Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers

Frontiers in Immunology

Immunology

Harpal Singh¹  Tulsidas G. Shrivastav²  Dinesh Kumar³  Mansi Kumari⁴  Subash Chandra Sonkar⁵  Bidhan Chandra Koner⁶  Harinder Singh Oberoi⁷  Prudhvi Lal Bhukya⁸ 

[1] Centre for Biomedical Engineering, Indian Institute of Technology Delhi (IIT-D), New Delhi, India;Department of Biomedical Engineering, All India Institute of Medical Sciences Delhi (AIIMS-D), New Delhi, India;Department of Reproductive Biomedicine, National Institute of Health and Family Welfare (NIHFW), New Delhi, India;Department of Reproductive Biomedicine, National Institute of Health and Family Welfare (NIHFW), New Delhi, India;Quality Assurance Division, Food Safety and Standards Authority of India (FSSAI), New Delhi, India;Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, India;Multidisciplinary Research Unit, Maulana Azad Medical College and Associated Hospital, New Delhi, India;Delhi School of Public Health, Institute of Eminence, University of Delhi, Delhi, India;Multidisciplinary Research Unit, Maulana Azad Medical College and Associated Hospital, New Delhi, India;Department of Biochemistry, Maulana Azad Medical College and Associated Hospital, New Delhi, India;Quality Assurance Division, Food Safety and Standards Authority of India (FSSAI), New Delhi, India;Rodent Experimentation Facility, Indian Council of Medical Research (ICMR)-National Animal Resource Facility for Biomedical Research (Indian Council of Medical Research (ICMR)-NARFBR), Hyderabad, India;

关键词: heterologous ELISA;    bridge/spacers;    prednisolone;    antibody;    horseradish peroxidase;    enzyme conjugates;    spacers;    immunogens;   

DOI  :  10.3389/fimmu.2023.1200328

 received in 2023-04-04, accepted in 2023-07-20,  发布年份 2023

来源: Frontiers

【 摘 要 】

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length.

【 授权许可】

Unknown   
Copyright © 2023 Kumar, Oberoi, Singh, Shrivastav, Bhukya, Kumari, Koner and Sonkar