Abstract
The use of peptides in immunoassays can be favored over the use of the full protein when more cost effective or less toxic approaches are needed, or when access to the full protein is lacking. Due to restricted access to recombinant bovine somatotropin (rbST), a protein enhancing growth and lactating performances of livestock, which use has been banned in the EU, Canada and Australia (amongst others), we developed a peptide-based biorecognition assay on an imaging planar array analyzer. For this, we identified the rbST epitope that is responsible for binding to the rbST-targeting monoclonal antibody 4H12 (MAb 4H12) to be 115DLEEGILALMR125. This linear peptide was synthesized and coupled to microspheres, after which it was tested in a biorecognition competitive inhibition assay format. We observed IC50 values of approximately 0.11 μg mL−1, which are lower than observed for the full rbST protein (IC50 = 0.20 μg mL−1). Importantly, there was no binding with the scrambled peptide. Preliminary results of directly coupled peptides in a microsphere biorecognition assay for detection of rbST are presented. Real-life applicability for detection of somatotropins (STs) in injection preparations of bovine-, porcine- and equine ST are shown. This newly developed immunoassay strongly supports future developments of peptide-based immunoassays to circumvent the limited access to the full protein.
Highlights
When immunoassays detect a full protein, the use of this full protein in assay building is commonplace
We tested whether attachment to the microsphere via either the C- or the N-terminus of the peptide was preferred, and whether the biorecognition would benefit from the presence of a hydrophilic or hydrophobic spacer between the peptide and the microspheres (Figure 2)
Compared to the full protein recombinant bovine somatotropin (rbST) Microsphere Peptide-Based Immunoassays (MIPA), the peptide-based MIPA was found to be more sensitive with IC50 of 0.20 and 0.11 μg mL−1, respectively
Summary
When immunoassays detect a full protein, the use of this full protein in assay building is commonplace. The use of peptides offers advantages in (i) cost effectiveness, (ii) toxicity, (iii) pathogenicity,. In the development of serological tests for COVID-19, the high costs for the S1-protein could be avoided using a. 12-mer peptide, while at the same time showing increased specificity, resulting in an assay with high diagnostic performance [1]. In development of neutralizing antibodies for toxin therapy of, e.g., venoms, bacteria toxins, etc., antigenic peptides are used instead of the toxic full protein [2,3,4]. Peptide-based assays provide insights on epitope-specific antibody binding [8,9], which is related to pathogenesis, its prognosis
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