Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers
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 se...
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Immunology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1200328/full |
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| author | Dinesh Kumar Dinesh Kumar Harinder Singh Oberoi Harpal Singh Harpal Singh Tulsidas G. Shrivastav Prudhvi Lal Bhukya Mansi Kumari Bidhan Chandra Koner Bidhan Chandra Koner Subash Chandra Sonkar Subash Chandra Sonkar |
| author_facet | Dinesh Kumar Dinesh Kumar Harinder Singh Oberoi Harpal Singh Harpal Singh Tulsidas G. Shrivastav Prudhvi Lal Bhukya Mansi Kumari Bidhan Chandra Koner Bidhan Chandra Koner Subash Chandra Sonkar Subash Chandra Sonkar |
| author_sort | Dinesh Kumar |
| collection | DOAJ |
| description | 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. |
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| institution | Directory Open Access Journal |
| issn | 1664-3224 |
| language | English |
| last_indexed | 2024-03-12T13:56:34Z |
| publishDate | 2023-08-01 |
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| spelling | doaj.art-1c7a653efe844a77b5a5974a3807c7b62023-08-22T12:45:26ZengFrontiers Media S.A.Frontiers in Immunology1664-32242023-08-011410.3389/fimmu.2023.12003281200328Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacersDinesh Kumar0Dinesh Kumar1Harinder Singh Oberoi2Harpal Singh3Harpal Singh4Tulsidas G. Shrivastav5Prudhvi Lal Bhukya6Mansi Kumari7Bidhan Chandra Koner8Bidhan Chandra Koner9Subash Chandra Sonkar10Subash Chandra Sonkar11Department of Reproductive Biomedicine, National Institute of Health and Family Welfare (NIHFW), New Delhi, IndiaQuality Assurance Division, Food Safety and Standards Authority of India (FSSAI), New Delhi, IndiaQuality Assurance Division, Food Safety and Standards Authority of India (FSSAI), New Delhi, IndiaCentre for Biomedical Engineering, Indian Institute of Technology Delhi (IIT-D), New Delhi, IndiaDepartment of Biomedical Engineering, All India Institute of Medical Sciences Delhi (AIIMS-D), New Delhi, IndiaDepartment of Reproductive Biomedicine, National Institute of Health and Family Welfare (NIHFW), New Delhi, IndiaRodent Experimentation Facility, Indian Council of Medical Research (ICMR)-National Animal Resource Facility for Biomedical Research (Indian Council of Medical Research (ICMR)-NARFBR), Hyderabad, IndiaDr. D. Y. Patil Biotechnology and Bioinformatics Institute, Dr. D. Y. Patil Vidyapeeth, Pune, IndiaMultidisciplinary Research Unit, Maulana Azad Medical College and Associated Hospital, New Delhi, IndiaDepartment of Biochemistry, Maulana Azad Medical College and Associated Hospital, New Delhi, IndiaMultidisciplinary Research Unit, Maulana Azad Medical College and Associated Hospital, New Delhi, IndiaDelhi School of Public Health, Institute of Eminence, University of Delhi, Delhi, IndiaThe 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.https://www.frontiersin.org/articles/10.3389/fimmu.2023.1200328/fullheterologous ELISAbridge/spacersprednisoloneantibodyhorseradish peroxidaseenzyme conjugates |
| spellingShingle | Dinesh Kumar Dinesh Kumar Harinder Singh Oberoi Harpal Singh Harpal Singh Tulsidas G. Shrivastav Prudhvi Lal Bhukya Mansi Kumari Bidhan Chandra Koner Bidhan Chandra Koner Subash Chandra Sonkar Subash Chandra Sonkar Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers Frontiers in Immunology heterologous ELISA bridge/spacers prednisolone antibody horseradish peroxidase enzyme conjugates |
| title | Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers |
| title_full | Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers |
| title_fullStr | Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers |
| title_full_unstemmed | Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers |
| title_short | Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers |
| title_sort | development and optimization of an in house heterologous elisa for detection of prednisolone drug in enzyme conjugates using spacers |
| topic | heterologous ELISA bridge/spacers prednisolone antibody horseradish peroxidase enzyme conjugates |
| url | https://www.frontiersin.org/articles/10.3389/fimmu.2023.1200328/full |
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