Abstract
BackgroundCell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component.ResultsTo develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples.ConclusionsCamelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0244-0) contains supplementary material, which is available to authorized users.
Highlights
Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors
Specificity of VHH B11 for chlorophyceaen algae To determine if VHH B11 recognizes all algae, or is restricted to Chlorophycean algae, we performed live-cell enzyme-linked immunosorbent assays (ELISAs) assays on two Heterokonts, Nannochloropsis oceanica and Thalassiosira pseudonana
When substituted for Chlamydomonas in the live-cell ELISA, none of these algae exhibited affinities above background levels (Figure 4)
Summary
Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. Detailed comparisons of cell wall compositions, synthesis and deposition between land plants and algae (and between different species of algae) are not presently possible. To help address this deficiency, we sought to develop techniques that would allow identification of cell surface-specific molecules in one particular alga, and in closely related algal species in a variety of environmental locations. As an initial proof-of-concept for this approach we chose to utilize Chlamydomonas reinhardtii (hereafter referred to as Chlamydomonas) as the alga whose cell wall is the most studied to date [3,5]
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