title: Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.
abstract: Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.
authors: Department of Biochemistry, University of Nebraska-Lincoln, 1901 Vine Street, Lincoln, NE, 68588, USA.
authors: Wenzhi
authors: W
authors: Jiang
authors: Julian N
authors: JN
authors: Rosenberg
authors: Akelia D
authors: AD
authors: Wauchope
authors: Jacqueline M
authors: JM
authors: Tremblay
authors: Charles B
authors: CB
authors: Shoemaker
authors: Donald P
authors: DP
authors: Weeks
authors: George A
authors: GA
authors: Oyler
mesh_terms: D000818:Animals; D000917:Antibody Formation / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D052179:Antigens, Plant / Q000235:genetics / Q000276:immunology*; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology*; D002455:Cell Division / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D046549:Plantibodies / Q000737:chemistry / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D052179:Antigens, Plant; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D046549:Plantibodies
keywords: VHH; algae; camelid antibodies; cell walls; sdAbs; technical advance