(C). into the wells with and without antigen coating, respectively, and incubated for 1 h at room temperature (RT). Unspecific phages were discarded after stringent washings steps with PBST (PBS containing 0.05% Tween-20, 10 times for 1st panning round, 20 times for 2nd and 3rd panning rounds). The remaining phages were collected after incubating the wells with TEA (100 L, 100 mM triethylamine solution, pH 11.0), and then immediately neutralized in 100 L Tris-HCl (1.0 M, pH 7.4). The enrichment for phages with antigen-specific Nbs was then determined by diluting 10 L of the resulted phage with 90 L of LB containing TG1 cells and plating on LB agar dishes. The remaining phage particles were allowed to infect fresh TG1 cells to amplify the phages used in the following panning round. 2.4. Screening of Specific Nbs To screen the Nbs positive in binding to the lupine protein extract, single colonies were randomly picked from the plates with enriched colonies after bio-panning and inoculated into 100 L 2 TY medium with supplementation of 100 g/mL ampicillin, 2% (WK6 cells. The Raphin1 resulted WK6 cells were cultured in Terrific Broth (TB) medium supplemented with 0.1% (= 10), which defined the limit of detection (LOD) as the concentration of the mean optical density plus 3 times the standard deviations (SD), and plus 10 times the SD for the limit of quantitation (LOQ) [25]. Then, the specificity of the established method was evaluated against peanut, macadamia and lupine proteins for cross-reactivity analysis with the steps described above. The acquired data was analyzed to reflect the specificity of the method for the surveillance of lupine proteins. 2.8.4. Detection of the Spiked Sample The effectiveness of the developed immunoassay was determined against dairy products of skim milk. The spiked milk sample was prepared after supplementing different concentration of general lupine protein extracts. The samples spiked with antigens were cleared from the cream and precipitation after centrifugation. The obtained supernatant was diluted in 1000 times with PBS prior the detecting and applied for the antigen quantification Rabbit polyclonal to PRKCH by the established method to determine the recovery rate and coefficient of variation (CV), which provided evidence of the applicability of the developed immunoassay. 3. Results 3.1. Construction of an Immune Nb Library Crude lupine protein extract was prepared with an acceptable efficiency. The molecular distribution of the proteins was visualized by Coomassie staining after SDS-PAGE (Figure S1), and their molecular mass ranged from 15 to 70 kDa, which fitted previous observations [26]. No significant variation was observed when proteins were separated under reducing Raphin1 or non-reducing conditions. The concentrated proteins with a size around 55 kDa could be observed and potentially reflected the distribution of conglutin, which has been identified as the main allergen source of lupinus. An immune Nb library was constructed after immunizing a young alpaca with the crude lupine protein extract. The VHH repertoire was amplified by two rounds of nested PCR. The fragments of VH-CH1-CH2 or VHH-CH2 were identified based on their size of ~900 or 700 bp, respectively and the fragments of 700 bp were extracted and served as template for the 2nd PCR to amplify VHH gene fragments. After Raphin1 sub-cloning the VHH into pMECS vectors, the recombinant phagemids were transformed into TG1 to prepare the immune Nb library of about 1.44 10colony forming units (CFU)/mL. The percentage of correct VHH insertion was determined by colony-PCR with randomly selected colonies to be 75% (data not shown). In summary, an adequate immune library against lupine protein extracts was constructed, which could be employed for retrieving lupine- specific Nbs. 3.2. Bio-Panning and Screening of Nbs Enrichment of Nbs recognizing lupine protein was accomplished by displaying Nbs at the tip of virus particles and followed by a round of bio-panning. The enrichment of each panning was evaluated by determining colony distribution from the phages collected from positive and negative, which demonstrated the strong increase of the enrichment from 8 to 40.6-fold (Figure 2A) reflecting an effective bio-panning for lupine protein specific Nbs. Open in a separate window Figure 2 Selection and sequence of selected Nbs from.
