Influence of Different Length Linker Containing Dhea-7-Cmo-Enzyme Conjugates on Sensitivity and Specificity of Dhea-17-Cmo-Antibody

Shrivastav, Tulsidas G.; Chaube, Shail K.; Kariya, Kiran P.; Singh, Rita; Kumar, Dinesh; Pandit, Deepa; Ujawane, Pragati; Kumari, Poonam; Pandey, Bhavana

doi:10.1080/15321819.2011.560325

Cited by 9 publications

(15 citation statements)

References 31 publications

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“…[40] The affinity constant of the HSA-antibody for the antigen was estimated by Scatchard plot according to method described elsewhere. [21] The sensitivity of the developed method was determined according to the method described elsewhere. [22] The statistical analyses-such as mean, standard deviations, and logit-log transformation-of the data were done using the Microsoft Excel computer program.…”

Section: Data Analysis-preparation Of Standard Curve Determination Omentioning

confidence: 99%

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

Shrivastav

Kariya²,

Prasad

et al. 2014

Journal of Immunoassay and Immunochemistry

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Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13-100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38-10.32 % and 4.22-11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.

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Section: Data Analysis-preparation Of Standard Curve Determination Omentioning

confidence: 99%

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

Shrivastav

Kariya²,

Prasad

et al. 2014

Journal of Immunoassay and Immunochemistry

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“…A remarkable improvement in the sensitivity of ELISA has been observed by various researchers using spacers between antigens and coupling protein when used as coating antigen in antigen immobilized format [14][15][16][17] or between antigen and enzyme when used as enzyme conjugate in antibody immobilized format 5,13 . It was observed that in the antigen-immobilized format, varying lengths of spacer arm of the coating antigen had signi cant effect on the sensitivity of ELISA [14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

“…In the antibody-immobilized format, spacers have been coupled between steroid derivative and label protein to minimize the conventional bridge recognition effects and to overcome steric constraints between the two high molecular weight proteins, i.e., antibody and label [5][6][7][8][9][10][11]14,19,20 . Shrivastav et al…”

Section: Introductionmentioning

confidence: 99%

“…The study revealed that the use of a spacer between the steroid and enzyme in enzyme-conjugate increases the sensitivity and speci city of the assay. It has also been reported that the spacer length does not bear any correlation with the assay sensitivity in ELISA of steroids 1,[5][6][7][8][9][10][11]14,19,20 . Further, the nature of the spacer (hydrophilic, hydrophobic, exible, or rigid) is related to assay sensitivity and not to spacer length [5][6][7][8][9][10][11]14 .…”

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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“…Recent studies have begun to focus on biosensors for determination of doping materials [22]. For the determination of DHEA or DHEAÀ S, immunoassay based ELISA (enzyme-linked immunosorbent assay) [23] and one aptamer-based biosensor [24] exists in literature however, an antibody-based biosensor with a conventional electrochemical system is yet to be designed.…”

Section: Introductionmentioning

confidence: 99%

An Electrochemical Biosensor Platform for Testing of Dehydroepiandrosterone 3‐Sulfate (DHEA−S) as a Model for Doping Materials

et al. 2019

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Endogenous steroids such as dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3‐sulfate (DHEA−S) have commonly used as doping materials by athletes and to date novel techniques are needed for detection of these molecules. In this study, antibody‐based electrochemical biosensor has developed for testing level of the DHEA−S. For this aim, gold surfaces were initially modified with cysteamine (Cys) and then, DHEA−S antibody was immobilized on the surface via glutaraldehyde (GA) as a crosslinking agent. The stepwise modification of electrode surface was monitored by using various electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Linear range was determined as 2.5–100 ng/mL DHEA−S using differential pulse voltammetry (DPV) technique, as well. Moreover, repeatability (±S.D.), coefficient of variation (%) and limit of detection (LOD) values were calculated as 0.033, 1.030 and 3.971, respectively. Also, DHEA−S in synthetic serum and urine samples were successfully determined with standard addition method and confirmation analysis were performed with liquid chromatography quadrupole‐time of flight mass spectrometry (LC‐QTOF/MS) system. The selectivity was studied with the addition of some interfering molecules (testosterone, bovine serum albumin (BSA), cholesterol, uric acid, lactic acid, codein (COD), ascorbic acid, DHEA). Consequently, this work is proposed as practical, innovative and cost‐effective technique that can be easily adapted for the miniaturized form for the analysis of other doping substances as well as DHEA−S for the future works.

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Influence of Different Length Linker Containing Dhea-7-Cmo-Enzyme Conjugates on Sensitivity and Specificity of Dhea-17-Cmo-Antibody

Cited by 9 publications

References 31 publications

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

Development of Enzyme-Linked Immunosorbent Assay for Estimation of Urinary Albumin

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

An Electrochemical Biosensor Platform for Testing of Dehydroepiandrosterone 3‐Sulfate (DHEA−S) as a Model for Doping Materials

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