What to do if you need a standard curve without know concentrations of standard?

On unique occasions, the absolute value for standard for a particular ELISA is not available for generation of a standard curve. For example there is no purified native CD36 available as an absolute standard for CD36 human ELISA. Frequently, the investigator in these cases will prepare dilutions of a calibrator sample to help in estimation of the test analyte. The calibrator is usually a pooled sample prepared from combining all the samples to be tested in an experiment with unknown quantities of the analyte. The calibrator should be included on each plate and as it aids in comparing the results between different plates and also in quantification of the relative amounts of analytes. As an example, Figure A. shows the raw data where the standard is unavailable and the calibrator is used for the analysis by pooling serum samples from 4 human subjects.

Figure A: Raw Data in Excel

Figure A: Raw Data in Excel

The analysis of data in this format is currently not supported by ELISAAnalysis since the user can only insert concentration values and not dilutions. A simple workaround solution for handling these types of data is suggested below:

1. The dilutions of the calibrator in Figure A can be multiplied by a factor to get whole number values. Figure B depicts the concentration values obtained upon multiplication of calibrator by an appropriate factor (i.e. 819200). Then the raw data will look like Figure C.

 

Figure B: Conversion of calibrator dilutions to absolute concentrations in Excel.

Figure B: Conversion of calibrator dilutions to absolute concentrations in Excel.

 

Figure C: Plate Layout of the converted data in Excel

Figure C: Plate Layout of the converted data in Excel

2. Subsequently, the OD values obtained for the calibrator are inserted in the marked “standard” wells & test samples in the “unknown” wells in a custom plate layout or one of the popular layout of ELISAAnalysis (Figure D). After this 4PL analysis should be performed (Figure E).

Figure D:  Plate Layout of the converted data in ELISAAnalysis

Figure D: Plate Layout of the converted data in ELISAAnalysis

Figure E. Results from 4PL analysis of converted data where the concentration values of unknown (Test) has been calculated relative to the calibrator.

Figure E. Results from 4PL analysis of converted data where the concentration values of unknown (Test) has been calculated relative to the calibrator.

3. To get back to the dilutions of the calibrator we can divide the predicted values by the factor (in this case 819200). However this type of analysis will provide fold change expression, more like a qualitative measure. This is because without defining the relationship between protein concentration and response (which is what a standard curve does) any measurements will simply be relative.