title: Specific detection of tetanus toxoid using an aptamer-based matrix.
abstract: Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
authors.forename: Harshvardhan B
authors.initials: HB
authors.lastname: Modh
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
authors.forename: Ankan K
authors.initials: AK
authors.lastname: Bhadra
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
authors.forename: Kinjal A
authors.initials: KA
authors.lastname: Patel
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
authors.forename: Rajeev K
authors.initials: RK
authors.lastname: Chaudhary
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India.
authors.forename: Nishant K
authors.initials: NK
authors.lastname: Jain
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Punjab 160 062, India. Electronic address: ipsita@niper.ac.in.
authors.forename: Ipsita
authors.initials: I
authors.lastname: Roy
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry*; D004355:Drug Stability; D004356:Drug Storage; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005456:Fluorescent Dyes; D057230:Limit of Detection; D015203:Reproducibility of Results; D013696:Temperature; D013745:Tetanus Toxoid / Q000032:analysis* / Q000737:chemistry; D014612:Vaccines
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D013745:Tetanus Toxoid; D014612:Vaccines
keywords: ALISA (aptamer-linked immobilized sorbent assay); Aptamers; ELISA (enzyme-linked immunosorbent assay); Tetanus toxoid