Isolation and characterization of a thermally stable recombinant anti-caffeine heavy-chain antibody fragment.
We have isolated and characterized a caffeine-specific, heavy-chain-only antibody fragment (V(HH)) from llama that is capable of being utilized to analyze caffeine in hot and cold beverages. Camelid species (llama and camel) were selected for immunization because of their potential to make heat-stable, heavy-chain-only antibodies. Llamas and camels were immunized with caffeine covalently linked to keyhole limpet hemocyanin, and recombinant antibody techniques were used to create phage displayed libraries of variable region fragments of the heavy-chain antibodies. Caffeine-specific V(HH) fragments were selected by their ability to bind to caffeine/bovine serum albumin (BSA) and confirmed by a positive reaction in a caffeine enzyme-linked immunosorbent assay (caffeine ELISA). One of these V(HH) fragments (VSA2) was expressed as a soluble protein and shown to recover its reactivity after exposure to temperatures up to 90 degrees C. In addition, VSA2 was able to bind caffeine at 70 degrees C. A competition caffeine ELISA was developed for the measurement of caffeine in beverages, and concentrations of caffeine obtained for coffee, Coca-Cola Classic, and Diet Coke agreed well with high performance liquid chromatography (HPLC) determination and literature values. VSA2 showed minimal cross reactivity with structurally related methylxanthines.
Ladenson
Ruth C
RC
Department of Pathology and Immunology, Division of Laboratory Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, Missouri 63110, USA.
Crimmins
Dan L
DL
Landt
Yvonne
Y
Ladenson
Jack H
JH
D000818:Animals; D001483:Base Sequence; D002110:Caffeine / Q000276:immunology*; D002162:Camelus; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D002110:Caffeine