I am using indirect ELISA to measure the antibody response against Aeromonas salmonicida in Atlantic salmon. I am using 3 dilutions of serum 1:50, 1:200, and 1:400.
The OD450 nm values measured at the 3 dilutions tend to increase and not decrease going from 1:50 to 1:400 dilution factor of serum. In addition, I am getting different results comparing the 3 serum dilutions.
Karen A. Darbinyan
Hello, in S Eliza system in linear regression as much OD as small analyte content. So it can cause by contamination, not proper pipetting, test kit problem etc.
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Bhairavi Vajaria
- The erroneous results might be due to matrix afffect. The matrix effect can arise from a number of matrix components including, but not limited to: interaction between endogenous biological components such as phospholipids, carbohydrates and endogenous metabolites or an interaction between the analyte of interest and the matrix, such as covalent binding to plasma proteins. This results in erroneous sample readings.Simply diluting the samples by 2 or 5 folds reduces the matrix effect, when diluting the samples remember to use the same diluent as used for standard curve.
- Make sure there is no hook affect and appropriate dilutions are within the detection range of ELISA kit.
- Take care of accurate pipetting, proper mixing of reagents in wells, no bubbles, proper washing and aspiration steps, and proper storage of serum (avoiding freeze thaw cycles) without contamination.
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Agnieszka Szczepek
Could you please write a few words about the standards? How does the standard curve look like?
What kind of substrate are you using for the read out? Is there an option of using two wavelengths for readout?
1 votes
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