KRIBIOLISA Neutralizing Antibodies to Pertuzumab (PERJETA) ELISA includes features like:
– Ready to use protocol with break-apart wells for ease of use
– Standardisation and High Reproducibility
– Lot to Lot Consistency
– Accuracy and Precision
Validated against seven points for a ?GOLD RING? Standard Quality ELISA – the benchmark sign for Krishgen Quality. KRIBIOLISA ELISA kit is used for assessing the specific biomarker in samples analytes which may be human serum, plasma, biological fluids and cell culture supernatant. The kit uses indirect sandwich assay with double antibodies / recombinant proteins – capture and detection to ensure a high degree of sensitivity and specificity in the estimation of the analyte. Extensive validation has been done on these kits. Please ask for our validation guide if not availableon our product page. Incase you wish us to customize the kit, connect with us at sales1@krishgen.com
Background: 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. Pertuzumab is a recombinant humanized monoclonal antibody that targets the extracellular dimerization domain (subdomain II) of the human epidermal growth factor receptor 2 protein (HER2). It consists of two heavy chains and two lights chains that have 448 and 214 residues respectively. It was first approved by the FDA in 2012 for use with docetaxel and another HER2-targeted monoclonal antibody, trastuzumab, in the treatment of metastatic HER2-positive breast cancer. Its indicated conditions have since expanded to include use as both a neoadjuvant therapy and an adjuvant therapy in the treatment of HER2-positive breast cancers at high risk of recurrence. Immunotherapy with antibodies directed at the programmed death 1 (PD-1) / PD ligand 1 (PD-L1) axis has shown remarkable activity in a variety of tumor types in clinical trials and has been approved worldwide for a number of malignancies (Robert et al. 2014; Weber et al. 2015; Fehrenbacher et al. 2016; Antonia et al. 2017; Apolo et al. 2017; Seiwert et al. 2016; Ansell et al. 2015; El-Khoueiry et al. 2017; Kaufman et al. 2016; Gong et al. 2018; Ahn et al. 2019; Khozin et al. 2019).
However, the production of ADAs is generally a treatment risk with therapeutic antibodies and can lead to an altered pharmacokinetic (PK) profile, which in turn may impact the safety and efficacy of therapeutic antibodies (Garcjs and Demengeot 2018; Bloem et al. 2017). 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). Thus, the presence of NAbs is concerning because they block the therapeutic function of the antibody and are likely to lead to reduced efficacy (Garcjs and Demengeot 2018; Bloem et al. 2017; Gunn et al. 2016). Furthermore, NAbs may lead to safety issues, such as hypersensitivity and anaphylaxis (Gunn et al. 2016; Krishna and Nadler 2016). Therefore, programs developing therapeutic antibodies or biosimlars to Pertuzumab need to develop and validate assays that adequately assess the presence of NAbs in the serum of patients treated with such therapeutic agents. 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).
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, non-cell-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 Pertuzumab 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.
Principle:
The method employs two step competitive inhibition sandwich ELISA technique. The protein-protein interaction between HRP-PD-L1 and PD1 can be blocked by Pertuzumab. The neutralizing antibodies against Pertuzumab bind to Pertuzumab and thus a competitive inhibition is created. Samples and controls are pipetted in a blank microtitre plate and incubated with Pertuzumab. Neutralizing Antibodies to Pertuxumab present in the samples / positive control will bind to Pertuzumab. This complex of bound and unbound Pertuzumab is then incubated with immobilized PD-1. The unbound Pertuzumab present will bind to PD-1. After washing, the detection conjugate of PD-L1:HRP is added. Post washing again, a substrate solution (TMB) is added to the microwells. Post incubation, color develops inversely proportionate to the amount of unbound Pertuzumab in the sample which inhibits PD-L1 to bind to the immobilized PD-1. Color development is then stopped by addition of stop solution. Absorbance is measured at 450 nm.
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