Secretory IgA ELISA Assay

Additional Information

Assay Background

Secretory IgA is comprised of two immunoglobulin A molecules, which are joined by a J-protein and a secretory component. The secretory component is synthesized by epithelial cells of the mucous membrane of gastrointestinal, respiratory and urogenital tracts. Secretory IgA is also produced by the saliva, tear and mammary glands. The plasma cells in the subendothelial area of mucous membranes are releasing a complex of two IgA-molecules, which are joined over the J-protein. This complex is binding to a secretory component, located at the surface of the epithelial cell. After binding, the sIgA is transported across the cell and excreted by exocytosis.  The determination of secretory IgA (sIgA) allows a first overview of the functionality of the gastrointestinal associated immune system (GALT). At this the secretory power and the degree of stimulation of the plasma cells of the intestinal submucosa is determined.
The Eagle Biosciences Secretory IgA (sIgA) ELISA kit allows for an easy, rapid and precise quantitative determination of secretory IgA in biological samples. The kit includes all reagents ready to use for preparation of the samples.

Assay Principle

The Secretory IgA (sIgA) ELISA kit determines human secretory IgA according to the “sandwich”-principle. sIgA 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 sIgA 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).

Typical Standard Curve