Correction of non-specific binding in RaPID assays


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Heterogeneous immunoassays are typically subject to a low level of direct binding of enzyme-antigen conjugate to the solid
support surface. This binding results in a very small amount of positive bias. Users can correct for this bias by using the
procedure presented below.

Standard curves which serve to estimate pesticide (antigen) concentration in immunoassays are usually sigmodial. By using
data transformations of one or both variables (X,Y) a suitable regression line can be fitted to the standard calibrators. In
some cases, correction for non-specific binding (NSB) of the enzyme-antigen conjugate in the data reduction can be used to
improve curve fitting around the calibrators or a cut-off level of interest. Color production related to NSB can be determined
using a sample with a high concentration of pesticide (excess antigen). Absorbance from NSB is subtracted from the
absorbance for the zero, standard calibrators and samples.

To determine the NSB, a sample with approximately fifty times the pesticide concentration of the highest calibrator should be
prepared. When using the RPA-I Photometric Analyzer, this sample is then be read as the 'reagent blank' and the NSB
absorbance corrections are performed automatically in the data reduction program.

For NSB correction in the Atrazine RaPID Assay (standard calibrators at 0.1, 1 and 5 ppb), prepare and analyze a sample
with 250 ppb atrazine in the assay run. Position this sample (one replicate tube) as the last tube in the rack.

Before reading results on the RPA-I, perform the following steps:

1. Select OPTION.
2. Scroll to 'ENTER/EDIT PROTOCOL'. Press ENTER.
3. Scroll to 'ATRAZINE'. Press ENTER.
4. Press ENTER 15 times to reach '# OF RGT BLANKS'.
5. Press '1'.
6. Press ENTER.
7. Press ENTER 3 times to reach 'LIST PROTOCOL'.
8. Press YES.
9. Confirm that '# RGT BLKS:1' is now on your printout (and that nothing else new prints out).
10. Press RUN to begin the assay.
11. Results which have been corrected for non-specific binding (NSB) will be printed for all controls and samples.

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