A major challenge of peptide quantification is to avoid matrix interference that can lead to biased results. Here we present a new sample preparation guide for CGRP quantification with Bertin Bioreagent CGRP ELISA kits.
General precautions
- All samples must be free from organic solvents prior to assay.
- Samples should be assayed immediately after collection or should be stored at -20°C or at -80°C prior the use with the assay.
Nervous tissue samples
Nervous tissues such as cerebrospinal fluid may be assayed directly if diluted more than 1/20 in ELISA buffer. Other nervous tissues such as spinal cord may be assayed after extraction procedure. Basically, the procedure [6] is to homogenize the tissue in 2N acetic acid (1 mg tissue in 4 mL acid), heat at 90°C for ten minutes, centrifuge, freeze-dry the supernatant (if freeze-drying is not possible a vacuum centrifugation with controlled temperature (+4°C) can be used.), and store under lyophilized form. Just before assay, reconstitute with ELISA buffer.
Plasma and serum samples
Plasma and serum samples should be measured according to one of these two methods:
- Either without an extraction procedure (option 1, left side of the scheme below); in which case the QC and standards should be reconstituted with CGRP-free plasma/serum.
- or with an extraction procedure (option 2, right side of the scheme below); in which case the QC and standards should be reconstituted in ELISA buffer.
Option 1: Without extraction procedure
In this first option, the common matrix for samples, standards and QC is plasma or serum. Therefore, CGRP Standard and Quality Control have to be reconstituted with plasma or serum that is free from CGRP (human), instead of the ELISA buffer as mentioned in reagent preparation section (CGRP standard and Quality Control). The dilutions of the CGRP Standard should also be prepared with plasma or serum that is free from CGRP (human).
If you don’t have plasma or serum that is free of CGRP (human), Bertin Bioreagent offers CGRP Affinity Sorbent containing anti-CGRP monoclonal antibody (the same as the one coated on the wells) as item Cat# A19482. To prepare CGRP-free plasma, use this Affinity Sorbent Cat# A19482 with a pool of 2 or 3 different sources of plasma or serum.
Option 2: With extraction procedure
In this option, Standard, QC and samples are assayed in ELISA buffer as a matrix. Please refer to the extraction protocol below to process your samples before the ELISA assay.
