TITLE: Test-serology-protocol
AUTHORS: Dr. John Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an assay describing indirect sandwich ELISA for RDP Ag of SARS-CoV-2. </div><div class = "text-block">Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda, Yadda, yadda, yadda.</div></div>

[STEPS]
?. [B. Preperation of Samples]
If the concentration of antigen in the sample potentially exceeds the highest point of the standard curve (i.e. > 1,000 pg/ml) prepare one or more dilutions of the sample using the standard diluent.
?. [A.Preparation of Standards]
Typically a standard curve may span concentrations from 0 to 1000 pg/ml but may go as high as 3000 pg/ml depending on the predicted amount of antigen in the sample and the amount of standard protein available. Typically two-fold or three-fold dilutions of the standard protein are prepared from the stock solution using the standard diluent. When preparing serial dilutions of a protein standard, use fresh tips after each dilution.
?. [C. Procedure]
Dilute the capture antibody to the appropriate concentration allowing sufficient volume for 50-100 	ul per well.
?. [C. Procedure]
Add the diluted capture antibody to the plate, cover and incubate for 2 hours at room temperature 	(RT).
?. [C. Procedure]
Remove the solution and wash the plate with 200 ul per well wash buffer for 3 x 5 minutes on a shaking platform.
?. [C. Procedure]
Add 300 ul blocking buffer per well, cover the plate and incubate for 1 hour at room temperature. Alternatively block overnight at 4uC.
?. [C. Procedure]
Prepare the samples and standards. The volume per well should be the same as the capture antibody used in step 1.
?. [C. Procedure]
Remove the blocking buffer and add the samples and standards. Cover the plate and incubate for 1 hour at RT.
?. [C. Procedure]
Remove the solution and wash the plate with 200 l per well wash buffer for 3 x 5 minutes on a 	haking platform.
?. [C. Procedure]
Dilute the biotinylated detection antibody to the appropriate concentration. The volume per well should be the same as the capture antibody used in step 1.
?. [C. Procedure]
Add the diluted detection antibody to the plate, cover and incubate for 1 hour at RT.
?. [C. Procedure]
Remove the solution and wash the plate with 200 l per well wash buffer for 3 x 5 minutes on a shaking platform.
?. [C. Procedure]
Dilute the enzyme conjugate to the appropriate concentration. The volume per well should be the 	ame as the capture antibody used in step 1.
?. [C. Procedure]
Add the diluted enzyme conjugate to the plate, cover and incubate for 1 hour at RT.
?. [C. Procedure]
Remove the solution and wash the plate with 200 l per well wash buffer for 6 x 5 minutes on a 	shaking platform
?. [C. Procedure]
Add substrate solution to the plate. The volume per well should be the same as the capture antibody used in step 1.
?. [C. Procedure]
Incubate the plate at RT until the desired color intensity is reached. Ideally a clear gradient will result for the standards.
?. [C. Procedure]
Stop the reaction by adding an equal amount of stop solution.
?. [C. Procedure]
If using TMB, measure the absorbance at 450 nm. For other substrates use the appropriate detection technique.
?. [C. Procedure]
Important: Do not allow the plate to dry at any point