EIA-5507 DRG Vitamin Drug Instruction Manual

EIA-5507 DRG Vitamin Drug

DRG Instruments GmbH, Germany Frauenbergstraße 18, 35039 Marburg Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50 Website: www.drg- diagnostics.de E-mail: drg@drg- diagnostics.de

DRG International, Inc., USA
841 Mountain Ave., Springfield, NJ 07081 Phone: (973) 564-7555, Fax: (973) 564-7556 Website: www.drg-international.com E-mail: [email protected]Please use only the valid version of the Instructions for Use provided with the kit.

PRINCIPLE OF THE TEST

After extraction and derivatization, Glutamate is quantitatively determined by ELISA. The competitive ELISA uses the microtiter plate format. The antigen is bound to the solid phase of the microtiter 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 in equilibrium, free antigen and free antigen-antibody complexes are removed by washing. The antibody bound to the solid phase is detected by an anti-rabbit IgGperoxidase 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.

PROCEDURAL CAUTIONS, GUIDELINES, WARNINGS AND LIMITATIONS

1. 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 instructions 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 the instructions provided.
2. This assay was validated for a certain type of sample as indicated in Intended Use (please refer to Chapter 1). Any off-label use of this kit is in the responsibility of the user and the manufacturer cannot be held liable.
3. The principles of Good Laboratory Practice (GLP) have to be followed.
4. To reduce exposure to potentially harmful substances, wear lab coats, disposable protective gloves and protective glasses where necessary.
5. 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.
6. For dilution or reconstitution purposes, use deionized, distilled, or ultra-pure water.
7. The microplate contains snap-off strips. Unused wells must be stored at 2 – 8 °C in the sealed foil pouch with desiccant and used in the frame provided.
8. Duplicate determination of sample is highly recommended to be able to identify potential pipetting errors.
9. Once the test has been started, all steps should be completed without interruption. Make sure that the required reagents, materials, and devices are prepared and ready at the appropriate time.
10. Incubation times do influence the results. All wells should be handled in the same order and time intervals.
11. To avoid cross-contamination of reagents, use new disposable pipette tips for dispensing each reagent, sample, standard, and control.
12. A standard curve must be established for each run.
13. 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.
14. Do not mix kit components with different lot numbers within a test and do not use reagents beyond the expiry date as shown on the kit labels.
15. Avoid contact with Stop Solution containing 0.25 M H2SO4. It may cause skin irritation and burns. In case of contact with eyes or skin, rinse off immediately with water.
16. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash your eyes with an abundant volume of water and your skin with soap and abundant water. Wash contaminated objects before reusing them.
17. For information on hazardous substances included in the kit please refer to the Safety Data Sheet (SDS). The Safety Data Sheet for this product is made available directly on the website of the manufacturer or upon request.
18. Kit reagents must be regarded as hazardous waste and disposed of according to national regulations.
19. The expected reference values reported in this test instruction are only indicative. It is recommended that each laboratory establishes its reference intervals.
20. In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing, at the latest, one week after receiving the kit. Severely damaged single components must not be used for a test run. They must be stored properly until the manufacturer decides what to do with them. If it is decided that they are no longer suitable for measurements, they must be disposed of under national regulations.
21. Any inappropriate handling of samples or modification of this test might influence the results.
22. Please note the sample preparation stabilization of the urine sample! It cannot be excluded that high acid concentrations lead to incorrect results. Up to 20 µl 6 N HCl per 1 ml urine, no influence on the results was observed.
23. There are no known substances (drugs, food) in which ingestion interferes with the measurement of glutamate level in the sample.
24. No hook effect was observed in this test.

STORAGE AND STABILITY

Store the unopened reagents at 2 – 8 ºC until the expiration date. Do not use components beyond the expiry date indicated on the kit labels. Once opened the reagents are stable for 2 months when stored at 2 – 8 °C. Once the resealable pouch has been opened, care should be taken to close it tightly with desiccant again.

MATERIALS

CONTENTS OF THE KIT

Standards and Controls – Ready to use

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)

SAMPLE COLLECTION AND STORAGE

Urine
Spontaneous urine (second-morning urine) stabilized with 10 µl 6 M 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.

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 microwell plate before usage to avoid any mix-up.
The binding of the antisera and of the enzyme conjugate and the activity of the enzyme are temperature dependent. The higher the temperature, the higher the absorption values will be. Varying incubation times will have similar influences on the absorbance. The optimal temperature during the Enzyme Immunoassay is between 20 – 25 °C. During the overnight incubation at 2 – 8 °C with the antiserum, the temperature should be uniform all over the ELISA plate to avoid any drift and edge effects.

PREPARATION OF REAGENTS

Wash Buffer
Dilute the 20 ml Wash Buffer Concentrate with water (deionized, distilled, or ultra-pure) to a final volume of 1000 ml. Storage: 2 months 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 2 months 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-free solution.

Glutamate Microtiter Strips
In rare cases residues of the blocking and stabilizing reagent can be seen in the wells as small, white dots or lines. These residues do not influence the quality of the product.

Extraction Plate
In rare cases residues of the cation exchanger can be seen in the wells as small, black dots or lines. These residues do not influence the quality of the product.

SAMPLES PREPARATION Extraction

1. Pipette 100 μl of the standards, controls and urine 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!

Derivatization

1. Pipette 25 μl of the extracted standards, controls and urine samples into the appropriate wells of the Reaction 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!

GLUTAMATE ELISA

1. Pipette 25 μl of the prepared standards, controls and urine samples into the appropriate wells of the Glutamate 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.
10. Read the absorbance of the solution in the wells within 10 minutes, using a microplate reader set to 450 nm (if available a reference wavelength between 620 nm and 650 nm is recommended).

CALCULATION OF RESULTS

• Measuring range
  • Urine
• Glutamate
  • 0.26 – 60 μg/ml

The standard 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. 4-parameter, Marquardt). 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 high concentrations of the analyte in the sample and have to be reported as being positive.

Urine samples and controls
The concentrations of the samples and controls can be read directly from the standard curve. Samples found with concentrations higher than the highest standard (Standard F) should be diluted accordingly with water (deionized, distilled, or ultra-pure) and have to be re-assayed.

Conversion
Glutamate (μg/ml) x 6.8 = Glutamate (μmol/l)

Expected reference values
It is strongly recommended that each laboratory should determine its own reference values.

Spontaneous urine

• 1034 – 7726 μg/g creatinine
• 7 – 52.5 μmol/g creatinine
• 0.8 – 5.9 mmol/mol creatinine

Values significantly outside the reference range should be assessed by a doctor.

QUALITY CONTROL
The confidence limits of the kit controls are indicated on the QC Report.

TYPICAL STANDARD CURVE
For example, do not use it for calculation!

ASSAY CHARACTERISTICS

Analytical Specificity (Cross Reactivity)

| Substance| Cross Reactivity (%)
---|---|---
Glutamate
L-Glutamine| < 0.4
Glycine| < 0.4
β-Alanine| < 0.4
L-Alanine| < 0.4
L-Aspartic Acid| < 0.4
GABA| < 0.4
5-Amino-n-valeric Acid| < 0.4
Precision

Intra- Assay| Inter- Assay
Sample| n| Mean ± SD

(µg/ml)

| CV (%)| Sample| n| Mean ± SD

(µg/ml)

| CV (%)
1| 10| 0.8 ± 0.1| 10.8| 1| 13| 1.7 ± 0.24| 14.3
2| 10| 1.3 ± 0.1| 8.7| 2| 14| 5.0 ± 0.57| 11.4
3| 10| 2.2 ± 0.1| 6.3| 3| 14| 10.6 ± 0.73| 6.9
4| 10| 4.8 ± 0.2| 4.0| 4| 13| 3.0 ± 0.43| 14.2
5| 10| 12.5 ± 0.6| 4.6| 5| 14| 5.6 ± 0.71| 12.5
6| 10| 39.7 ± 2.2| 5.6| 6| 14| 10.0 ± 0.87| 8.7
Linearity| | Serial dilution up to| Range (%)| Mean (%)
---|---|---|---|---
Urine| 1:64| 94 – 113| 105
Recovery| | Range (µg/ml)| Range (%)| Mean (%)
---|---|---|---|---
Urine| 1.25 – 41.0| 97 – 108| 102

SYMBOLS USED

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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