Tag: ELISA

Using the Ratio of Phosphorylated to Non-phosphorylated Forms of Stress Kinase PKR as a Potential Diagnostic Test for ME/CFS

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex illness characterized by a set of mainly neurological symptoms lasting for over 6 months. Currently, there is no definitive laboratory diagnostic test readily accessible to all clinicians and patients, and so clinical diagnosis occurs only after an exhaustive process of exclusion of all other possible causes of the varied symptoms experienced by the patient.

Here we present the development of a method that uses specific antibodies able to identify a changed ratio of phosphorylated and active protein kinase R in the peripheral blood monocyte cells (PBMCs) and neutrophil cells from a small group of ME/CFS sufferers, compared to age and sex-matched controls.

Protein kinase R (PKR) is an RNA-activated immune protein and stress kinase that has been observed to be present in its cleaved, auto-phosphorylated, and active form in past ME/CFS studies. After further validation, the activation status of PKR detected via specific antibodies in an ELISA format has potential for a simple readily accessible diagnostic tool for the early acute stage of ME/CFS illness, or as a long-term measure to evaluate the disease status.

Source: Sweetman E, Tate WP. Using the Ratio of Phosphorylated to Non-phosphorylated Forms of Stress Kinase PKR as a Potential Diagnostic Test for ME/CFS. Methods Mol Biol. 2025;2920:13-28. doi: 10.1007/978-1-0716-4498-0_2. PMID: 40372675. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_2

Measuring Biomarkers of Oxidative Stress in ME/CFS Patients

Abstract:

Patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) have a deficiency in energy production as a result of dysfunctions in their mitochondrial metabolism, defects in the complexes of the electron transport chain, and in the regulation of reactive oxygen species (ROS). This can lead to an imbalance and excess of these species with subsequent modifications of proteins, lipids, and DNA.

Oxidative stress is defined as an accumulation of ROS due to a loss of regulation and the subsequent inability to detoxify them. The modifications to the cellular macromolecules by ROS can be used as biomarkers of oxidative stress and so have the potential to monitor the disease course of a condition like ME/CFS.

Proteins are especially vulnerable to oxidative stress as amino acid residues are naturally modified as part of cell signaling so, in an imbalance between ROS and antioxidants, proteins become modified at multiple sites potentially altering structure and function. Protein carbonyl modifications are stable and can be measured using 2,4-dinitrophenylhydrazine using a commercial ELISA assay. This has been applied here to immune cell proteins and plasma from ME/CFS patients who had moderate functional activity before and during an exercise protocol, and was shown to have potential as a marker of oxidative stress in these patients. The methods used to measure the DNA modification, 8-hydroxy-2′-deoxyguanosine (8-OHdG) are known to give varied results depending on the technology used.

Here, a commercial ELISA assay did not have the sensitivity to detect the modifications in the DNA before and during the exercise protocol of these ME/CFS patients.

Source: Walker M. Measuring Biomarkers of Oxidative Stress in ME/CFS Patients. Methods Mol Biol. 2025;2920:225-244. doi: 10.1007/978-1-0716-4498-0_13. PMID: 40372686. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_13

Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method

Abstract:

A commercial line blot using recombinant antigens was compared with a commercial ELISA and ‘in-house’ IFA (reference test). Two panels were evaluated: Panel A was selected to distinguish between primary infections (89), past infections (20) and seronegatives (8) in immunocompetent individuals. In panel B, patients with a high number of reactivations were included: immunosuppressed patients (37), lymphoma (19), nasopharyngeal carcinoma (10), chronic fatigue syndrome (14). Blood donors (43) and cross-reactive sera (29) were added as controls.

Line blot and IFA were concordant in 94% of primary infections, 100% of seronegatives and 100% of past infections, similar to ELISA. Results differed significantly with regard to reactivations. When compared with IFA, the incidence of reactivations was overestimated by the blot, 24 and 58% in blood donors and cross-reactive sera, respectively. ELISA showed a similar problems with 21 and 34% indeterminate results, respectively.

The line blot is easy to carry out, has a good concordance with the reference IFA for primary infections, and is, therefore, a sufficient choice for distinguishing primary infection from seronegative and past infection. EBV reactivation assessment will require other methods such as EBV viral load.

 

Source: Gärtner BC, Fischinger JM, Roemer K, Mak M, Fleurent B, Mueller-Lantzsch N. Evaluation of a recombinant line blot for diagnosis of Epstein-Barr Virus compared with ELISA, using immunofluorescence as reference method. J Virol Methods. 2001 Apr;93(1-2):89-96. http://www.ncbi.nlm.nih.gov/pubmed/11311347