ELISA test were run to optimise the appropriate antigen concentration and serum dilution factor. I had triplicates of substrate control, negative control and blank for my analysis. Since this is a unique study I was not able to compare it with a positive control and determine the exact antibody concentration of the serum. But the OD values between diluted serum samples and controls showed significant difference.
I'm not sure if I 100% understand your question, but you could do an endpoint ELISA... report antibody titres as the dilution required to give an OD as low as that of a 'negative' or 'blank' well +3 standard deviations. To do this you would need to run many negative samples to get this negative value, and then serial dilutions of your test samples.
Dear Sheela Devi, please, let us know full description of your research problems. I guess, you ask about those antiserum, which you planned to study in cytotoxic test (as it has appeared in your previous question). Describe the antigen and, especially, methods which were used for its purification. What animals were used to obtain antiserum? Did you analyze the specificity of antiserum by Western blot? If the antigen is associated with cell membrane and contains hydrophobic tail, it will be difficult to prepare the samples suitable for ELISA. In this case cytotoxic test will be more appropriate to compare different antisera. If the antigen is soluble protein, it may be, simple titration in Ouchterlony double radial immunodiffusion will be sufficient to determine (approximately) the zone of equivalence.
Test systems from large diagnostic test manufacturers doing this. They have a large experience with the stability of the assay including controls.
I would never suggest to leave this (and the other controls) in a new or house-made assay. You can forget the blank, it's just for the photometer only and will be added as the intercept to your calibration function. The photometer can be blanked against the negative control (in a non-competitive test set up).
Choose the negative control OD value plus the three SD to assess your positive responses. Choosing a negative control should be a proper one in that case. lalitha kabilan
You cant fix exact concentration without positive controls or standards.
Pls always use control and standard to ensure the realiablity of the result. This should also be carried out 2ce.
Dear Sheela Devi,
I'm not sure if I exactly understand your question, I think you should do a checkerboard titration of your antigen, you will get an optimum concentration of your antigen. Then you should do an endpoint ELISA to get an antibody titer which is the dilution that give an OD as low as mean of a negative or blank well +3 standard deviations or use a mathematics equation from this paper "A statistically defined endpoint titer determination method for immunoassays" [Authors: Andreas Frey, James Di Canzio, and David Zurakowski, published in 1998]
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