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Glutamate ELISA Assay Kit

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 Product Description

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Glutamate ELISA Assay Kit: 
For Research Use Only

Size:  1 x 96 wells
Sensitivity: 0.3 µg/ml
Dynamic Range: 0.6-60 µg/ml
Incubation Time:  3 hours

Sample Type:  Serum, EDTA-Plasma, Urine
Sample Size: 100 µl

Intended Use

The Eagle Biosciences Glutamate ELISA assay kit if for the quantitative determination of L-Glutamate in human Glutamate in serum, EDTA-plama and/or urine. The Eagle Biosciences Glutamate ELISA assay kit is for research use only and should not be used for diagnostic procedures.

Assay Principle

The Eagle Biosciences Glutamate ELISA assay kit employs the competitive quantitative sandwich enzyme immunoassay technique. An antigen has been pre-coated onto a microtiter plate. After extraction and derivatization, controls, standards or samples are pipetted into the wells together with specific Glutamate antiserum. The standards, controls and samples and the solid phase bound analyte antigen compete 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. 

Following a washing to remove unbound substances, anti-rabbit IgG conjugated to Peroxidase is added to each well and incubated. After washing away any unbound antibody conjugate, a substrate solution (TMB) is added to the wells and color develops in inverse-proportion to the amount of glutamate present in the samples. The color development is stopped by the addition of STOP solution and the intensity of the color is measured at a wavelength of 450nm ±2nm. The concentration of glutamate in the sample is then determined by comparing the O.D of samples to the standard curve.

Assay Procedure

1. Add 100 μl of standards, controls and diluted samples (Serum/plasma 1:5) into the Extraction Plate.
2. Add 100 μl of the Diluent into each well. Cover the plate with adhesive foil and incubate for 10 minutes at RT on a microplate shaker. (600 rpm)
3. Use 25 μl for the subsequent derivatization

1. Add 25 μl of extracted standards, controls and samples in duplicate into the Reaction Plate.
2. Add 10 μl of NaOH into each well.
3. Add 50 μl of the Equalizing Reagent into all wells and shake at 600 rpm on a microplate shaker for 1 min.
4. Add 10 μl of the D-Reagent into all wells.
5. Cover the wells with adhesive foil and incubate for 2 hours at RT on a microplate shaker at 600rpm.
6. Add 75 μl of the Q-Buffer into all wells. Shake at 600 rpm on a microplate shaker for 10 min at RT.
7. Use 25 μl for the ELISA assay.

Glutamate ELISA Procedure
All materials should be equilibrated to room temperature (RT) before use. Standards, samples and controls should be assayed in duplicates.

1. Remove excess microtiter strips from the plate frame (Antigen-coated microplate), return them to the foil pouch containing the desiccant pack, and reseal it.
2. Add 25 μl of the derivatized standards, controls and samples in duplicates into the Glutamate-coated microtiter strips (Antigen-coated microplate).
3. Add 50 μl of the Glutamate antiserum into each well, mix shortly.
4. Cover the wells with adhesive foil and incubate for 15-20 hours at 2-8°C (or incubate on a microplate shaker (600 rpm) for 2 hours at RT).
5. Remove the foil and discard. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1× Wash Buffer (300 μl) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating, decanting or blotting against clean paper towels.
6. Add 100 μl of the Anti-rabbit IgG peroxidase conjugate into each well. Incubate on a microplate shaker (600 rpm) for 30 mins at RT.
7. Aspirate each well and wash as step 5.
8. Add 100 μl of TMB Reagent to each well. Incubate on a microplate shaker (600 rpm) for 20-30 mins at RT in dark.
9. Add 100 μl of Stop Solution to each well and shake lightly to ensure homogeneous mixing.
10. Read the OD with a microplate reader at 450nm (with a reference wavelength between 620nm and 650nm) within 10 minutes.


Standard Curve: 

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Eagle Biosciences, Inc.
20A NW Blvd., Suite 112
Nashua, NH 03063
Email: info@eaglebio.com
Toll Free: 866-411-8023
International: +617-419-2019

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