Antigen Heterologous Enzyme Linked Immunosorbent Assay for the Measurement of Dhea in Serum

Shrivastav, Tulsidas G.; Chaube, Shail K.; Kariya, Kiran P.; Kumar, Dinesh; Singh, Rita

doi:10.1080/15321819.2011.570117

Cited by 5 publications

(5 citation statements)

References 41 publications

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“…antibody and label. It has also been previously reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA [1][2][3][4][5]11,19,20 as it does have an effect in some other immunoassay and auxiliary binding systems [9][10][11]20 . The nding of present study also support the studies carried out by Nara Possibly the use of a hydrophilic and rigid spacer helped in projecting the hapten away from enzyme, leading to enhanced antibody binding signal and improved sensitivity of the assay [9][10][11]20 .…”

Section: Discussionmentioning

confidence: 99%

“…It is not always easy to develop speci c and sensitive ELISA for small molecule like prednisolone, a synthetic steroid which is not naturally present in the body. One of the most important factors that determine the sensitivity and speci city is the combination of antibody and enzyme conjugate used in the assay [1][2][3][4] .…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, homologous combination does not provide satisfactory sensitivity, because the binding a nity of the labeled antigen to antibody is higher than that of the antigen to be measured 12,13 . But few sensitive homologous assays for steroids have been developed [1][2][3][4]12,13 .…”

Section: Introductionmentioning

confidence: 99%

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ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

Sonkar

Kumar

Oberoi³

et al. 2023

Preprint

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The introduction of spacers in coating steroid protein complex and / or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers between enzyme and prednisolone (PSL) moiety having atomic length 3 to 10 and studied their influence on functional parameters such as sensitivity and specificity of PSL enzyme immunoassays. In this study, four enzyme conjugates of PSL-21-HS and HRP were prepared by N-Hydroxy Succinimide mediated carbodiimide reaction, these were PSL-21-HS-adipic acid dihydrazide (ADH)-HRP, PSL-21-HS- carbohydrazide (CH)-HRP, PSL-21-HS-ehylenediamine (EDA)-HRP and PSL-21-HS- urea (U)-HRP. The assays were developed using these enzymes conjugate with antibody raised against PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of bridge in enzyme conjugate were 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL and 0.018 ng/mL with ADH, CH, EDA and urea as spacer respectively. Among all four combinations, PSL-21-HS-BSA antibody and PSL-21-HS-U-HRP enzyme conjugate gave better sensitivity and showed cross-reaction with less number of steroids. 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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

Sonkar

Kumar

Oberoi³

et al. 2023

Preprint

Self Cite

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show abstract

“…It is not always easy to develop a specific and sensitive ELISA for a small molecule such as prednisolone, a synthetic steroid that is not naturally present in the body. One of the most important factors that determine the sensitivity and specificity is the combination of antibody and enzyme conjugate used in the assay (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

“…However, the homologous combination does not provide satisfactory sensitivity, because the binding affinity of the labeled antigen to the antibody is higher than that of the antigen to be measured (18,19). Few sensitive homologous assays for steroids have been developed (1)(2)(3)(4)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Section: Introductionmentioning

confidence: 99%

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

Kumar,

Oberoi,

Singh

et al. 2023

Front. Immunol.

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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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Label-free impedimetric immunosensor based on arginine-functionalized gold nanoparticles for detection of DHEAS, a biomarker of pediatric adrenocortical carcinoma

Lima

Inaba

Lopes

et al. 2019

Biosensors and Bioelectronics

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Antigen Heterologous Enzyme Linked Immunosorbent Assay for the Measurement of Dhea in Serum

Cited by 5 publications

References 41 publications

ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

ELISA Assay for estimation of Prednisolone Drug Using Bridge Heterology in Enzyme Conjugates

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

Label-free impedimetric immunosensor based on arginine-functionalized gold nanoparticles for detection of DHEAS, a biomarker of pediatric adrenocortical carcinoma

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