Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Nara, Seema; Tripathi, Vineeta; Chaube, Shail K.; Rangari, Kiran; Singh, Harpal; Kariya, Kiran P.; Shrivastav, Tulsidas G.

doi:10.1016/j.ab.2007.10.042

Cited by 20 publications

(18 citation statements)

References 30 publications

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“…The insertion of hydrophilic spacers in OP-Pep2-HRP conjugate enhanced the sensitivity significantly. The finding was also supported by study carried out by Nara 19 , et al in which insertion of hydrophilic spacer resulted in the enhancement of sensitivity and specificity of assay. Possibly the use of a hydrophilic spacer helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.…”

Section: Resultssupporting

confidence: 75%

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Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

et al. 2016

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The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC 50 ), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC 50 value achieved was 0.01 µg mL −1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.

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Section: Resultssupporting

confidence: 75%

“…The Effect of ELISA format and spacer of enzyme conjugate both were found to play an important role in order to improve the sensitivity of immunoassay [19][20] . There are two kinds of assay formats, with either immobilised antigen or antibody.…”

Section: Introductionmentioning

confidence: 99%

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

et al. 2016

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“…This means that the sensitivity and ED 50 of the assay depend partly on the nature of the steroid and spacer arm link to carrier protein and the enzyme. [35] CONCLUSION From the present study, we conclude that the physicochemical nature of the bridge and steroid is determinant in defining the assay parameters. Urea being a 3 atom homo-bifunctional non-aliphatic molecule, cheaply available, and hydrophobic in nature, could be tried for other steroids= hapten assays, and its role as a spacer needs to be studied more deeply at the structural level.…”

Section: Downloaded By [Anadolu University] At 06:35 26 December 2014mentioning

confidence: 82%

“…This differential behavior of different linkers toward the sensitivity and ED 50 of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates. [35] The incorporation of spacer arms might have resulted in minimization of the conventional bridge recognition effects, decrease in the influence of local environment, and overcoming steric constraints between the two high molecular weight proteins, i.e., antibody and label. [15][16][17] The specificities of the antibodies obtained depend partly on the nature of the steroid link to the protein carrier, in particular the position on the steroid nucleus at which the linker is attached, since this determines the orientation of the steroid in the antibody combining site.…”

Section: Precisionmentioning

confidence: 99%

Influence of Different Length Linker Containing Dhea-7-Cmo-Enzyme Conjugates on Sensitivity and Specificity of Dhea-3-Hs-Antibody

Shrivastav

Chaube

Kariya³

et al. 2012

Journal of Immunoassay and Immunochemistry

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The introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced different homobifunctional spacers having varying atomic length (3 to 10) between enzyme and dehydroepiandrosterone (DHEA) moiety and studied their effects on functional parameters such as sensitivity and specificity of DHEA enzyme immunoassays. DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-carboxymethyloxime (DHEA-7-CMO) as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as an enzyme label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that DHEA moiety having a 3-hemisuccinate carboxyl arm that is hydrophilic in nature and spacer arm urea that is also hydrophilic in nature when used for the link to the protein carrier and enzyme for the preparation of immunogen and enzyme conjugate respectively resulted in development of assay having comparable sensitivity and lowest ED(50) as compared to other spacers. Thus sensitivity and ED(50) of the assay depend partly on the nature of the steroid and spacer arm link to the carrier protein and the enzyme.

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“…Van Weemen and Schuurs [41] observed that hapten and bridge heterology have a lesser effect on sensitivity compared with site-heterologous systems. In later studies, it has been reported that both bridge and hapten heterology improved sensitivity of EIAs for cortisol, [23,35,44] testosterone, [17,45] progesterone, [22,24,46] and DHEA. [34] In these heterologous assays, an EIA becomes specific and sensitive due to the use of structurally different hapten derivatives.…”

Section: Discussionmentioning

confidence: 95%

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

Shrivastav

Chaube

Kariya³

et al. 2011

Journal of Immunoassay and Immunochemistry

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Homologous and heterologous combinations of enzyme conjugate and antibody in steroid enzyme immunoassay (EIA) influences unlabeled steroid recognition by antibody that affects sensitivity of the assay. To develop dehydroepiandrosterone (DHEA) antigen heterologous enzyme linked immunosorbent assay (ELISA), antibodies were generated against DHEA-3-hemisuccinate-bovine serum albumin (DHEA-3-HS-BSA), DHEA-7-carboxymethyloxime-bovine serum albumin (DHEA-7-CMO-BSA), and DHEA-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA). Five horseradish peroxidase (HRP) enzyme conjugates were prepared using five testosterone derivatives [testosterone-3-CMO (T-3-CMO), testosterone-17-HS (T-17-HS), testosterone-17-glucuronoside (T-17-G), testosterone-19-carboxymethylether (T-19-CME), and testosterone-11-HS (T-11-HS)]. Fifteen antigen heterologous combinations of antibody and enzyme conjugates were evaluated in the standard binding assay; only two combinations showed binding. The use of antigen heterologous combination (different antigen in label than the immunogen) resulted in development of a simple, direct, and convenient assay as it permits the direct addition of the serum sample into the assay and it requires only 1.5 h to complete.

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Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay

Cited by 20 publications

References 30 publications

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

Influence of Different Length Linker Containing Dhea-7-Cmo-Enzyme Conjugates on Sensitivity and Specificity of Dhea-3-Hs-Antibody

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

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