Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Thompson, Roisin; Creavin, Aileen; O’Connell, Michael P.; O’Connor, Brendan; Clarke, Paul

doi:10.1016/j.ab.2011.02.013

Cited by 57 publications

(49 citation statements)

References 43 publications

(51 reference statements)

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“…ELLAs have reaction formats similar to those of enzyme-linked immunosorbent assay (ELISA), using 96-well plates and stepwise reactions with target substances. Unlike ELISAs, which are based on antibody reactions and are widely used, defects in lectin reaction conditions have hindered the use of ELLAs for diagnosis (15). Recently, the use of oxidized BSA (16,18) and polymer polyvinyl alcohol (15) has substantially improved the specificity and sensitivity of ELLAs.…”

Section: Lewismentioning

confidence: 99%

“…In the present study, alterations in glycosylation patterns were A lectin-based approach to detecting carcinogenesis in breast tissue (14). ELLA is the simplest method to analyze lectins; however lectin blotting and lectin microarrays may also be used (15). To date, ELLA systems have been used to investigate a limited number of materials, with purified glycoproteins analyzed most commonly (16,17).…”

Section: Introductionmentioning

confidence: 98%

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A lectin-based approach to detecting carcinogenesis in breast tissue

Moon

Kim

et al. 2016

Oncology Letters

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Abstract. It has been suggested that the diversity of glycosylation structures that form during cancer progression and the sensitivity with which they are able to be detected have great potential for cancer screening. However, the large majority of breast cancer research has instead focused on the development of protein or nucleic acid markers. In the present study, alterations in glycosylation in breast cancer tissue were analyzed using enzyme-linked lectin assays (ELLAs), which have potential for high-throughput screening. Cancer tissues (CCs) and normal tissues (CNs) were collected from women with breast cancer ranging from stage 0 to IIIA. The specimens were divided into two groups, stage 0-I and stage II-III, and the levels of four types of lectin in stage 0-I and stage II-III CCs and CNs were compared by ELLA. The results demonstrated that, relative to CNs, the CCs contained significantly enhanced levels of mannosylation (stage 0-I, P<0.001; stage II-III, P<0.001), galactosylation (stage 0-I, P<0.05; stage II-III, P<0.001), sialylation (stage 0-I, P<0.001; stage II-III, P<0.01) and fucosylation (stage 0-I, P<0.01; stage II-III, P<0.01). Furthermore, stage II-III CCs had higher levels of mannosylation (P<0.05) and galactosylation (P<0.01) than stage 0-I CCs. The sensitivity of the ELLA system ranged from 71-100% when specificity was set at 100%. These results demonstrate that enhanced glycosylation levels identified by ELLA are associated with the development of breast tumors, and provide evidence of the exceptional sensitivity and specificity of the ELLA system in the detection of breast cancer. This approach is anticipated to contribute highly to the development of reliable diagnostic procedures for breast cancer.

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Section: Lewismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

A lectin-based approach to detecting carcinogenesis in breast tissue

Moon

Kim

et al. 2016

Oncology Letters

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“…This is not an issue when measuring other high-abundance serum glycoproteins, such as transferrin [59] or haptoglobin [63], as dilution of the serum sample can lower the background noise to a minimal level. For low-abundance glycoproteins, for which sample dilution is not an option, more rigorous washing and blocking steps are required [66]. …”

Section: Lectin-based Methodsmentioning

confidence: 99%

The sweet and sour of serological glycoprotein tumor biomarker quantification

Kuzmanov

Kosanam

Diamandis

2013

BMC Med

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Aberrant and dysregulated protein glycosylation is a well-established event in the process of oncogenesis and cancer progression. Years of study on the glycobiology of cancer have been focused on the development of clinically viable diagnostic applications of this knowledge. However, for a number of reasons, there has been only sparse and varied success. The causes of this range from technical to biological issues that arise when studying protein glycosylation and attempting to apply it to practical applications. This review focuses on the pitfalls, advances, and future directions to be taken in the development of clinically applicable quantitative assays using glycan moieties from serum-based proteins as analytes. Topics covered include the development and progress of applications of lectins, mass spectrometry, and other technologies towards this purpose. Slowly but surely, novel applications of established and development of new technologies will eventually provide us with the tools to reach the ultimate goal of quantification of the full scope of heterogeneity associated with the glycosylation of biomarker candidate glycoproteins in a clinically applicable fashion.

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“…These Western blots were probed with digoxigenin-labeled GNA, which specifically binds mannose residues [Tytgat et al, 2015;Van Damme and Peumans, 1988]. To reduce background, polyvinylalcohol was used as a blocking agent [Thompson et al, 2011]. Spots reacting with the lectin were matched to the Sypro ® -stained gels and picked for identification.…”

Section: Image Analysis and Spot Pickingmentioning

confidence: 99%

Systematic Exploration of the Glycoproteome of the Beneficial Gut Isolate <b><i>Lactobacillus rhamnosus </i></b>GG

Tytgat

Schoofs²,

Vanderleyden³

et al. 2016

Microb Physiol

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Glycoproteins form an interesting class of macromolecules involved in bacterial-host interactions, but they are not yet widely explored in Gram-positive and beneficial species. Here, an integrated and widely applicable approach was followed to identify putative bacterial glycoproteins, combining proteome fractionation with 2D protein and glycostained gels and lectin blots. This approach was validated for the microbiota isolate Lactobacillus rhamnosus GG. The approach resulted in a list of putative glycosylated proteins receiving a ‘glycosylation score'. Ultimately, we could identify 41 unique glycosylated proteins in L. rhamnosus GG (6 top-confidence, 10 high-confidence and 25 putative hits; classification based on glycosylation score). Most glycoproteins are associated with the cell wall and membrane. Identified glycoproteins include proteins involved in transport, translation, and sugar metabolism processes. A robust screening resulted in a comprehensive mapping of glycoproteins in L. rhamnosus GG. Our results reflect the glycosylation of sugar metabolism enzymes, transporters, and other proteins crucial for cell physiology. We hypothesize that protein glycosylation can confer an extra level of regulation, for example by affecting enzyme functions. This is the first systematic study of the glycoproteome of a probiotic and beneficial gut isolate.

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Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Cited by 57 publications

References 43 publications

A lectin-based approach to detecting carcinogenesis in breast tissue

A lectin-based approach to detecting carcinogenesis in breast tissue

The sweet and sour of serological glycoprotein tumor biomarker quantification

Systematic Exploration of the Glycoproteome of the Beneficial Gut Isolate <b><i>Lactobacillus rhamnosus </i></b>GG

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