I want to check interferon beta protein concentration in mouse tissue sample like heart, liver, lung and kidney. Can anyone please help how can I make the sample from the tissue for ELISA?
You must homogenize the tissue sample, generally the methodology of the kit specifies in which it should be homogenized. You can take for example 1 g of sample in 5 ml of buffer or whatever the technique suggests.
Mr./Miss/Mrs. Rajbongshi,
Despite, the fact that the ELISA based quantification in tissue has been accepted as a routine procedure in the clinical practice, mainly, due to low price of the ELISA kit, as well as, relatively low concentration LODs of analytes at about ng.(mL)-1, I should mention that:
(i) The accepted error contribution to the concentration LODs and LOQs is very high (at about 20 %;) and
(ii) the difference in values between ELISA analyses and, for instance, HPLC-MS/MS ones can be up to 30 %.
These results indicate that ELISA assay does not provide reliable analytical information, owing to the fact that the metod of mass spectrometry yields to an absolute (or exact) analytical information in chemometric terms at significantly lower concentration LODs and LOQs of pg.(mL)-1.
In the latter context, I should mention that the currently employed in tha analytical practice mass spectrometric protocols operate at lower (and unreal) capability of the method, due to the fact that they do not account precisely for the fluctuations of the experimental variables. These protocols quantify the so-called total intensity values of the MS peak of the analyte; and an average m/z-value per the whole time of the experimental measurement, which is conceptually wrong. There is a stochastic nature of the mass spectrometric phenomena.
In order to clarify my statements, please consider our innovative quantitative approach to treat the mass specrometric variable 'intensity' and new authored (to me and my co-author) stochastic dynamic formulas for quantification of the analytes in solution showing reliability of the analysis at /r/ = 1 between our theory and the experiment, studying analytes in mixture at pg.(mL)-1 and ng.(L)-1 concentration levels. It is given as references to the following discussion [A] and its subdiscussion, shown therein.
[A] [https://www.researchgate.net/post/What_would_be_the_internal_standard_for_Remdesivir_in_bioanalytical_method_by_UHPLC-MS_MS]
There is detailed briefly aspects of mass spectrometric phenomena; and the employment of internal standard for quantification by mass spectrometry, as well.
As can be seen, currently, accurate, precise, selective and sensitive quantitative analytical information about analytes in complex samples, including tissues can be achieved only by mass spectrometry; furthermore, within the framework of our innovative theory and model formulas.
Since, as aforementioned, the theory and the equations are our own-authored contribution to the quantitative mass spectrometry, if you have any questions, then, please, do not hesitate to ask them, herein. We shall provide to you a comprehensive guideline.
Please look at the following below links which may help you in your analysis:
https://docs.abcam.com/pdf/protocols/elisa-sample-preparation-guide.pdf
http://tools.thermofisher.com/content/sfs/manuals/ELISA_guide_man.pdf
Thanks
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