Instructions for use

Glycine ELISABA E-2100

Glycine Urine ELISA
1.

Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of Glycine in urine.
After derivatization Glycine is quantitatively determined by ELISA.
The competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phase of themicrotiter plate. The derivatized standards, controls and samples and the solid phase bound analytecompete for a fixed number of 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 isdetected by an anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The reaction is monitored
at 450 nm.
Quantification of unknown samples is achieved by comparing their absorbance with a reference curve
prepared with known standards.

2.

Precautions, Guidelines and Warnings
This kit is intended for professional use only. Users should have a thorough understanding of this protocol
for the successful use of this kit. Only the test instruction provided with the kit is valid and has to be used
to run the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
Reagents of this kit which contain human serum or plasma have been tested and confirmed negative for
HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as
potential biohazards in use and for disposal.
The principles of Good Laboratory Practice (GLP) have to be followed.
In order to reduce exposure to potentially harmful substances, wear lab coats, disposable latex glovesand protective glasses where necessary.
All kit reagents and specimens should be brought to room temperature and mixed gently but thoroughly
before use. Avoid repeated freezing and thawing of reagents and specimens.
For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
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.
Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
Once the test has been started, all steps should be completed without interruption. Make sure that therequired reagents, materials and devices are prepared ready at the appropriate time.
Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,sample, standard and control.
A calibrator curve must be established for each run.
The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report.
Do not mix kit components with different lot numbers within a test and do not use reagents beyond
expiry date as shown on the kit labels.
Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In caseof contact with eyes or skin, rinse off immediately with water.
Some reagents contain sodium azide (NaN3) as preservatives. In case of contact with eyes or skin, rinse
off immediately with water. NaN3 may react with lead and copper plumbing to form explosive metalazides. When disposing reagents, flush with a large volume of water to avoid azide build-up.
TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them.
For information on hazardous substances included in the kit please refer to Material Safety Data Sheets(MSDS). The Material Safety Data Sheet for this product is made available directly on the website of themanufacturer or upon request.
Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.

3.

Storage and stability
Store the reagents at 2 - 8 °C until expiration date. Do not use components beyond the expiration dateindicated on the kit labels. Do not mix various lots of any kit component within an individual assay.

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4.

Materials

4.1

Contents of the kit
SymbolReagent

ContentColour CodeBA D-0024

Reaction Plate

1 x 96 wells

BA D-0090

Adhesive Foil

2x4

BA E-0030

1 x 20 ml light purple

concentrated

BA E-0040

Wash Buffer
Concentrate
Enzyme Conjugate

1 x 12 ml red

ready for use, anti-rabbit
IgG conjugated withperoxidase

BA E-0055

Substrate

1 x 12 ml black

ready for use, containing asolution of TMB

BA E-0080

Stop Solution

1 x 12 ml light grey

ready for use

BA E-2413

Assay Buffer

1 x 20 ml yellow

ready for use

BA E-2428

Equalizing Reagent

BA E-2446

D-Reagent

1 x 4 mlready for use

BA E-2101

Standard A

1 x 4 ml white

ready for use

BA E-2102

Standard B

1 x 4 ml light yellow

ready for use

BA E-2103

Standard C

1 x 4 ml orange

ready for use

BA E-2104

Standard D

1 x 4 ml dark blue

ready for use

1 x brown

ready for use
ready for use

lyophilized

BA E-2105

Standard E

1 x 4 ml light grey

ready for use

BA E-2106

Standard F

1 x 4 ml black

ready for use

BA E-2110

Glycine Antiserum1 x 6 ml blue

ready for use, from rabbit,
blue coloured

BA E-2131

Glycine Microtiter
StripsBA E-2129

1 x 1 ml pink

BA E-2151

Reducing
Concentrate
Control 1

12 strips, 8 wells each,break apart, pre-coatedconcentrated

1 x 4 ml light green

ready for use

BA E-2152

Control 2

1 x 4 ml dark red

ready for use

4.2

Additional materials and equipment required but not provided with the kit-5.

1 x 96 wells

Calibrated precision pipettes to dispense volumes between 20 µl – 300 µl; 2,5 ml; 12,5 ml
Polystyrene or polypropylene tubes and suitable rack
Microtiter plate washing device (manual, semi-automated or automated)
ELISA reader capable of reading absorbance at 450 nm (reference wavelength 620 - 650 nm)
Shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Water (deionized, distilled, or ultra-pure)
Vortex mixer

Sample collection and storage
Urine
Spontaneous or 24-hour urine, collected in a bottle containing 10 - 15 ml of 6 M HCl, should be used.
Storage: up to 48 hours at 2 - 8 °C or for a longer period (up to 6 months) at -20 °C.
More than 3 freeze-thaw cycles should be avoided.

6.

Test procedure
Allow all reagents to reach room temperature and mix thoroughly by gentle inversion before use.
Duplicate determinations are recommended.

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6.1Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volumeof 1000 ml.
Storage: up to 6 months 2 - 8 °C.
Equalizing Reagent
Reconstitute the Equalizing Reagent with 12.5 ml of Assay Buffer.
Reconstituted Equalizing Reagent which is not used immediately has to be stored in aliquotes at -20 °C
and may be thawed only once.
Reducing Solution
Dilute Reducing Concentrate 1:100 with water (deionized, distilled, or ultra-pure) and mix thoroughly.
Use immediately!

Examples for the preparation of Reducing Solution:
Reducing Concentrate
Water
6.2

40 µl
3,96 ml

50 µl
4,95 ml

80 µl
7,92 ml

160 µl
15,84 ml

Dilution1.

Pipette 20 µl of standards, controls and samples into the respective tubes.

2.

Add 2,5 ml of water (deionized, distilled, or ultra-pure) to all tubes and mix thoroughly (vortex).
Take 100 µl for the derivatization.

6.3

Derivatization

1.

Pipette 100 µl of the diluted standards, controls and samples into the appropriate wells of theReaction Plate.

2.

Add 50 µl of the Equalizing Reagent to all wells.

4.

Add 10 µl of the D-Reagent to all wells.

5.

Cover plate with Adhesive Foil and shake for 2 h at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

6.

Pipette 150 µl Reducing Solution (refer to 6.1) into all wells.
The Reducing Solution should be prepared directly prior to use!

7.

Shake for 30 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).
Take 25 µl for the ELISA!

6.4

Glycine ELISA

1.

Pipette 25 µl of the prepared standards, controls and samples into the appropriate wells of theGlycine Microtiter Strips.

2.

Pipette 50 µl of the Glycine Antiserum into all wells and mix shortly.

3.

Cover plate with Adhesive Foil and incubate for 15 - 20 h (overnight) at 2 - 8 °C.

4.

Remove the foil. Discard or aspirate the contents of the wells. Wash the plate 3 x by adding 300 µl of
Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate on
absorbent material.

5.

Pipette 100 µl of the Enzyme Conjugate into all wells.

6.

Incubate for 30 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

7.

Remove the foil. Discard or aspirate the contents of the wells. Wash the plate 3 x by adding 300 µl of
Wash Buffer, discarding the content and blotting dry each time by tapping the inverted plate onabsorbent material.

8.

Pipette 100 µl of the Substrate into all wells and incubate for 20-30 min at RT (20 - 25 °C) on a
shaker (approx. 600 rpm). Avoid exposure to direct sunlight!

9.

Add 100 µl of the Stop Solution to each well and shake the microtiter plate to ensure ahomogeneous distribution of the solution.
Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to450 nm and a reference wavelength between 620 nm and 650 nm.

10.

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7.

Calculation of results

Concentration of the standards
StandardAB

C

D

E

F

Glycine (µg/ml)

0

1030

100

300

1 000

The calibration curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of
the standards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use non-linear regression for curve fitting (e.g. spline, 4- parameter, akima).
The concentrations of the samples and controls can be read directly from the standard curve.

7.1

Quality control
It is recommended to use control samples according to national regulations. Use controls at both normal
and pathological levels. The kit or other commercial controls should fall within established confidencelimits. The confidence limits of the kit controls are indicated on the QC-Report.

7.2

Calibration
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the extinction values may vary if a thermostat is not used. The higher the temperature,
the higher the extinction values will be. Corresponding variations also apply to the incubation times. The
optimal temperature during the Enzyme Immunoassay is between 20 - 25 °C.
In case of overflow, read the absorbance of the solution in the wells within 10 minutes, using amicroplate reader set to 405 nm

7.3

Typical calibration curve

Example, do not use for calculation!
Glycine
100
90
80
70
OD

60
50
40
30
20
100
1101001000

Gly cine µg/ml

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8.

Assay characteristics

GlycineAnalytical Sensitivity(Limit of Detection)

3,3 µg/ml

Cross Reactivity (%)

Substance

GlycineAnalytical Specificity(Cross Reactivity)

Glycine
D-Serin
L-CysteinBeta-Alanin
GABA
L-Aspartic Acid
L-Glutamat
Taurin100
3,7
1,80,70,8<0,1<0,1<0,1Precision
Intra-Assay

Inter-Assay

SampleRange (µg/ml)

CV (%)

SampleRange (µg/ml)

CV (%)

1 (n = 20)
2 (n = 20)
3 (n = 20)

66,7 ± 4,294,0 ± 3,5217 ± 11,0

6,2
3,75,11 (n = 23)
2 (n = 23)
3 (n = 23)

63,3 ± 7,991,1 ± 9,2
211 ± 9,4

13
109,4Linearity

RecoveryRange Linearity %94 - 116

Serial dilution up to1:128

Mean Linearity %100

Mean Creatinine (mg/dl)
38,8
102
135

Mean Recovery (%)
108
95108

Mean Linearity (%)
96 – 116
93 – 96
105 – 114

Urine

Sample 1
Sample 2
Sample 3

For updated literature, information about clinical significance or any other information pleasecontact your local supplier.
Symbols:

Version: 10.0

Storagetemperature

Manufacturer

Contains sufficient for<n> tests

Expiry dateBatch code

For in-vitro diagnostic
use only!

Consult instructions
for useContentCE labelled

CautionCatalogue
number

For research use
only!

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