Rat IgM ELISA Assay

Additional Information

Assay Principle

The determination of rat IgM is carried out as direct sandwich ELISA. An antibody specific for rat IgM has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IgM present is bound. After washing away any unbound substances, an enzyme-linked antibody is added. Following a wash, a substrate solution is added to the wells and color develops in proportion to the amount of antibody conjugate. The absorption at 450 nm is proportional to the IgM con­cen­tration.

Assay Procedure

A. Preparation

1. Bring all reagents to room temperature
2. Dilute wash buffer 1:10
3. Prepare the standard curve from a 1:2-dilution series of standard concentrate with diluent
4. Dilute samples with diluent
5. Dilute freshly HRP conjugate 1:100 with diluent

B. Performance

1. Pipette 100 µl of samples, standards, diluent (blank) into the wells
2. Incubate for 1 hour at room temperature with shaking (or 2 hrs without shaking)
3. Wash three times with 300 µl of wash buffer
4. Add 100 µl of HRP conjugate to each well
5. Incubate for 1 hour at room temperature with shaking (or 2 hrs without shaking)
6. Wash three times with 300 µl of wash buffer
7. Dispense 100 µl of TMB substrate solution
8. Incubate for 20 minutes at room temperature in the dark
9. Add 100 µl of stop solution
10. Measure absorption at 450 nm

Typical Standard Curve