Quantification of functional cTA12 recognising native BoNT/A1 by a sandwich immunoassay.

<p>Native BoNT/A1 from culture supernatant (100 µl at 300 LD100) was incubated in 96-well microtitre plates coated with different dilutions of purified TA12 (from 0.05 to 1.5 µg/ml). TA13-labeled AChE was used as the tracer antibody, and absorbance was measured at 414 nm after incubation with AChE substrate. cTA12 purified from ascitic fluids and cTA12 purified from Sf9 supernatant were compared using mTA12 as reference (□ purified mTA12; ▪ cTA12 purified from ascitic fluids; ▪ cTA12 purified from S<i>f9</i> supernatant).</p

Detection of <i>Y. pestis</i> strains of various biovars by the Pla45/Pla35

<p>* <b>immunoassay.</b> Six different strains of <i>Y. pestis</i> were grown at 28°C and 2×10⁷ cfu/ml of each strain were used as antigen in the sandwich immunoassay. Absorbances at 414 nm were normalized using the absorbance of CO92 as reference (100%). Biovars were A: Antiqua, M: Medievalis and O, Orientalis. A <i>Y. pestis</i> strain cured of pPla (6/69ΔpPla) was used as a negative control.</p

Specificity of the Pla45/Pla35* mAb pair for PLA.

<p>(<b>A</b>) Sandwich immunoassay against <i>Salmonella enterica</i> serovar Typhimurium, <i>Erwinia pyrifoliae</i>, <i>Escherichia coli</i> and BL21(<i>pla</i>). (<b>B</b>) Sandwich immunoassay against <i>Y. pestis</i> CO92 and six strains of <i>Y. pseudotuberculosis</i> of various serotypes (for serotypes I to VI). (<b>C</b>) Reactivity of the Pla45/Pla35* pair against <i>Y. pestis</i> strain CO92 harboring pPla, and strain 6/69ΔpPla cured of the plasmid. (<b>D</b>) Western-blotting with Pla35 against recombinant PLA (lane 1, 1 µg), whole cell extracts of <i>Y. pestis</i> CO92 at a concentration of 4×10⁶ cfu/well (lane 2), or 4×10⁵ cfu/well (lane 3), IP516 at a concentration of 4×10⁶ cfu/well (lane 4), or 4×10⁵ cfu/well (lane 5), and 6/69ΔpPla (lane 6, 4×10⁷ cfu/well), <i>Y. pseudotuberculosi</i>s IP32953 (lane 7, 4×10⁷ cfu/well), and <i>E coli</i> BL21 (lane 8, 4×10⁷ cfu/well). Numbers on the left indicate the molecular weight markers (in kDa). Greek letters on the right indicate the various forms of PLA.</p

Lateral flow immunoassays.

<p>(<b>A</b>) Specificity of the PLA-dipstick for <i>Y. pestis</i> harboring pPla. Both CO92 and 6/69ΔpPla were grown at 28°C and serially diluted. The bacterial suspensions were incubated for 10 min with colloidal gold-labeled Pla35 mAb, and the PLA-dipsticks were then dipped for 30 min into 100 µl of the bacterial suspensions for upward migration of the liquid. Numbers below the dipsticks indicate the number of cfu/well. (<b>B</b>). Influence of the temperature of culture on CO92 detection. Bacteria were grown at 20°C, 28°C or 37°C, and the tests were performed as described in (A).</p

Test of the four most sensitive mAbs pairs against BL21(<i>pla</i>).

<p>Sensitivity in two-site immunoassays was determined using 10 fold serial dilutions of BL21(<i>pla</i>) or BL21 as negative control. The * indicates the tracer antibody.</p

Production of cTA12 in <i>Sf9</i> cells by infection of a recombinant baculovirus at different multiplicities of infection (MOI).

<p>1.35×10⁷ cells seeded in T75 flasks were infected at different MOI (•: 0.04; ▪: 0.12; <b>▴</b>0.4; ⧫1.2) with the recombinant baculovirus stock. cTA12 containing supernatants were harvested at different times and antibody was quantified by enzyme immunoassay.</p

Impact of the growth temperature on PLA detection by sandwich ELISA.

<p>(<b>A</b>) <i>Y. pestis</i> CO92 (blue lines) and <i>Y. pseudotuberculosis</i> IP32953 (orange lines) were grown at three temperatures: 37°C (▪), 28°C (•) and 20°C (▴). Ten-fold serial dilutions of the cultures were used as antigens for the Pla45/Pla35* sandwich ELISA. (<b>B</b>) Comparison of <i>Y. pestis</i> detection using an anti-PLA or an anti-F1 tracer antibody. 10⁹ cfu/ml of <i>Y. pestis</i> cultivated at three different growth temperatures (20°C, 28°C and 37°C), were incubated with Pla45 as capture antibody, and with either Pla35* (dark blue), or anti-F1* (light blue) conjugates.</p

Epitope mapping of Pla45 and Pla35 mAbs.

<p>(<b>A</b>) Pepscan epitope mapping of Pla45 and Pla35 mAbs was performed with synthetic peptides covering the 5 extracellular loops (L1 to L5) and the amino acids bordering these loops in PLA. The epitopes recognized by Pla45 are shown in a pale pink box, and those recognized by Pla35 in a darker pink box. (<b>B</b>) Amino acid sequence alignment of loop 5 from PLA, Epo, PgtE, OmpT and OmpP. Epitopes recognized by Pla45 and Pla35 mAbs on PLA, and the corresponding epitopes on the other molecules are in pink boxes.</p

Western blot analysis of cTA12 produced in insect cells and mammalian cells.

<p>Samples of TA12 were denatured at 95°C in Laemmli buffer and electrophoresed in 13% SDS-PAGE; in non-reducing conditions (A), lane 1: cTA12 purified from ascitic fluids (clone 16G7) and lane 2: cTA12 purified from Sf9 supernatant; and in reducing conditions (B) Lane 1: Sf9 supernatant, lane 2: cTA12 purified from Sf9 supernatant, lane 3: cTA12 purified from ascitic fluids (clone 16G7), lane 4: unpurified ascitic fluids (clone 16G7). After transfer, membranes were incubated with a polyclonal rabbit anti-human antibody, detected with HRP-conjugated anti-rabbit IgG and revealed using chemiluminescence.</p

Sensitivity of the optimized Pla45/Pla35* immunoassay.

<p><i>Y. pestis</i> CO92 was grown at 37°C (dark blue), 28°C (medium blue), or 20°C (light blue), and subjected to the optimized sandwich ELISA. The graphs represent the lowest AU values. The limit of detection (LoD) is represented by a horizontal black line. Vertical lines represent the standard deviation of duplicates.</p