Alpha-1 Antitrypsin ELISA Assay

Additional Information

Assay Principle

The Alpha-1 Antitrypsin ELISA assay kit determines human alpha-1-antitrypsin according to the “sandwich”-principle. Alpha-1 Antitrypsin in sample, standard and controls binds to antibodies, which are coated to the microtiter plate. After a washing step a peroxidase labeled detection antibody is added. A second washing step is followed by the addition of the substrate which is converted to a colored product by the peroxidase. The reaction is terminated by the addition of an acidic stop solution. The optical densities are read at 450 nm (against the reference wavelength 620 nm) in a microtiter plate reader. The α-1-AT concentration can be calculated from the standard curve.

Assay Procedure

1. Washing step:
   Take out the needed strips of the microtiter plate and wash 1x with 250 µl diluted WASHBUF. Remove residual buffer by tapping the plate on absorbent paper after the washing step.
2. Incubation samples:
   Pipette 100µl STD, CTRL and samples in duplicate in the microtiter plate.
   The strips are covered and incubated by shaking for 60 min at room temperature (18-26 °C).
   The reaction in each well starts immediately. Pipetting should be performed as quickly as possible. When processing many samples at once the samples should be pipetted to a separate microtiter plate (150 µl) and transferred simultaneously using a multichannel pipette.
3. Washing step:
   Discard the content of the microwells and wash 5x with 250 µl diluted WASHBUF. Remove residual buffer by tapping the plate on absorbent paper after the last washing step.
4. Incubation conjugate:
   Pipette 100 µl CONJ in each microwell.
   The strips are covered and incubated by shaking for 60 min at room temperature (18-26 °C).
5. Washing step:
   Discard the content of the microwells and wash 5x with 250 µl diluted WASHBUF. Remove residual buffer by tapping the plate on absorbent paper after the last washing step.
6. Incubation substrate:
   Pipette 100 µl SUB in each microwell.
   Incubate for 10-15 min at room temperature (18-26 °C) in the dark.
7. Stopping reaction:
   Pipette 50 µl STOP in each microwell, mix well.
8. Reading:
   Read the absorbance at 450 nm. If the microtiter plate reader allows to use a reference wavelength use 620 or 690 nm as reference wavelength.
   Reading should be done within 5 min after stopping reaction.
   In case that the highest standard exceeds the range of the reader the reading should be done at 405 nm against 620 nm (690 nm).