if the kit controls are within the
ranges specified on the QC Certificate. Otherwise, the test should be
repeated.
SDMA vet ELISA
Page 13
9 Assay Characteristics
Expected Values (Serum, EDTA-Plasma)
Dogs: 0.30 – 0.65 µmol/l (6.0 – 13 µg/dl)
Cats: 0.30 – 0.75 µmol/l (6.0 – 15 µg/dl)
The reference ranges given above should only be taken as a guideline. It is
recommended that each laboratory should establish its own reference values.
Sensitivity
0.03 µmol/l (0.6 µg/dl)
Recovery
9.3.1 Recovery Cat
Increasing amounts of SDMA were added to a cat serum sample. Each spiked
sample was assayed. The analytical recovery of SDMA was estimated at 10
different concentrations by using the theoretically expected and the actually
measured values. The mean recovery from all concentrations was 101%.
added
[µmol/l]
measured
[µmol/l]
expected
[µmol/l]
% recovery
0.00
0.58
0.12
0.68
0.70
97
0.24
0.77
0.82
91
0.35
0.86
0.93
92
0.45
0.91
1.03
88
0.55
0.99
1.13
88
0.65
1.24
1.23
101
0.77
1.71
1.35
127
1.04
1.90
1.62
117
1.35
1.95
1.93
101
1.65
2.29
2.23
103
mean value
101
SDMA vet ELISA
Page 14
9.3.2 Recovery Dog
Increasing amounts of SDMA were added to a dog serum sample. Each spiked
sample was assayed. The analytical recovery of SDMA was estimated at 10
different concentrations by using the theoretically expected and the actually
measured values. The mean recovery from all concentrations was 104 %.
added
[µmol/l]
measured
[µmol/l]
expected
[µmol/l]
% recovery
0.00
0.54
0.12
0.74
0.66
112
0.24
0.74
0.78
95
0.35
0.86
0.89
97
0.45
0.94
0.99
95
0.55
1.01
1.09
93
0.65
1.19
1.18
101
0.77
1.51
1.31
115
1.04
1.73
1.58
109
1.35
2.19
1.89
116
1.65
2.23
2.19
102
mean value
104
SDMA vet ELISA
Page 15
Linearity
9.4.1 Linearity Cat
The linearity of the ELISA method was investigated using seven different
dilutions of a cat serum sample. The mean linearity from all dilutions was 96%.
dilution
measured
[µmol/l]
recalculated
value [µmol/l]
recovery %
orig.
2.09
4 + 1
1.73
2.16
103
2 + 1
1.30
1.95
93
1 + 1
0.95
1.90
91
1 + 2
0.59
1.77
85
1 + 3
0.52
2.08
100
1 + 5
0.33
1.98
95
1 + 7
0.28
2.24
107
mean recovery
96
9.4.2 Linearity Dog
The linearity of the ELISA method was investigated using seven different
dilutions of a dog serum sample. The mean linearity from all dilutions was
92%.
dilution
measured
[µmol/l]
recalculated
value [µmol/l]
recovery %
orig.
1.88
4 + 1
1.30
1.63
87
2 + 1
1.21
1.82
97
1 + 1
0.92
1.84
98
1 + 2
0.59
1.77
94
1 + 3
0.43
1.72
91
1 + 5
0.28
1.68
89
1 + 7
0.20
1.60
85
mean recovery
92
SDMA vet ELISA
Page 16
Specificity (Cross Reactivity)
Structural related components were tested for possible interference with the
antisera against SDMA used in the ELISA method. The tested compounds were
Arginine, Homoarginine, Monomethylarginine (NMMA) and ADMA.
Substance
ED-50-Value (µmol/l)
Cross Reactivity (%)
SDMA
1.39
100
ADMA
84
1.2
NMMA
182
0.76
Homoarginine
2807
0.05
Arginine
8574
0.016
Reproducibility
The reproducibility of the ELISA method was investigated by determining the
intra- and inter-assay-coefficient of variation (cv) by repeated measurements
of different serum samples with different SDMA concentrations
(concentrations in µmol/l):
9.6.1 Intra-Assay Cat
sample
n =
mean value
sd
cv (%)
K1
40
0.837
0.064
7.6
K2
40
0.862
0.049
5.7
K3
40
0.770
0.058
7.7
9.6.2 Intra-Assay Dog
sample
n =
mean value
sd
cv (%)
H1
40
0.531
0.061
11.5
H2
40
0.804
0.068
8.5
H3
40
0.776
0.046
5.9
SDMA vet ELISA
Page 17
9.6.3 Inter-Assay Cat
sample
n =
mean value
sd
cv (%)
K1
32
0.49
0.037
7.5
K2
32
0.59
0.043
7.3
K3
32
0.72
0.079
11.0
K4
32
0.74
0.058
7.8
K5
32
0.84
0.068
8.1
K6
32
0.84
0.982
9.7
9.6.4 Inter-Assay Dog
sample
n =
mean value
sd
cv (%)
H1
32
0.49
0.042
8.5
H2
32
0.66
0.046
7.0
H3
32
0.76
0.063
8.2
H4
32
0.93
0.069
7.4
H5
32
1.21
0.128
10.6
H6
32
1.35
0.152
11.3
SDMA vet ELISA
Page 18
Correlation ELISA to LC-MS/MS
Correlation ELISA to the LC-MS/MS method
SDMA vet ELISA
Page 19
10 Literature
Josipa Kuleš, Petra Bilić, Blanka Beer Ljubić, Jelena Gotić, Martina Crnogaj,
Mirna Brkljačić, Vladimir Mrljak
Glomerular and tubular kidney damage markers in canine babesiosis
caused by Babesia canis
Ticks and Tick-borne Diseases (2018) 9 1508 - 1517
Bode-Böger S.M., Scalera F., Kielstein J.T., Martens-Lobenhoffer J.,
Breithardt G., Fobker M., Reinecke H.
Symmetrical Dimethylarginine: A new combined parameter for renal
function and extent of coronary artery disease
J. Am. Soc. Nephrol. (2006) 17: 1128-1134
Kielstein J.T., Salpeter S.R.; Bode-Böger S.M., Cooke J.P., Fliser D.
Symmetric dimethylarginine (SDMA) as endogenous marker of renal
function – a meta-analysis
Nephrol. Dial. Transplant (2006) 21: 2446 – 2451
11 Changes to declare
Version _15: Changes are highlighted in gray.
Version _14: Changes in section 4 are highlighted in gray.
Version _13: Additions/Changes are highlighted in gray.
Version _12: Hazard symbol “Warning” was removed from POD Conjugate in
section 4, as no longer required. Grey highlighting as in version _11 has been
removed.
Version _11: IFU has been re-formatted. Precautions and Calculation of the
Results have been complemented (highlighted in grey). Component names as
printed on labels were included in pipetting schemes to provide greater
clarity. No changes have been made to components or execution of protocols.
SDMA vet ELISA
Page 20
Pipetting Scheme - Sample Preparation
Standards
Controls
Samples
ACYL-PLATE:
CAL 1 - 6
µl
20
CON 1 & 2
µl
20
Sample
µl
20
ACYL-BUFF
µl
20
20
20
recon. EQUA-REAG
µl
200
200
200
Shake for 10 seconds
recon. ACYL-REAG
(fresh)
µl
50
50
50
Incubate for 20 minutes at room temperature on an orbital shaker
Take 20 µl each for ELISA
Pipetting Scheme - ELISA
Acyl.
Standards
Acyl.
Controls
Acyl.
Samples
STRIPS:
Transfer from
ACYL-PLATE into STRIPS
µl
20
20
20
AS
µl
50
50
50
Cover frame with FOIL and incubate on an orbital shaker
for 90 minutes at room temperature
Wash 4 x with 300 µl WASH per well
CONJ
µl
100
100
100
Incubate for 30 minutes at room temperature on an orbital shaker
Wash 4 x with 300 µl WASH per well
SUB
µl
100
100
100
Incubate for 25 ± 5 minutes at room temperature on an orbital shaker
STOP
µl
100
100
100
Shake for 30 seconds
Within 15 minutes, read absorbance at 450 nm (ref. 570 – 650 nm)
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