Instructions for use

Glutamate ELISABA E-2400R

Glutamate ELISA
1. Introduction
1.1 Intended use and principle of the test
Enzyme Immunoassay for the quantitative determination of L-Glutamate in urine and various biologicalsamples.
After extraction and derivatisation Glutamate 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 analyte concentrations in the standards, controls and samples and the solid
phase bound analyte compete for a fixed number of antibody binding sites. When the system is inequilibrium, free antigen and free antigen-antibody complexes are removed by washing. The antibody boundto the solid phase is detected 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 standard curve
prepared with known standards.
2. Procedural cautions, guidelines, warnings and limitations
2.1 Procedural cautions, guidelines and warnings(1) This kit is intended for research use only. Users should have a thorough understanding of this protocol forthe successful use of this kit. Only the test instruction provided with the kit is valid and has to be used torun the assay. Reliable performance will only be attained by strict and careful adherence to theinstructions provided.
(2) The principles of Good Laboratory Practice (GLP) have to be followed.
(3) In order to reduce exposure to potentially harmful substances, wear lab coats, disposable protective
gloves and protective glasses where necessary.
(4) 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.
(5) For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
(6) The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch with desiccant and used in the frame provided.
(7) Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
(8) 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.
(9) Incubation times do influence the results. All wells should be handled in the same order and timeintervals.
(10) To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent,sample, standard and control.
(11) A standard curve must be established for each run.
(12) The controls should be included in each run and fall within established confidence limits. The confidence
limits are listed in the QC-Report provided with the kit.
(13) 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.
(14) 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.
(15) 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.
(16) For information on hazardous substances included in the kit please refer to Safety Data Sheet (SDS). TheSafety Data Sheet for this product is made available directly on the website of the manufacturer or uponrequest.
(17) Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
(18) In case of any severe damage to the test kit or components, LDN has to be informed in writing, at thelatest, one week after receiving the kit. Severely damaged single components should not be used for atest run. They have to be stored until a final solution has been found. After this, they should be disposed
according to the official regulations
2.2 Limitations
Any inappropriate handling of samples or modification of this test might influence the results.
2.2.1 Interfering substances
Avoid excess of acid: excess of acid might exceed the buffer capacity of the dilution buffer.
A pH of 5.0 during the extraction is mandatory.
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2.2.2 Drug interferences
There are no known substances (drugs, food) which ingestion interferes with the measurement of glutamate
level in the sample.
2.2.3 High-Dose-Hook effectNo hook effect was observed in this test.
3. Storage and stability
Store the unopened reagents at 2 - 8 C until expiration date. Do not use components beyond the expiry
date indicated on the kit labels. Once opened the reagents are stable for 1 month when stored at
2 – 8 °C. Once the resealable pouch has been opened, care should be taken to close it tightly with desiccantagain.
4. Materials
4.1 Contents of the kit
Adhesive Foil - Ready to use

BA D-0090
Contents:

Adhesive Foils in a resealable pouch

Volume:1 x 4 foils

BA D-0024
Contents:
BA E-2442
Contents:
BA E-0030

Reaction Plate - Ready to use
1 x 96 well plate, empty in a resealable pouch
Extraction Plate - Ready to use
2 x 48 well plate, precoated with cation exchanger in a resealable pouch
Wash Buffer Concentrate - Concentrated 50x

Contents:

Buffer with a non-ionic detergent and physiological pH

Volume:1 x 20 ml/vial, light purple cap

BA E-0040

Enzyme Conjugate - Ready to use

Contents:

Goat anti-rabbit immunoglobulins conjugated with peroxidase

Volume:1 x 12 ml/vial, red cap

BA E-0055

Substrate - Ready to use

Contents:

Chromogenic substrate containing tetramethylbenzidine, substrate buffer and hydrogen
peroxide

Volume:1 x 12 ml/black vial, black cap

BA E-0080

Stop Solution - Ready to use

Contents:

0.25 M sulfuric acid

Volume:1 x 12 ml/vial, light grey cap

Hazards
identification:
H290 May be corrosive to metals.
BA E-2431

Glutamate Microtiter Strips - Ready to use

Contents:

1 x 96 well (12x8) antigen precoated microwell plate in a resealable foil pouch withdesiccant

BA E-2410

Glutamate Antiserum - Ready to use

Contents:

Rabbit anti- glutamate antibody, blue coloured

Volume:1 x 6 ml/vial, blue cap

BA E-2413

Assay Buffer - Ready to use

Contents:

Buffer with alkaline pH

Volume:1 x 20 ml/vial, yellow cap

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Equalizing Reagent - Lyophilized

BA E-2428
Contents:

Lyophilized protein

Volume:1 vial, brown cap

Standards and Controls - Ready to use
Cat. no.

Component

BA E-2401

Colour/Cap

Concentrationµg/ml

Concentrationµmol/lVolume/Vial04 mlwhite0

BA E-2402

light yellow

0.6

4.08

4 ml

BA E-2403

orange213.6

4 ml

BA E-2404

dark blue

640.8

4 ml

BA E-2405

light grey

20136

4 ml

BA E-2406

black
light green

60408

BA E-2451
BA E-2452

dark red

Refer to QC-Report for expected value andacceptable range!

4 ml
4 ml
4 ml

Conversion:

Glutamate (µg/ml) x 6.8 = Glutamate (µmol/l)Contents:

Acidic buffer with non-mercury preservative, spiked with defined quantity of Glutamate
D-Reagent - Ready to use

BA E-2446
Contents:

Crosslinking agent in dimethylsulfoxide

Volume:1 x 4 ml/vial, brown cap

Hazards
identification:
H317 May cause an allergic skin reaction.
Q-Buffer - Ready to use

BA E-2458
Contents:

TRIS buffer

Volume:1 x 20 ml/vial, white cap

BA E-2460

Diluent - Ready to use

Contents:

Buffer with sodium acetate

Volume:1 x 20 ml/vial, dark green cap

BA E-2787

NaOH - Ready to use

Contents:

Sodium hydroxide solution

Volume:1 x 2 ml/vial, purple cap

Hazards
identification:
H290 May be corrosive to metals.
H315 Causes skin irritation.
H319 Causes serious eye irritation.
4.2 Additional materials and equipment required but not provided in the kit




Calibrated precision pipettes to dispense volumes between 10 – 100 µl; 12.5 ml
Microtiter plate shaker (shaking amplitude 3 mm; approx. 600 rpm)
ELISA reader capable of reading absorbance at 450 nm and if possible 620 - 650 nm
Shaker (shaking amplitude 3 mm; approx. 600 rpm)
Absorbent material (paper towel)
Vortex mixer
Water (deionized, distilled, or ultra-pure)

5. Sample collection and storage
Various biological samples can be used for L-Glutamate determination. The assay was validated for urinesamples.
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UrineUrine stabilized with 10 µl 6N HCl per 1 ml of urine sample can be used.
Storage: up to 6 hours (18 – 25 °C); up to 14 days (2 – 8 °C); up to 6 months (<-15 °C).
Repeated freezing and thawing should be avoided. Avoid exposure to direct sunlight.
6. Test procedure
Allow all reagents and samples to reach room temperature and mix thoroughly by gentle inversion before
use. Duplicate determinations are recommended. It is recommended to number the strips of the microwellplate before usage to avoid any mix-up.
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperaturedependent, and the absorbance 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 a microplate
reader set to 405 nm.
6.1 Preparation of reagents
Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of1000 ml.
Storage: 1 month at 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 aliquots for max
1 month at -20 C and may be thawed only once.
D-Reagent
The D-Reagent has a freezing point of 18.5 °C. To ensure that the D-Reagent is liquid when being used, it
must be ensured that the D-Reagent has reached room temperature and forms a homogeneous, crystal-freesolution.
Glutamate Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots orlines. These residues do not influence the quality of the product.
6.2 Preparation of samples
The Glutamate ELISA is a flexible test system for various biological sample types and volumes. It is notpossible to give a general advice how to prepare the samples. However, the following basics should help theresearcher to adapt the protocol to his specific needs:

Avoid excess of acid: excess of acid might exceed the buffer capacity of the dilution buffer.
A pH of 5.0 during the extraction is mandatory.
It is advisable to perform a Proof of Principle to determine the recovery of glutamate from the samples.
Prepare a stock solution of glutamate. Add small amounts (to change the native sample matrix as less aspossible) of the stock solutions to the sample matrix and check the recovery.
The sample volume determines the sensitivity of this test. Determine the sample volume needed todetermine glutamate in your sample by testing different amounts of sample volumes.
If a sample volume < 100 µl is used, water (deionized, distilled, or ultra-pure) has to be added to a final
volume of 100 µl.

If you need any support in establishing a protocol for your specific purposes, do not hesitate to contact themanufacturer directly!
Extraction
1.

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

2.

Add 100 µl of the Diluent to all wells. Cover plate with Adhesive Foil and shake for 10 min at RT(20 - 25 °C) on a shaker (approx. 600 rpm).

3.

Use 25 µl for the subsequent derivatization!

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Derivatization
1.

Pipette 25 µl of the extracted standards, controls and samples into the appropriate wells of theReaction Plate.

2.

Pipette 10 µl of NaOH into all wells.

3.

Pipette 50 µl of the Equalizing Reagent into all wells.

4.

Pipette 10 µl of the D-Reagent into 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 75 µl of the Q-Buffer into all wells.

7.

Shake for 10 min at RT (20 - 25 °C) on a shaker (approx. 600 rpm).

8.

Use 25 µl for the ELISA!

6.3 Glutamate ELISA
1.

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

2.

Pipette 50 µl of the Glutamate 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 content 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.

Discard or aspirate the contents of the wells and 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.

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 a homogeneous
distribution of the solution.
Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

10.

7. Calculation of results
Glutamate0.26 – 60 µg/ml

Measuring range

The standard curve is obtained by plotting the absorbance readings (calculate the mean absorbance) of thestandards (linear, y-axis) against the corresponding standard concentrations (logarithmic, x-axis).
Use non-linear regression for curve fitting (e.g. spline, 4- parameter, akima).
This assay is a competitive assay. This means: the OD-values are decreasing with increasing
concentrations of the analyte. OD-values found below the standard curve correspond to highconcentrations of the analyte in the sample and have to be reported as being positive.
Samples and Controls
The concentrations of the samples (100 µl undiluted sample used) and controls can be read directly from thestandard curve.
In case < 100 µl sample volume was used, concentrations of the samples taken from the standard curve
have to be multiplied by a correction factor:
100 µl (volume of standards)
Correction factor =sample volume (µl)

In case samples were pre-diluted correct the read values for the pre-dilution
Conversion
Glutamate (µg/ml) x 6.8 = Glutamate (µmol/l)Version: 15.0-r

Effective:2020-02-26

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7.1 Quality control
The confidence limits of the kit controls are indicated on the QC-Report.
7.2 Typical standard curve
Example, do not use for calculation!

OD

Glutamatµg/ml

8. Assay characteristics
Various biological samples can be used for L-Glutamate determination. The assay was validated for urine
samples.
Analytical Sensitivity

Glutamate

Limit of Blank (LOB)

0.11 µg/ml

Limit of Detection (LOD)

0.17 µg/ml

Limit of Quantification (LOQ)

0.26 µg/ml
Substance

Cross Reactivity (%)
Glutamate

Analytical Specificity(Cross Reactivity)

Version: 15.0-r

L-Glutamine
Glycineβ-Alanine
L-Alanine
L-Aspartic Acid
GABA
5-Amino-n-valeric Acid

Effective:2020-02-26

< 0.4
< 0.4
< 0.4
< 0.4
< 0.4
< 0.4
< 0.4

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Precision
Intra-Assay
Sample

n12
34
56

1010
10101010

Inter-Assay
Mean ± SD(µg/ml)
0.8 ± 0.1
1.3 ± 0.1
2.2 ± 0.1
4.8 ± 0.2
12.5 ± 0.6
39.7 ± 2.2

Linearity

RecoveryCV (%)

Sample

n10.8
8.7
6.3
4.04.65.6123456131414131414

Mean ± SD(µg/ml)
1.7 ± 0.24
5.0 ± 0.57
10.6 ± 0.73
3.0 ± 0.43
5.6 ± 0.71
10.0 ± 0.87

CV (%)
14.3
11.4
6.9
14.2
12.5
8.7

Urine

Serial dilution up to
1:64

Range (%)
94 - 113

Mean (%)
105

Urine

Range (µg/ml)
1.25 - 41.0

Range (%)
97 - 108

Mean (%)
102

For literature or any other information please contact your local supplier.
Symbols:

Version: 15.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContentCaution

Catalogue
numberEffective:2020-02-26

Contains sufficient for<n> tests

For research use
only!

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