Hi everyone,
While doing sandwich ELISA, every-time I face the problem of positive readings in negative control wells regardless of the sample type or protein targets against which primary antibody (monoclonal) used.
Coating buffer composition: 0.1M sodium carbonate pH 9.5 (7.13g NaHCO3, 1.59g Na2CO3 in 1L of milli-Q water)
Details for the negative control wells that show readings are as follows:
1) wells that lack capture antibody in coating buffer are showing readings close to the test wells
2) wells that lack sample also shows similar kind of readings
But, wells containing either no primary or secondary antibodies show no readings.
Please suggest some solutions.
thanks Margit,
For blocking, I am using 5% BSA in 1X PBS for 2 to 3 hours at room temperature.
Possible reasons:
1) The most likely reason is that your primary antibody might have cross-reaction with BSA (try to remove BSA from your blocking and washing buffers, try TBST from Pierce)
2) Sometimes BSA can give very high background (especially at 5% concentration) because it might react with some antibodies and it might have some contaminant proteins (try TBST from Pierce)
As some one already mentioned, your primary or secondary antibody may be cross reacting with your blocking buffer. So use alternative blocking reagent (other than BSA) to see whether you can get rid of high background.
What is your diluent for your sample and conjugate? What washing solution are you using and how are you washing your plate? What are you capturing and what is your conjugate? What grade of BSA are you using (Fatty acid free, IgG free)? Have you titred your conjugate as high conjugate concentrations can cause high backgrounds? Without these details the only suggestion is similar to the others and try other blocking agents casein, ovalbumen , fish gelatin or a non-protein blocking agent e.g. PVP
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