Production of a recombinant anti-morphine-3-glucuronide single-chain variable fragment (scFv) antibody for the development of a "real-time" biosensor-based immunoassay.
A recombinant single-chain variable fragment (scFv) antibody to morphine-3-glucuronide (M3G) was produced using genetic material obtained from the spleen cells of mice immunised with a morphine-3-glucuronide-bovine serum albumin (M3G-BSA) conjugate. Immunoglobulin light (V(L)) and heavy (V(H)) chain genes were amplified and cloned into pAK vectors for generation of recombinant antibody fragments in Escherichia coli. A competition ELISA assay was developed in PBS to characterise the ability of the antibody fragments to recognise free drug and the detection limits were found to be as low as 3 ng ml(-1). Surface plasmon resonance-based inhibition immunoassays were developed. The recombinant antibody was pre-incubated with various concentrations of free drug followed by injection over a morphine-3-glucuronide-thyroglobulin (M3G-THY) immobilised surface. The response of antibody binding to the surface of the chip was inversely proportional to the amount of free drug in solution. Regeneration conditions for antibody binding to the surface were optimised resulting in a binding-regeneration capacity of at least 30 cycles. The inhibition assay for M3G was tested with assay ranges between 3 and 195 ng ml(-1) and 3 and 97 ng ml(-1) in PBS and urine, respectively.
School of Biotechnology, Dublin City University, 9, Dublin, Ireland. paul.dillon@dcu.ie
Paul P
PP
Dillon
Bernadette M
BM
Manning
Stephen J
SJ
Daly
Anthony J
AJ
Killard
Richard
R
O'Kennedy
D000595:Amino Acid Sequence; D000818:Animals; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D009022:Morphine Derivatives / Q000276:immunology* / Q000652:urine; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D020349:Surface Plasmon Resonance / Q000379:methods*; D013997:Time Factors
D007135:Immunoglobulin Variable Region; D009022:Morphine Derivatives; D011994:Recombinant Proteins; C018108:morphine-3-glucuronide