Hi
can Someone explain to me the protocol to estrone extraction from the different biological matrix before immunological analysis (by RIA, CLIA)
Thank you so much.
Sarah Canon
A general guideline procedure can be performed as follows:
Extraction
- Add Acidified Water: Adjust the sample pH to acidic (~3) by adding acidified water. This step helps in releasing estrone from binding proteins.
- Add Solvent: Add an organic solvent (like ether or ethyl acetate) to the sample in a 1:1 or 2:1 ratio (solvent:sample).
- Mix Thoroughly: Vortex the mixture for a few minutes to ensure thorough mixing.
- Centrifugation: Centrifuge the mixture to separate the aqueous and organic phases. The estrone will partition into the organic phase.
Separation
- Collect Organic Phase: Carefully collect the upper organic phase (where estrone is present) into a clean glass test tube.
- Repeat Extraction: Perform the extraction step multiple times (usually 2-3 times) to ensure maximum recovery of estrone.
Evaporation
- Evaporate Solvent: Evaporate the collected organic solvent under a stream of nitrogen gas or using a rotary evaporator. Avoid overheating to prevent degradation of estrone.
- Reconstitution: Reconstitute the dried residue in a small volume of assay buffer (usually provided with RIA or CLIA kits).
Purification (if necessary)
- Solid-Phase Extraction (SPE): For additional purification, you can use SPE columns. Pass the reconstituted sample through the column, elute estrone, and then evaporate and reconstitute again.
Immunological Analysis
- RIA or CLIA: Follow the manufacturer’s protocol for the immunological assay. Typically, this involves adding the sample to a microplate or tube, incubating with a specific antibody, and measuring the signal generated by estrone binding.