Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates.
A small albumin-binding domain (ABD) of 46 amino acids derived from streptococcal protein G was employed for the directed attachment of recombinant immunoglobulin (Ig) fragments to microtitre plates that had been coated with human serum albumin (HSA). Generic vectors were constructed in order to produce the Fv or Fab fragments fused with the ABD in Escherichia coli. Using the anti-lysozyme antibody D1.3 as the capture antibody fragment it was possible to quantify the non-radioactively labelled antigen with high sensitivity in a sandwich ELISA. The new strategy avoids denaturation or an unfavourable orientation of the Ig fragment, which can occur during direct adsorption to the microtitre plate. The HSA that serves to complex the ABD ensures efficient saturation of reactive binding sites on the plastic surface as well so that no additional blocking steps are necessary and the assay can be quickly performed.
König
T
T
Institut für Biochemie, Technische Universität, Darmstadt, Germany.
Skerra
A
A
D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000032:analysis*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005813:Genes, Synthetic; D005822:Genetic Vectors; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D007164:Immunosorbents; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012709:Serum Albumin / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007128:Immunoglobulin Fragments; D007164:Immunosorbents; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; C055966:immunoglobulin Fv