GABA (Gamma-aminobutyric acid) ELISA Assay Kit

Additional Information

Assay Principle

The Eagle Biosciences GABA ELISA Assay Kit employs the competitive enzyme immunoassay technique. An antigen GABA has been pre-coated onto a microtiter plate. Standards or samples are pipetted into the wells and any GABA present compete for the fixed number of antibody binding site. After washing away any unbound substances, the antibody bound to the solid phase is detected by using TMB as a substrate. The reaction is monitored at 450 nm±2 nm. Quantification of unknown samples is achieved by comparing their absorbance with a reference curve prepared with known standards.

Assay Procedure

Extraction (Urine)

1. Add 100 μl of standards (standard range between 75 – 7500 ng/ml), controls and samples into the Extraction Plate.
2. Add 100 μl of the Diluent into each well. Incubate for 15 minutes at RT.
3. Discard and blot dry by tapping the inversed plate on absorbent material. Wash each well with 500 μl of distilled water for 5min. at RT. After washing, discard and blot dry by tapping the inversed plate on absorbent material.
4. Add 400 μl of Elution Buffer to each well. Incubate for 10 minutes at room temperature.

Derivatization (Urine)

1. Add 100 μl of extracted standards, controls and samples in duplicate into the Macrotiter Plate.
2. Add 10 μl of NaOH into each well.
3. Add 50 μl of the Equalizing Reagent into all wells and shake 600rpm for 1 min.
4. Add 10 μl of the D-Reagent into all wells.
5. Cover the wells and incubate for 2 hours at RT.
6. Add 200 μl of the Q-Buffer into all wells. Shake 600rpm for 10 min. at RT.

GABA ELISA (Urine)

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

1. Add 50 μl of the derivatized standards, controls and samples into the GABA-coated microtiter strips.
2. Add 50 μl of the GABA antiserum into each well.
3. Cover the wells and incubate for 15-20 hours at 2-8°C (or incubate for 2 hours at RT).
4. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1× Wash Buffer (350 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.
5. Add 100 μl of the Enzyme-Conjugate Antibody into each well. Incubate for 30 min. at RT.
6. Aspirate each well and wash as step 4.
7. Add 100 μl of TMB Reagent to each well. Incubate for 20 minutes at room temperature in dark.
8. Add 100 μl of Stop Solution to each well.
9. Read the OD with a microplate reader at 450 nm immediately.

Extraction (Serum/Plasma)

1. Dilute standards and controls 1:3 with distilled water. (e.g. 100 μl standard + 200 μl distilled water, in turn to get standard range between 25 – 2500 ng/ml.)
2. Add 300 μl of diluted standards, controls and undiluted samples into the Extraction Plate.
3. Add 300 μl of the Diluent into each well. Incubate for 30 minutes at RT.
4. Discard and blot dry by tapping the inversed plate on absorbent material. Wash each well with 1 ml of I-Buffer for 5min. at RT. After washing, discard and blot dry by tapping the inversed plate on absorbent material. Repeat this step 2 times.
5. Add 250 μl of Elution Buffer to each well. Incubate for 10 minutes at room temperature

Derivatization (Serum/Plasma)

1. Add 100 μl of extracted standards, controls and samples in duplicate into the Macrotiter Plate.
2. Add 10 μl of NaOH into each well.
3. Add 50 μl of the Equalizing Reagent into all wells and shake 600rpm for 1 min.
4. Add 10 μl of the D-Reagent into all wells.
5. Cover the wells and incubate for 2 hours at RT.
6. Add 150 μl of the Q-Buffer into all wells. Shake 600rpm for 10 min. at RT.

GABA ELISA (Serum/Plasma)

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

1. Add 25 μl of the derivatized standards, controls and samples into the GABA-coated microtiter strips.
2. Add 50 μl of the GABA antiserum into each well.
3. Cover the wells and incubate for 15-20 hours at 2-8°C (or incubate for 2 hours at RT).
4. Aspirate each well and wash, repeating the process 2 times for a total 3 washes. Wash by filling each well with 1× Wash Buffer (350 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.
5. Add 100 μl of the Enzyme-Conjugate Antibody into each well. Incubate for 30 min. at RT.
6. Aspirate each well and wash as step 4.
7. Add 100 μl of TMB Reagent to each well. Incubate for 20 minutes at room temperature in dark.
8. Add 100 μl of Stop Solution to each well.
9. Read the OD with a microplate reader at 450 nm immediately.