Development and optimization of a high-throughput assay to measure neutralizing antibodies against Clostridium difficile binary toxin

Abstract

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.

FIG 1

Identification and characterization of purified CDTa and GST_ProCDTb. (A) SDS-PAGE of CDTa and 1mCDTa. Proteins were stained with Coomassie blue. (B) Western blot of CDTa and 1mCDTa using anti-CDTa monoclonal antibody. (C) SDS-PAGE of GST_ProCDTb and a_CDTb. Proteins were stained with Coomassie blue. (D) Western blot of GST_ProCDTb and a_CDTb using anti-CDTb monoclonal antibody. Some degradation of GST_ProCDTb was seen in the Western blot but not in the Coomassie blue-stained gel. Two micrograms of each protein was loaded onto the SDS-PAGE gels. M.W., molecular weight, in thousands.

FIG 2

Measurement of binary toxin cytotoxicity of Vero cells in a 384-well plate using an ImageXpress Velos scanning cytometer. Shown are the representative well images of Vero cells treated with different reagents. (A) Medium only (100%); (B) binary toxin consisting of 484 pM of CDTa with a 1:7 CDTa-to-a_CDTb molar ratio (1%). F-actin filaments were completely depolymerized by the binary toxin. (C) Binary toxin in panel B was preincubated with an equal volume of 1- to 640-fold diluted hyperimmune monkey serum. The cells were partially protected from binary toxin cytotoxicity (16%). (D) Binary toxin in panel B was preincubated with hyperimmune monkey serum. The cells were fully protected from binary toxin cytotoxicity (98%). Percentages listed in the figure legend refer to the amount of the cell area that is fluorescently labeled relative to that of the medium-only well.

FIG 3

Optimization of the binary toxin cytotoxicity assay. (A) SDS-PAGE gel of GST_ProCDTb treated with immobilized chymotrypsin for different times from 0 min to 240 min. (B) TC₅₀ of CDTa in binary toxin. CDTa was mixed with a_CDTb activated with different chymotrypsin activation times at a 1:7 molar ratio. The midpoint lines indicate the geometric mean value of two independent tests. (C) TC₅₀ of 1mCDTa at 1:4, 1:7, and 1:10 1mCDTa-to-a_CDTb molar ratios in the DOE. The decreasing trend in TC₅₀ with increasing molar ratio was statistically significant (P = 0.002). The error bars indicate the 95% confidence interval of the geometric mean. (D) TC₅₀ of 1mCDTa in binary toxin with a 1:7 1mCDTa-to-a_CDTb molar ratio at different cell-seeding densities in the DOE. The increasing trend in TC₅₀ with increasing cell-seeding density was statistically significant (P < 0.001). The midpoint lines indicate the geometric mean values. The error bars indicate the 95% confidence interval of the geometric mean.

FIG 4

Optimization of binary toxin NAb assay using design-of-experiment methodology. (A) Effect of binary toxin concentration on the NAb titers of the monkey serum. The decreasing trend in the NAb titer of the monkey serum with increasing concentration of binary toxin was statistically significant (P < 0.001). (B) Effect of binary toxin concentration on the signal-to-noise ratio. The increasing trend in the signal-to-noise ratio with increasing concentration of binary toxin was statistically significant (P < 0.001). The midpoint line indicates the geometric mean value. (C) Effect of serum-toxin preincubation time on the NAb titers of monkey serum. No statistically significant difference was observed among the three time points (P = 0.65). The relatively large spread on monkey serum titers within each preincubation time is due to the effect of toxin concentration. Each point in panels A and C represents the geometric mean titer of duplicate data from one of the DOE runs. The midpoint lines in panels A to C indicate the geometric mean value, and the error bars indicate the 95% confidence interval of the geometric mean. (D) Correlation of NAb titer with the concentration of spiked monkey serum in negative human serum with 484 pM of CDTa in the binary toxin NAb assay. Each point represents the geometric mean titer ± standard deviation of eight runs. The signal-to-noise ratio is the ratio of the medium-only response to the toxin-only response.

FIG 5

Measurement of anti-binary toxin NAb titers in hamster serum. (A) Preimmune (Pre), post-dose 1 (PD1), PD2, and PD3 sera were pooled for the testing. Each point at PD4 in the vaccine group represents the geometric mean titer ± standard deviation of the 10 hamster serum samples. LCT, large clostridial toxin, i.e., 5mTcdA and 5mTcdB. (B) Correlation of endpoint titers using a traditional visual endpoint titer assay with NAb titers (ED₅₀) using the imaging-based assay for anti-binary toxin NAb assay. Each point represents the geometric mean NAb titer of one of the 30 hamster serum samples analyzed independently by two analysts.