Materials and reagents were obtained from Sigma-Aldrich (St Charles, MO) except where indicated. LF, EF, PA83, and PA63 were obtained from List Biological Laboratories (Campbell, CA). Normal North American (NNA) human ten-donor-pooled plasma and serum and plasma from 100 individual NNA donors were obtained from Interstate Blood Bank (Memphis, TN). AIGIV was obtained from the Division of Strategic National Stockpile, CDC (Atlanta, GA).

The inhalation anthrax study in 24 rhesus macaques (Macaca mulatta) was conducted at the Battelle Biomedical Research Center (Columbus, OH) as previously described and approved by both Battelle and the Centers for Disease Control and Prevention Institutional Animal Care and Use Committees [25]. This two-part study was approved in 2006 (CDC IACUC protocol no. 1459BOYMONX and Battelle MREF protocol no. 570) and carried out in 2006–2007. Sufficient sample volumes generated from this approved protocol were remaining and used for this study. Animals were challenged with 275 ± 72 LD₅₀ aerosolized B. anthracis Ames spores by inhalation (target dose 200 LD₅₀). Whole blood and plasma were collected in EDTA tubes at −30 days and 12, 18, 24, 36, 48, 72, 96, and 120 h, and serum was collected for five animals as described previously [25]. Plasma/serum was filter sterilized and culture confirmed before shipment. Culture and PCR of the protective antigen gene (pagA) were described previously [25]. Total LF and LTx analysis was determined in two animals, 16N and 89N, over the course of infection. An additional 22 post-spore exposure samples were analyzed. Pre- and post-antibiotic treatment samples were available for six animals treated with Ciprofloxacin at 20 mg/kg twice daily for 14 days in surviving animals. Treatment commenced at 48 h post-challenge in four animals and at 72 h in two. Four of six survived.

Anti-PA mouse monoclonal antibodies (mAb) AVR1046 and two anti-LF mAb (AVR1674 and AVR1675) were linked to Tosyl-activated magnetic beads (MB; Life Technologies, Grand Island, NY) according to the manufacturer’s instructions yielding anti-PA and dual anti-LF MBs. Kingfisher 96 magnetic particle processor (Thermo-Fisher Scientific, Waltham, MA) was used for all MB handling.

Specificity of anti-PA and anti-LF MBs was demonstrated for LTx, LF, and PA. PA63/LF were mixed at a w/w ratio of 10:1 to form LTx complex. PA83 and LF were mixed as a non-complexed control. Mixtures were incubated at room temperature (RT) for 30 min. LF, PA63, PA83, LF/PA63, and LF/PA83 at 0.5 μg LF and 5 μg PA83 or PA63 were immunoprecipitated with anti-LF and anti-PA MBs, eluted with 50 % acetonitrile, 2 mM HCl for 1 min, dried by centrifugal evaporation, reconstituted in sample buffer, analyzed by 5–14 % SDS-polyacrylamide gel electrophoresis (PAGE; Life Technologies), and visualized by silver stain (SS; Protea Biosciences, Morgantown, WV).

LTx was formed by mixing PA63 and LF at w/w ratios of 0:1 to 60:1 PA63/LF, incubating at RT for 30 min. Mixtures were analyzed for free LF levels by non-denaturing PAGE (Life Tech) and SS. A ratio of 50:1 adsorbed the maximum amount of LF. The final LTx standard material was similarly prepared with 25 μg PA63/0.5 μg LF for 500 ng/μL PA63 and 10 ng/μL LF as LTx, then diluted as described below to make standard and quality control materials.

The LTx standard material was analyzed by non-denaturing PAGE to quantify the “free” LF and correct standards. Gel lanes contained 7.5 μg PA63, 25, 12.5, 6.25, and 3.125 ng LF and the LTx standard material with 150 ng LF and 7.5 μg PA63 to visualize and quantify the small amount of unbound LF. The gel was scanned and bands intensities quantified (Softmax Pro, Molecular Devices, Sunnydale, CA). Free LF intensities were fitted to a linear regression (r² = 0.996), and free LF in the LTx lane was calculated from the linear regression.

The qualitative efficiency of anti-PA purification of the LTx standard material in plasma in the presence of the excess PA63 was determined by comparison with anti-LF purification. The LTx standard material at 100, 50, 25, and 12.5 ng LF and 50 times PA63 each in plasma sample were immunoprecipitated by anti-PA and anti-LF MBs and eluted and analyzed by SDS-PAGE/SS.

Standard and quality control (QC) materials in plasma were prepared from the characterized material with free LF corrected values of 24.3 to 0.0025 ng/mL and three QCs with 5.82 ng/mL (QCH), 0.582 ng/mL (QCM), and 0.0582 ng/mL (QCL) LTx.

For LTx analysis, 50 μL of sample was diluted 1:10 in 450 μL phosphate-buffered saline with 0.05 % Tween20 (PBST), mixed with 6.25 μL anti-PA-MBs for 1 h, washed two times 2 min each in 1.0 mL PBST, then in 200 and 100 μL dH₂O for 1 min each, resuspended in 30 μL reaction buffer with the peptide substrate and reacted for 2 and 18 h at 37 °C. After the 2-h reaction, 3 μL of sample was mixed with α-cyano-4-hydroxycinnamic acid (CHCA) at 5 mg/mL in 50 % acetonitrile, 0.1 % trifluoroacetic acid, and 1 mM ammonium phosphate (CHCA-MALDI matrix) and isotopic internal standard peptides (ISTD) and analyzed by ID-MALDI-TOF/MS as described previously [22, 23]. The remaining reaction was incubated for analysis at 18 h. The LTx cleaved peptide MS peak area/ISTD peak area (area ratio) was plotted versus LTx standard concentration with dual log₁₀ transformation and 5PL robust quantification.

Recovery for this method compared activities for 0.5 ng LF and LTx (0.5 ng LF + 1 ng PA63) both without capture and with anti-PA purification. Precision, accuracy, and LOD were determined from 25 standards/QC analyses with precision as the coefficient of variation (%CV) and accuracy as the percent error (%error). Acceptable criteria described previously [29] place accuracy at ≤20 % error and precision at ≤20 %CV except at the upper (≤15 %) and lower limit of quantitation (<25 %). Linearity was acceptable with a slope ≥0.8 and ≤1.2 and an r-squared ≥0.85. Quantifiable ranges were determined using these criteria for both 2- and 18-h reactions. Limits of detection (LOD) were determined for the method including all steps (sample preparation, enzymatic reaction, and MALDI-TOF analysis) by plotting standard deviations (SD) versus concentration for the plasma blank and three low standards, one above, one near, and one below the estimated LOD for 30 runs. The y-intercept and slope from the linear regression, the SD, and mean of the blank (SD_b and mean_b) were used to calculate the LOD concentration as follows [30]:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\mathrm{Conc}}_{\mathrm{LOD}}=\left({\mathrm{mean}}_{\mathrm{b}}+1.645\left({s}_{\mathrm{b}}+\mathrm{i}\mathrm{n}\mathrm{t}\right)\right)/\left(1-1.645\left(\mathrm{slope}\right)\right) $$\end{document}ConcLOD=meanb+1.645sb+int/1−1.645slopewhere b is blank, s is standard deviation, and int is y-intercept.

Specificity was assessed from 100 individual NNA plasma samples and 41 archived human serum samples that included elderly Argentinian’s, children from San Diego without routine immunizations, and other infections (Staphylococcus aureus, Legionella pneumophila, Chlamydophila pneumoniae). Sensitivity was assessed for samples from 24 rhesus macaques (19 plasma collected, 5 serum collected) with inhalation anthrax, and in samples from three inhalation anthrax cases (CDC IRB Protocol no. 5343.0, Use of Residual Human Specimens for Laboratory Diagnostic Research) [26, 27, 31]. Samples were analyzed with two or more replicates at one or more dilutions for both 2- and 18-h reactions. Lower-level samples requiring higher sample volumes were analyzed singly due to limited sample volumes. For these, means were from both 2- and 18-h results.

Accuracy was assessed for plasma samples with 7.5, 0.75, and 0.075 ng/mL LTx co-spiked with PA83, LF, and EF at 50 ng/mL each, analyzed in triplicate. For matrix accuracy, serum and plasma from five individual donors each were spiked with LTx at 2.5 ng/mL and analyzed in duplicate. Matrix-dependent activation of PA83 to PA63 and formation of LTx was assessed using ten-donor pooled serum and plasma spiked with either 5 ng/mL PA83 or PA63 and then 2.5 ng/mL LF, incubated for 1 h then analyzed for LTx.

For utility with anthrax therapeutics, AIG interference was assessed by spiking AIG at an equivalent of 200 μg/mL anti-PA IgG into all 15 standards, plasma blank, and QC samples over the entire range from 24.3 to 0.0024 ng/mL. These were prepared, spiked, and analyzed, on two separate days alongside control (no AIG) standards/blank/QCs spiked with PBS instead of AIG. A clinical anthrax sample with previously quantified LTx of 88 ng/mL was analyzed in triplicate without and with AIG spiked at 200 μg/mL anti-PA IgG. After spiking, samples were mixed for 1 h then analyzed for LTx. Interferences from antimicrobial treatment were assessed by analyzing LTx before and after treatment for animal and clinical inhalation anthrax described above.

Analyses and formulas were described above where appropriate. Standard calculations, means, standard deviations, and linear regressions were performed in excel. Custom visual basic programs calculated values for LTx from the 5PL regressions. LTx levels were reported in nanograms per milliliter. Grading for culture results were described previously [25].

As described in the “Introduction,” the method has three primary steps, magnetic immunoprecipitation, substrate cleavage reaction, and MALDI-TOF MS quantification. The following results include characterization of LTx complex formation from PA63 and LF for standards material, characterization of the ability of the magnetic-bead-coated antibodies to selectively purify LTx, LTx reaction with substrate, and MALDI-TOF MS quantification of LTx reaction products.

Since the measurement of LTx relies on detection of LF activity in LTx complex, the aim was to create standards with a minimum of free LF. Activated PA63 exists as a heptamer/octamer that binds up to four LF molecules forming the LTx complex, with optimal molecular ratios represented by a weight/weight ratio of 1.5:1 PA63/LF [12, 13, 32]. However, optimum binding of LF in these standards materials required 50:1 PA63/LF as visualized by non-denaturing gel electrophoresis and higher PA63 levels did not bind more LF (not shown). PA63 alone with multimer and monomer (Fig. 2a, lane 1) was compared with the LTx mixture (lane 6) which shows two additional primary bands that migrate lower than the non-complexed multimer. These bands represent the LTx complex. A significant amount of PA63 multimer and monomer remains uncomplexed. The free LF band in lane 6 was visible, and its gel-band intensity was quantified against a linear regression derived from the LF-only lanes 2–5. The amount of free LF in the standards was quantified at 4.56 ng, which was 3 % of the total 150 ng LF loaded (Fig. 2a). LTx standard concentrations were corrected by subtracting the 3 % free LF.Fig. 2

LTx and antibody characterization. LTx for standard material was analyzed by non-denaturing gel electrophoresis. The gel was scanned, LF band intensities were quantified, and the amount of free LF was estimated from the linear curve (a). Specificity of anti-LF and anti-PA for LF, PA83, and PA63 alone (b), PA63/LF (LTx) and PA83/LF (c), and LTx standards material; A 100 ng LTx, B 50 ng, C 25 ng, and D 12.5 ng in plasma (d) was assessed by magnetic immunoprecipitation and SDS-PAGE

Antibody specificities demonstrated by magnetic immunoprecipitation of individual toxin components showed that anti-LF purified LF but not PA83 or PA63 and anti-PA purified PA83 and PA63 but not LF (Fig. 2b). Specificity for pre-formed LTx complex was observed for both anti-PA and anti-LF since both PA63 and LF bands were extracted and present (Fig. 2c). With PA83 + LF, complex was not formed and anti-LF extracted LF but not PA83 and anti-PA extracted PA83 but not LF. In all, the data support the individual specificities of anti-PA and anti-LF mAbs for LF and PA and the ability of both to purify LTx complex.

Since LTx standards were prepared with 50 times more PA63 than LF, it was determined whether the excess PA63 interfered with purification of LTx. Immunoprecipitation of 100 to 12.5 ng LTx standard material from plasma showed that the amount of LF in complex extracted by anti-PA and anti-LF MBs were visually similar (Fig. 2d). Additionally, the PA63 bands purified by anti-PA were more intense (lanes 5–8). Combined, this indicated that anti-PA levels on the beads were sufficient to capture LTx and excess non-complexed PA63. Thus, PA did not saturate the beads at these levels. Furthermore, much less material is used for LTx calibrators. The highest standard is at 24 ng/mL with only 50 μL used for analysis. In summary, anti-PA was suitable for purification of LTx and the excess PA63 did not interfere with its purification.

LTx activity is quantified by cleavage of a peptide substrate yielding two products (Fig. 3a). MALDI-TOF MS of a representative positive reaction show the substrate at 2804.2 m/z, two products at 1232.8 and 1589.8 m/z and ISTD peptide peaks at 1239.8 and 1596.8 m/z, respectively (Fig. 3b). Narrowing the x-axis shows the isotopic detail of the CT-product and ISTD. The CT isotopic area divided by the CT-ISTD area yields the area ratio that is directly proportional to the amount of LTx present. For a negative reaction processed from the plasma blank only the substrate and ISTD peaks are present (Fig. 3c). Area ratios were plotted versus concentration and both axes were log₁₀ transformed generating a sigmoidal curve. Quantitative accuracy was optimal using 5PL regression with robust weighting. Custom visual basic programs integrated MS peaks and applied the 5PL fits to quantify standards and samples (Fig. 3d).Fig. 3

ID-MALDI-TOF MS of LTx catalytic activity and quantification. The LTx substrate peptide sequence shown in standard amino acid codes except for non-standard amino acid norleucine depicted as an X. Magnetic-affinity-purified LTx hydrolyzes the LF peptide substrate between the p1 proline and p1' tyrosine, yielding two smaller peptides (a). The cleavage reaction is mixed with ISTD peptides (+7 m/z) and analyzed by ID-MALDI-TOF MS. Spectra show a positive reaction with LTx in plasma (b) and negative reaction without LTx in plasma (c). Output for the 5PL regression generated by custom visual basic software for an 18-h reaction. The orange line indicates the estimated LOD (three times the plasma blank area ratio) for each analysis (c). Linearity of LTx quantification for 25 independent analyses of standards levels showing means and SD (error bars) for 2- and 18-h reactions (e)