Description
Introduction:
The KRIBIOLISA™ ELISA kits are used for assessing the specific biomarker in samples analytes which may be serum and cell culture supernatant as validated with the kit. The kit employs a blocking ELISA technique which engages the blocking pathway to estimate the neutralizing antibodies. Peptides have also attracted attention in the field of tumor diagnosis and treatment because of their small size, high affinity, easy modification, and low immunogenicity. Semaglutide is a glucagon-like peptide 1 (GLP-1) analog used to manage type 2 diabetes along with lifestyle changes, such as dietary restrictions and increased physical activity. GLP-1, a major incretin hormone in humans, acts by numerous mechanisms like augmented insulin secretion (glucose-dependent), inhibition of glucagon release and suppressed hepatic gluconeogenesis. It also causes delayed gastric emptying, reduced appetite and energy intake. Reduction of HbA1c level along with body weight without any risk of hypoglycaemia, provides it a special status for the
treatment of obese type 2 diabetic patients. Semaglutide uses three strategies to improve the residence time of the peptide. First, stearic diacid is used instead of palmitic acid to increase the molecules affinity for albumin. Second, an extended spacer consisting of two 8-amino3,6- dioxaoctanoic acid (ADO) moieties and a glutamic acid residue further improves albumin binding stability. Third, the peptides second residue is mutated to aminoisobutyric acid to confer greater DPP IV resistance. These modifications result in a half-life of 165 hours in humans, making it suitable for once weekly administration. The fatty di-acid chain attached to the GLP-1R agonist provides semaglutide with a higher affinity for albumin.
Immunogenicity is also a critical concern with this class of therapeutics. Prolonged exposure to the drug could initiate the development of neutralizing antidrug antibodies, which reduces safety and efficacy. The production of ADAs is generally a treatment risk with therapeutic peptides and can lead to an altered pharmacokinetic (PK) profile, which in turn may impact the safety and efficacy of therapeutic peptides (Wang, L., Wang, N., Zhang, W. et al. Therapeutic peptides: current applications and future directions.). ADAs can be binding, leading to minimal or no impact; clearing, leading to impact through an altered PK profile; sustaining, leading to a prolonged exposure that may change the efficacy and/or safety; or neutralizing, leading to reduced pharmacological activity of the therapeutic antibody (Garcjs and Demengeot 2018; Bloem et al. 2017). Regulatory agencies prefer cell-based assays because they rely on cellular responses to NAb-mediated inhibition of therapeutic antibodies (Wu et al. 2016). Because of their mode of detection, these assays have been considered more biologically relevant than noncell-based assays (Bloem et al. 2017; Liao et al. 2012; Cong et al. 2015; Jolicoeur and Tacey 2012). However, cell-based assays are labor intensive and highly variable and exhibit low serum tolerance and poor drug tolerance (Bloem et al. 2017; Liao et al. 2012; Cong et al. 2015; Jolicoeur and Tacey 2012; Wu et al. 2016).
In contrast to cell-based assays, noncell-based assays often rely on binding of the drug and target for signal detection and quantitation. Competitive ligand-binding assays, used to characterize NAbs, have been shown to provide higher sensitivity, a wider dynamic range, increased precision, and better matrix tolerance than cell-based assays (Wu et al. 2018). Although regulatory agencies generally prefer cell-based assays, the United States Food and Drug Administration and the European Medicines Agency recognize both cell-based and noncell-based competitive ligand-binding assays as valid measurements of NAbs in some cases (Jolicoeur and Tacey 2012; Wu et al. 2016). The selection of either cell based or noncell-based assays is driven by several different variables, including therapeutic mechanism of action (MOA), assay performance characteristics, and risk of immunogenicity, with therapeutic MOA being the primary determinant (Wu et al. 2016).
The KRIBIOLISA™ Neutralizing Antibodies to Semaglutide is a competitive ligand-binding assay that is specific for the detection of anti-Pertuzumab NAbs in human serum. The kit is validated for assay precision, sensitivity, hook effect, selectivity, robustness, stability, and system suitability
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Principle:
The method employs two step competitive inhibition sandwich ELISA technique. The protein-protein interaction between Biotin-Anti-GLP-1 and GLP-1 can be blocked by Semaglutide. The neutralizing antibodies against Semaglutide bind to Semaglutide and thus a competitive inhibition is created. Samples and controls are pipetted in a blank microtitre plate and incubated with Semaglutide. Neutralizing Antibodies to Semaglutide present in the samples / positive control will bind to Semaglutide. This complex of
bound and unbound Semaglutide is then incubated with immobilized Anti-GLP-1. The unbound Semaglutide present will bind to Anti-GLP-1. After washing, the detection conjugate of Biotin GLP-1 is added. Post washing Streptavidin:HRP is pipetted. After incubation and washing, a substrate solution (TMB) is added to the microwells. Post incubation, color develops inversely proportionate to the amount of unbound Semaglutide in the sample which inhibits GLP-1 to bind to the immobilized Anti-GLP-1. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.
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