1. Make sure that correct buffers and reagents were used to wash the plate and dilute the samples. 2. Make sure that the 1X ELISA Wash Buffer was prepared according to the instructions listed in the protocol. 3. Make sure that all components were stored at 4 °C.

We guarantee the integrity of kit components for up to 6 months from date of purchase.

The components are shipped on blue ice. Upon arrival, immediately put all the kit components to 4 °C.

Negative Control contains human negative serum (heat-inactivated) diluted 1:50 in Sample Diluent. Positive Control contains human negative serum (heat-inactivated) diluted 1:50 in Sample Diluent spiked with an antibody directed against the coated antigen. Cut-off Control contains an antibody directed against the coated antigen diluted in Sample Diluent.

1. Allow all reagents to warm up to room temperature for 30 minutes prior to starting. 2. Make sure that all controls have been mixed well before adding them to the plate. 3. Stick to the order of the protocol and add the controls after the samples right before incubating the plate at 37 °C for 1 h. 4. After adding the TMB Substrate, make sure that the Stop Solution is added after 10 minutes. Determine OD immediately after adding the Stop Solution. 5. Double-check the absorbance filters used in the plate reader. Use 450 nm for measuring with a reference filter between 560 nm and 650 nm.