Comparison of Enzyme-Linked Lectin Sorbent Assay and Flow Cytometry for Profiling Microbial Glycans

Dragačević, Luka; Lopandić, Zorana; Gavrović-Jankulović, Marija; Živković, Irena; Blagojević, Veljko; Polović, Natalija; Minić, Rajna

doi:10.1007/s12010-021-03772-w

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“…In addition, we have previously detected that ELISA format is more suitable for measuring specific binding levels to bacteria due to higher precision. 30 The obtained flow cytometry results have to be corroborated further by isolating the bacterial protein responsible for producing the high signal, which was in the case presented here available from literature data.…”

Section: Discussionsupporting

confidence: 53%

“…Therefore, we conclude that the flow cytometric analysis presented here is primarily useful in screening large numbers of microorganisms for initial detection of the presence of IgA binding proteins, or eventually in exploring pathological IgA response, as the procedure is laborious and the signals obtained though Fab specific binding are generally not of high intensity. In addition, we have previously detected that ELISA format is more suitable for measuring specific binding levels to bacteria due to higher precision 30 . The obtained flow cytometry results have to be corroborated further by isolating the bacterial protein responsible for producing the high signal, which was in the case presented here available from literature data.…”

Section: Discussionmentioning

confidence: 53%

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Selectivity of polyclonal repertoire of anti‐microbial IgA and its subclasses in saliva and serum in humans

et al. 2022

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Increased interest in microbiota calls for the thorough analysis of antibody reactivity to different microorganisms. As salivary IgA represents the first line of defence against microorganisms contacting mucosal surfaces, we explored the binding and specificity of salivary IgA by testing the binding of purified, FITC‐labelled salivary IgA to different microorganisms in flow cytometry and conclude that this kind of analysis enables the differentiation of species/strains with high IgA binding capacity, which should be corroborated on a larger sample size. Further we compare, with in‐house ELISA, the binding of polyclonal salivary IgA with the binding of polyclonal serum IgA from the same individuals to whole microbial cells and to purified microbial components. High correlations were obtained in total salivary IgA binding to Lactobacillus rhamnosus and Escherichia coli, very distant bacterial species, as well as to isolated bacterial components (r = .70–.97). The binding of total salivary IgA resembled the binding of both salivary IgA1 and IgA2, with IgA2 predominating. For serum polyclonal IgA repertoire, substantially higher specificity was obtained. Serum IgA binding to E. coli correlated best with serum IgA binding to lipopolysaccharide (r = .86), and serum IgA against L. rhamnosus correlated best with the anti‐peptidoglycan IgA levels (r = .88). We have also detected that total serum IgA response is governed by either IgA1 or IgA2 response, depending on the nature of the antigen/s. We conclude that steady state salivary IgA repertoire, unlike serum IgA repertoire, consists of polyreactive antibodies with innate specificity, questioning its capacity to select resident microbiota.

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Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 53%