Detecting total immunoglobulins in diverse animal species with a novel split enzymatic assay

Abstract

BackgroundTotal immunolobulin G concentration is a useful, albeit underutilized, diagnostic parameter for health assessments of non-domestic animal species, due to a lack of functional diagnostic tools. Traditional assays, including enzyme-linked immunosorbent assay or radial immunodiffusion, require development of specific reagents (e.g., polyclonal antisera and appropriate protocols) for each animal species, precluding wide and easy adoption in wildlife welfare. As an alternative, bacterial virulence factors able to bind IgGs in antigen-independent manner can be used. To further simplify the diagnostic procedure and increase the number of species recognized by an assay, in this study a recently developed Split Trehalase immunoglobulin assay (STIGA) with bIBPs as a sensing elements was used to detect antibodies in 29 species from 9 orders. Three bacterial immunoglobulin binding proteins (protein G, protein A and protein L) were incorporated into STIGA reagents to increase the number of species recognized.ResultsIgG concentrations were detected through glucose production and produced signals were categorized in 4 categories, from not active to strong signal. Activation was detected in almost all tested animal species, apart from birds. Incorporation of Protein G, Protein A and Protein L allowed detection of IgGs in 62, 15.5 and 6.9% of species with a strong signal, respectively. Assays combining 2 bacterial immunoglobulin binding proteins as sensing element generally gave poorer performance than assays with the same bacterial immunoglobulin binding proteins fused to both trehalase fragments.ConclusionsSTIGA assays have potential to be further developed into an easily adoptable diagnostic test for total amount of IgGs in almost any serum sample, independent of species.

Determining Immunoglobulin Content of Bovine Colostrum and Factors Affecting the Outcome: A Review

“…The glucose formed can be detected with a colorimetric assay or a glucometer. Based on the glucose, the IgG concentration in the colostrum can then be indirectly inferred [ 27 , 63 ].…”

Engineering Protein Switches for Rapid Diagnostic Tests

“…Reporters that tap into this established technology have been used in protein switches. Trehalase reporters generate glucose so activity can be indirectly measured with a glucometer, although background glucose in the biological matrix complicates measurements. ,, This is avoided if glucose dehydrogenase (GDH) is itself used as a reporter, with a saturating concentration of glucose substrate. GDH is well developed and offers advantages in respect to its ease of manufacture, high stability, turnover rate, and functionality in biological matrices. ,,,, Nevertheless, detection of glucose oxidation by GDH protein switches has largely been done colorimetrically, and sensitivity is very low when measured electrochemically with a glucometer. ,,,, Standard glucometers are set up to measure turnover by fully active GDH, and adaptations are needed to improve sensitivity for protein-switch measurements.…”

“…In proximity switches, analyte binding induces colocalization of sensor components, which results in a change in response (Figure ). The most common approach is split-enzyme complementation, in which an enzyme is split into two inactive fragments, usually directly but sometimes via mutations at a dimerization interface. ,,− ,, Each fragment is attached to recognition elements that bind nonoverlapping epitopes on the analyte, such that binding colocalizes the fragments and induces reconstitution of the active enzyme. ,,− ,, This approach is insensitive to the exact geometry of the analyte and binding domains, so long as linkers between the binder and fragment are sufficient. Once colocalized, the high effective concentration and residual affinity between fragments should drive reconstitution, regardless of exact orientations.…”

“…The NanoBit system was linked to antibodies to form the basis of the recent Lumit cell-lysate immunoassay . To utilize NanoBit for diagnostic assays, both Dixon et al and Ohmuro-Matsuyama et al further split the system, to give a polypeptide and two peptide fragments. , The two small peptide fragments could be produced more easily in fusion with recognition elements and the polypeptide could be held at a separately high concentration in the assay. , The sensitivity (pM), response dynamic range (up to 100 000%), and analyte linear range (>3 orders of magnitude) of these systems are superior to other in vitro split-enzyme assays. ,,− ,, These qualities are a testament to the engineering effort that enabled low background complementation and high luminescent activity recovery. Nevertheless, tests were in a maximum 5% serum matrix, and use as a general format for wide ranging analytes is yet to be shown.…”

“…There are wide opportunities for protein switch antibody tests: those developed for infectious disease serology could be widened to autoimmune, allergy, and broader immunology applications, ,,,, and those for therapeutic drug monitoring can guide dosing in the ever-growing biologics market. ,,, Small molecule tests have also been developed for drug monitoring, , as well as metabolic , and disease biomarker assays. , Tests for a range of proteins have also been reported, and the scope expands as new biomarkers are discovered. ,,, Human health applications dominate the literature, but the significant opportunities for quantitative on-site tests should be explored in broader sectors. Some protein switch assays have been developed for food standards, agricultural disease monitoring, and veterinary immunology applications, , but there are much wider prospects in these settings and others, including environmental monitoring, illicit drug detection, and sports medicine …”