Abstract
Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.
Highlights
CIT (Fig 1) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium [1], Aspergillus [2] and Monascus [3], originally isolated in 1931
The UV spectra of CIT, Bovine serum albumin (BSA) and CIT-BSA are shown in Fig 2 The ultraviolet absorption peaks of CIT-BSA conjugate were at 280 nm and 330nm, while that of CIT and BSA were at 319 nm and 280 nm, respectively
SDS-PAGE analysis of BSA and CIT-BSA is in Fig 3, showed that the molecular weight (MW) of CIT-BSA is higher than that of BSA
Summary
CIT (Fig 1) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium [1], Aspergillus [2] and Monascus [3], originally isolated in 1931. The second round of in vitro selection was performed with almost the same conditions, except that the microtiter plates were coated with the CIT-OVA conjugate and different primers were used.
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