Abstract
Background:
It has been known for decades that many cytokines, such as IL-2, IL-6, and IL-12, bind to heparin. Even though some enzyme-linked immunosorbent assays (ELISA) use antibody-recognizing epitopes not affected by this binding, ELISA manufacturers often warn that heparinized plasma or serum fractions containing more than 3 IU (international units)/mL of heparin should not be used in assays so as to prevent heparin interference in the reaction. In addition, enzyme-based nucleic acid amplifications from heparinized samples have been shown defective by several research groups. The aim of this study was to determine optimal degradation and/or removal of heparin from heparinized blood samples to best turn them into fractions for appropriate ELISA and RT-PCR analysis.
Methods and Results:
A colorimetric reporter assay based on the metachromatic effect of the binding of heparin to toluidine blue was shown to be a low-cost effective method to discriminate assay compatible blood fractions with heparin levels below 3 IU/mL. Heparin removal from human blood fractions was best achieved by treatment with either Bacteroides Heparinase II or the less expensive Heparinase I at a final concentration of 0.1 U/μL and incubations at 30°C for a period between 30 min and 4 h, or by adsorption to Ecteola slurries at a concentration of 20 mg/mL for 20 min at room temperature (RT). The fact that both enzymatic and resin-based optimized treatments allowed for replication of the readings obtained with heparin-free equivalent fractions in both ELISA and RT-PCR assays indicates they should be appropriate for quantitative studies such as expression profiling at both the protein and nucleic acid level.
Conclusions:
The cost-effective protocols developed in this study could make heparinized, otherwise unusable, blood-derived collections suitable for analysis by ELISA and RT-PCR amplifications, among other analyses, enhancing the possibilities for studying valuable bio-banked heparinized human samples.
Get full access to this article
View all access options for this article.
References
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
