Molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin

Abstract

Haptoglobin (Hp) is a positive acute-phase protein and a valuable marker of inflammation in both human and veterinary medicine. The aim of this study was to validate the molecular characterization of Hp in dolphins and to validate commercially available Hp measurement methods such as Hp-ELISA (originally designed for pigs) and Hp-hemoglobin (Hb) binding assay. The dolphin Hp (dHp) amino acid sequence appeared most similar to pig Hp by sequence homology and phylogenetic clustering. Amino acid sequence analysis revealed that dHp comprises the Hp1 form of α1 and β chains. The anti-pig Hp antibody cross-reacted with both recombinant dHp, expressed by Escherichia coli, and dHp from serum. The intra- and inter-assay levels of imprecision of pig Hp-ELISA and the Hp-Hb binding assay were found to be tolerable for the determination of Hp in dolphin, and there was no significant discrepancy between the two determination methods. The ability of the assay to differentiate between healthy and inflammation groups was investigated, and a significant increase in Hp concentration was detected in inflammatory conditions. Thus, Hp is a useful inflammation marker for dolphin, and the Hp concentration in dolphin serum samples can be reliably measured using commercially available pig Hp-ELISA and Hp-Hb binding assay.

Acute Phase Proteins in Marine Mammals: State of Art, Perspectives and Challenges

“…The only clinical significance of these alteration was a higher ability to detect chronic inflammation for Hp. Regarding Hp, Segawa and colleagues validated commercially available Hp-ELISA and Hp-hemoglobin binding assay in bottlenose dolphins with ‘’acceptable” intra- and inter-assay imprecision (CV: 3.3% healthy dolphins and 3.5% inflamed dolphins; CV: 10.4% healthy dolphins and 21.7% inflamed dolphins) and demonstrated that Hp levels in the serum increase under inflammatory conditions (62).…”

“…This means the development of a de-novo method, often a time-consuming and expensive approach, or the validation of a commercial available assay used in other species (65). The latter approach is surely the most used in veterinary medicine, in which some human assays were validated for dogs, cats and horses (62). However, even if some APPs appeared highly conserved among species, an accurate validation of antibody cross reactivity is needed as well as species specific standards and control material (54).…”

“…In veterinary medicine, the validation studies do not always follow all these steps, mainly due to the lack of resources or technical limitation (44, 65). Also in marine mammals, the majority of studies had performed only some steps (44, 54, 56, 61, 62). This is mainly due to the limitation in species-specific reagents, the number of samples from animal with known health status and, last but not least, the capability to generate appropriate reference intervals, hampered the possibility to perform complete validation studies.…”

The plasma phospholipidome of Tursiops truncatus: From physiological insight to the design of prospective tools for managed cetacean monitorization

“…In humans, whole blood, plasma (the liquid portion of blood) and serum (plasma minus the clotting factors) biochemical analyses are still the most common way to assess physiological status or make an early disease diagnostic (Quinn et al, 2016;Sharma et al, 2018;Yu et al, 2011). That is also true for marine mammals, namely those in captivity (Hunt et al, 2013;Segawa et al, 2013;Terasawa et al, 2002). In humans, plasma lipid composition has been previously thoroughly characterized and it was found to contain free fatty acids (FAs), triglycerides, cholesterol (free and esterified), phospholipids, and sphingolipids, sterols, and prenols (Quehenberger et al, 2010;Quehenberger & Dennis, 2011).…”

Untitled

“…Alguns estabelecimentos da União Européia já estão em processo de implementação de sistemas de quantificação da haptoglobina de suínos e bovinos durante o abate, objetivando identificar animais portadores de infecções que representam risco à segurança alimentar e saúde pública, de forma a orientar as ações executadas na inspeção post-mortem (Blagojevic et al, 2011;Soler et al, 2013). A análise laboratorial pode ser conduzida por meio de ensaios imunológicos ou bioquímicos, os quais já estão disponíveis comercialmente (Cooke & Arthington 2012;Segawa et al, 2013;Czopowicz et al, 2017), e como material biológico podem ser utilizadas amostras de sangue, saliva e urina, assim como amostras de carne, que após trituradas liberam seus fluidos incorporados (Soler et al, 2013).…”