Purification Of Human Single-chain Variable Fragment Of Anti-digoxin Antibody(ADAscFv) Expressed In Bacteria

Objective To date ,Dexin Liu et al,have obtained a cell line E.coli HB2151 that could secrete human single-chain variable fragment of ADAs (ADAscFv) as soluble protein into culture medium .The ADAscFv can react with Dig and Dig 's analog. Whether ADAscFv serves as measurement of digoxin in plasma following intoxication or clinical treatment, supernatant of the culture medium including soluble ADAscFv should be separated and purified according to it's own features, Nevertheless there is no all-purpose protocol for proteins purification . In order to obtain highly purified ADAscFv and pave the ground for future reseach in which the reagent for diagnosing Dig toxication can be produced and the antagonism of ADAscFv to Dig toxication can be clarified,This work was to explore different methods for separating and purifying supernatant containing soluble ADAscFv.Methods ①collected supernatant after having cultured E.coli strain HB2151 in culture medium which include soluble ADAscFv, then analysed the antigen-binding activity and purity of ADAscFv respectively with Enzyme-linked immunosorbent assay(ELISA) and Sodium dodecyl sulphate- polyacrylamide gel electrophoresis(SDS-PAGE).②salted-out supernatant with ammonium sulfate of different saturation and analysed the yielded sample respectively with ELISA and SDS-PAGE. ③selected column HiTrap Q HP(1cm×5cm) and eluted it with 0.05mol/L Tris-HCl buffer of different pH, NaCl concentration to purify the salted-out sample through Anion exchange chromatography (AEC),then analysed the purified sample respectively with ELISA and SDS-PAGE.④in order to enhance efficiency, purified the salted-out sample through AEC in Fast performance liquid chromatography (FPLC), analysed the purified sample respectively with ELISA and SDS-PAGE.⑤purified the salted-out sample through gel filtration chromatography (GFC) in FPLC which was performed through using XK16/60 empty column .The column was filled with matrix superdex x75 and eluted with different eluting fluids and flow rates,etc,analysed the purified elution respectively with ELISA and SDS-PAGE.Results ①ELISA showed that E.coli strain HB2151 could secrete soluble protein ADAscFv stably,but the cultured supernatant was of many contaminated proteins.②cultured supernatant was seperated through ammonium sulfate gradient-based salting-out,then collected sample to be analysed bioactivity through ELISA and be determined the nature through SDS-PAGE which showed that the supernatant salted-out by ammonium sulfate of 0-50% saturation was of 36kDa lane that accorded with ADAscFv. ③the elution collected through AEC was analysed by ELISA and SDS-PAGE.The result showed that the pH 9.0, 0.05mol/L Tris-HCl buffer containing 0.2mol/L NaCl eluted the column was a comparatively ideal .④Supported by above-step, the column was step-wise washed with pH 9.0, 0.05mol/L Tris-HCl buffer containing NaCl with AEC in FPLC. The elution profile showed six protein peaks and the elution was analysed by ELISA and SDS-PAGE. The presence of the ADAscFv in the fraction corresponded to the second peak. ⑤tried purifying the salted-out sample through Gel filtration chromatography (GFC) in FPLC and observed eluting peaks.Collected elutions respectively and then analysed by ELISA and SDS-PAGE.The eluting peaks was group-peaks.Discussion In view of our results,we could obtain purified ADAscFv through the following steps which salted-out supernatant contained soluble ADAscFv with 0-50% saturated ammonium sulfate ,then selected column HiTrap Q HP(1cmx5cm) connected with FPLC and AEC step-wise elute the column with pH 9.0,0.05mol/L Tris-HCl (NaCl) buffer .Yielded the second peak elution that contained isolated ADAscFv which has a high purity . Consequently,from this work we can get helpful purification datas and make a good preparation for continued study.