Glutamate ELISA Assay Kit
For Research Use Only
Size: 1×96 wells
Sensitivity: 0.3 µg/ml
Standard Range: 0.6-60 µg/ml
Incubation Time: overnight, 2 x 30 min
Sample Type: Serum, EDTA-Plasma, Urine
Sample Size: 100 µl
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 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 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.
- Add 100 μl of standards, controls and diluted samples (Serum/plasma 1:5) into the Extraction Plate.
- 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)
- Use 25 μl for the subsequent derivatization.
- Add 25 μl of extracted standards, controls and samples in duplicate into the Reaction Plate.
- Add 10 μl of NaOH into each well.
- Add 50 μl of the Equalizing Reagent into all wells and shake at 600 rpm on a microplate shaker for 1 min.
- Add 10 μl of the D-Reagent into all wells.
- Cover the wells with adhesive foil and incubate for 2 hours at RT on a microplate shaker at 600rpm.
- Add 75 μl of the Q-Buffer into all wells. Shake at 600 rpm on a microplate shaker for 10 min at RT.
- 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.
- Remove excess microtiter strips from the plate frame (Antigen-coated microplate), return them to the foil pouch containing the desiccant pack, and reseal it.
- Add 25 μl of the derivatized standards, controls and samples in duplicates into the Glutamate-coated microtiter strips (Antigen-coated microplate).
- Add 50 μl of the Glutamate antiserum into each well, mix shortly.
- 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).
- 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.
- 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.
- Aspirate each well and wash as step 5.
- Add 100 μl of TMB Reagent to each well. Incubate on a microplate shaker (600 rpm) for 20-30 mins at RT in dark.
- Add 100 μl of Stop Solution to each well and shake lightly to ensure homogeneous mixing.
- Read the OD with a microplate reader at 450nm (with a reference wavelength between 620nm and 650nm) within 10 minutes.
Typical Standard Curve