Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins

Kim, Bokhee; Sig, Geun; Meyerhoff, Mark E.

doi:10.1021/ac00223a003

Cited by 19 publications

(13 citation statements)

References 23 publications

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“…Among the enzymes tested, G6PDH has been extensively used for labeling in homogeneous EMIT assays . There are two main methods for conjugating G6PDH and analytes.…”

Section: Discussionmentioning

confidence: 99%

Glucose‐6‐phosphate dehydrogenase mutant D306C has a higher inhibition rate for enzyme multiplied immunoassay of cholyglycine

Qi¹,

Xiao²,

Zhang³

et al. 2019

Biotech and App Biochem

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Glucose-6-phosphate dehydrogenase (G6PDH) has been used in enzyme multiplied immunoassay technique (EMIT) assays for detecting small molecule metabolites such as cholyglycine (CG). A key parameter for successful EMIT CG assay development is the inhibition rate of the G6PDH-CG conjugate, measured as the decrease in enzyme activity upon CG antibody binding. Several commonly used G6PDH cysteine mutants including A45C and K55C have been labeled with CG-maleimide derivative, but inhibition rates of are unsatisfactory. Herein, we investigated whether other mutation sites can achieve better inhibition rates. We generated eight cysteine mutants (K106C, Y155C, A201C, T258C, D306C, D375C, G426C, and D480C) of G6PDH, measured their inhibition rates, and evaluated the performance of the D306C mutant using EMIT CG assays. One of the eight mutants (D306C) displayed improved inhibition rate, whereas all others exhibited inhibition similar to or lower than that of A45C and K55C. The enhanced inhibition rate of D306C improved the EMIT CG assay calibration curve, using an Abbott c16000 automated biochemical analyzer, resulting in better repeatability, precision, and linearity than with K55C assays and a commercially available EMIT CG kit. The G6PDH mutant D306C has a higher inhibition rate in EMIT CG assays and improves assay performance.

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“…Among the enzymes tested, G6PDH has been extensively used for labeling in homogeneous EMIT assays . There are two main methods for conjugating G6PDH and analytes.…”

Section: Discussionmentioning

confidence: 99%

Glucose‐6‐phosphate dehydrogenase mutant D306C has a higher inhibition rate for enzyme multiplied immunoassay of cholyglycine

Qi¹,

Xiao²,

Zhang³

et al. 2019

Biotech and App Biochem

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“…Lectin based recognition: Lectins constitute a broad family of proteins involved in diverse biological processes, occasionally having potent toxic properties. Lectins generally exhibit strong binding to specific carbohydrate moieties known as glycans, and this property has been extensively exploited as a basis for biosensor design 15 . Furthermore, particular structural profiles of glycans and their recognition by lectins have been attributed to disease progression, making analysis of saccharide-lectin binding processes important as a diagnostic tool.…”

Section: Fig 5: Schematic Representation Of the Preparation Of Molecmentioning

confidence: 99%

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2013

IJPSR

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Biosensors have revolutionized the way of diagnosis by allowing detection of diseases at earlier stage & detection of altered amount of biomarkers in the body. All biosensors usually involve minimal sample preparation as the biological sensing component is highly selective for the analyte concerned. There are numerous biosensors available now-a-days such as electrochemical biosensors, optical biosensors, DNA biosensors, microbial biosensors, mass based biosensors, calorimetric biosensors etc, which enable reliable & fast detection of analyte & provide accurate results at earliest. Biosensors have many uses in clinical analysis, general health care monitoring, veterinary and agricultural applications, industrial processing and monitoring, and environmental pollution control. Biosensor technology has the potential to provide fast and accurate detection, reliable imaging of cancer cells, and monitoring of angiogenesis and cancer metastasis, and the ability to determine the effectiveness of anticancer chemotherapy agents. INTRODUCTION: A biosensor is an analytical device, used for the detection of an analyte that combines a biological component with a physicochemical detector. It consists of three parts. A) Sensitive biological element (biological material (e.g. tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derived material or biomimic component) that interacts (binds or recognizes) the analyte under study. The biologically sensitive elements can also be created by biological engineering.

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“…Lectins generally exhibit strong binding to specific carbohydrate moieties known as glycans, and this property has been extensively exploited as a basis for biosensor design (Kim et al, 1990). Furthermore, particular structural profiles of glycans and their recognition by lectins have been attributed to disease progression, making analysis of saccharide-lectin binding processes important as a diagnostic tool.…”

Section: Lectin Based Recognitionmentioning

confidence: 99%