Development of a high-throughput format for solid-phase extraction of enantiomers using an immunosorbent in 384-well plates.
An antibody-based solid-phase extraction method for filtered 384-well plates was developed for a medical drug candidate having two enantiomeric forms in order to demonstrate the potential of the use of recombinant antibody fragments as specific and efficient immunosorbents. An immobilization method using a six-histidine tag of the antibody fragment and mild oxidation was applied in order to immobilize antibody fragments in an oriented and kinetically stable way that ensured high capacity of the antibody support. Phosphate buffer or plasma spiked with enantiomers were used as samples. Selective solid-phase extraction was followed by liquid chromatography-mass spectrometry analysis. Average recoveries for buffer and plasma samples ranged from 79 to 122% and 80 to 108%, respectively. Good linearity was observed in the concentration range of 30-3000 ng/mL of the enantiomer.
Nevanen
Tarja K
TK
VTT Biotechnology, Tietotie 2, 02044 VTT, Espoo, Finland. tarja.nevanen@vtt.fi
Simolin
Helena
H
Suortti
Tapani
T
Koivula
Anu
A
Söderlund
Hans
H
D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D002021:Buffers; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006639:Histidine / Q000737:chemistry; D007163:Immunosorbent Techniques; D013058:Mass Spectrometry / Q000379:methods; D010084:Oxidation-Reduction; D010710:Phosphates / Q000737:chemistry; D010949:Plasma; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D000906:Antibodies; D002021:Buffers; D010710:Phosphates; D011994:Recombinant Proteins; D006639:Histidine