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. 2023 Aug 22:14:1200328.
doi: 10.3389/fimmu.2023.1200328. eCollection 2023.

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

Dinesh Kumar  1   2 , Harinder Singh Oberoi  2 , Harpal Singh  3   4 , Tulsidas G Shrivastav  1 , Prudhvi Lal Bhukya  5 , Mansi Kumari  6 , Bidhan Chandra Koner  7   8 , Subash Chandra Sonkar  7   9
Affiliations

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

Dinesh Kumar et al. Front Immunol. .

Abstract

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.

Keywords: antibody; bridge/spacers; enzyme conjugates; heterologous ELISA; horseradish peroxidase; immunogens; prednisolone; spacers.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Scheme 1
Scheme 1
Conjugation of PSL-21-HS (a carboxylic derivative of prednisolone) to BSA.
Scheme 2
Scheme 2
Blocking of the amino group and coupling of spacers (ADH, CH, EDA, and U) to HRP.
Scheme 3
Scheme 3
Coupling of prednisolone to spacer-HRP.
Figure 1
Figure 1
Mass spectra of PSL-21-HS, BSA, and PSL-21-HS-BSA by MALDI-TOF.
Figure 2
Figure 2
Mass spectra of PSL-21-HS, HRP and enzyme conjugate with spacers by MALDI-TOF.
Figure 3
Figure 3
Composite dose-response, logit-log, and Scatchard plot curves of the homologous and bridge heterologous ELISA of prednisolone using PSL-21-HS-BSA antibody PSL-21-HS-HRP with (ADH, CH, EDA, and U) different length spacer–containing enzyme conjugates.
Figure 4
Figure 4
Regression graph of the correlation between the serums prednisolone concentrations as estimated by the developed ELISA and an established ELISA kit.
Figure 5
Figure 5
Box-and-Whisker diagram of level of prednisolone in human serum samples estimated by developed ELISA.
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