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Fig. 3 | BMC Biotechnology

Fig. 3

From: Identification of a novel fully human anti-toxic shock syndrome toxin (TSST)-1 single-chain variable fragment antibody averting TSST-1-induced mitogenesis and cytokine secretion

Fig. 3

The significant binding of purified MS473 scFv to TSST-1. (A) Soluble MS473 scFv was purified by IMAC and analyzed by SDS-PAGE. A single protein band at approximately 27 kDa, corresponding to the expected molecular weight of the MS473 scFv, is seen on a 12% SDS-PAGE gel stained with Coomassie® blue. IgG: human immunoglobulin G, Lane M: unstained protein marker. The original image can be found in Additional file 3: Fig. S3. (B) Purified MS473 scFv was analyzed for aggregate content by SEC. The purified scFv was loaded onto the TSKgel G3000PWXL column, followed by elution with PBS. The eluted protein was detected by UV absorbance at 214 and 280 nm. (C) SDS-PAGE analysis of the SEC elution profile of MS473 scFv shows the presence of a highly pure band of 27 kDa, corresponding to the scFv protein. Lane M: pre-stained protein marker. The original image can be found in Additional file 4: Fig. S4. (D) The binding ability of purified MS473 scFv to TSST-1 was assessed by ELISA. The wells coated with TSST-1 or bovine serum albumin (BSA) (as control) were individually incubated with MS473 or SP220, followed by mouse anti-human scFv polyclonal antibody (MPAb) or unimmunized mouse sera (UnMsera; control). Empty wells were used as the control. Next, the wells were incubated with goat anti-mouse IgG-horseradish peroxidase (HRP)-conjugated antibody (GtAb), followed by the addition of the TMB solution. Other control groups include the TSST-1-coated wells, BSA-coated wells, and empty wells incubated directly with anti-staphylococcal TSST-1 mAb (STmAb) followed by GtAb; incubated directly with MPAb, followed by GtAb; incubated directly with UnMsera, followed by GtAb; or incubated directly with GtAb. The samples were run in triplicate, and the results are expressed as mean ± SEM of at least three individual experiments. Statistical comparisons were carried out with the one-way analysis of variance (ANOVA). *P < 0.05

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