Journal: Clinical and Vaccine Immunology : CVI

Article Title: Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

doi: 10.1128/CVI.00046-13

Figure Lengend Snippet: Immune sera generated from different species and anthrax vaccines a

Article Snippet: The anti-rLF sera were pooled from female CD-1 mice that received two 0.5-ml intraperitoneal (i.p.) injections of 100 μg rLF-A with 750 μg Alhydrogel (Alhydrogel 2%; Brenntag Biosector, Frederikssund, Denmark) and 100 μg CPG 7909 on days 0 and 14.

Techniques: Generated, Vaccines, Recombinase Polymerase Amplification, Antiviral Assay, Injection

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

doi: 10.1128/CVI.00046-13

Figure Lengend Snippet: (A to F) Serum TNA potency values obtained in the presence of rLF-A were in agreement with those obtained with 40 ng/ml rLF-HMAL. Anthrax LT neutralization potency (ED50 and NF50) of immune sera from mice (n = 32) immunized with different anthrax vaccines (AVA, AVA plus CPG 7909, or rPA plus Alhydrogel; Table 2 describes the serum samples) were evaluated in the TNA using 50 ng/ml rPA in the presence of rLF-HMAL at 40 ng/ml or rLF-A at 4, 5, or 6 ng/ml, respectively. Negative-control wells showed that 4 ng/ml rLF-A was less cytotoxic (greater OD value) than rLF-HMAL, whereas 5 and 6 ng/ml rLF-A were equivalent in cytotoxicity to that of rLF-HMAL (Fig. 5A). NF50 values are the quotient of test sample ED50 values divided by the reference serum ED50 values obtained from the respective TNA plate (lot MS011211; ED50 mean and SD, 2,130 ± 214). The dotted line represents the Deming linear regression (orthogonal least-squares estimates) of log10(ED50) and log10(NF50) values obtained with rLF-A and rLF-HMAL, and the solid line represents the line of equivalence (slope, 1; intercept, 0). The degree of agreement between the log10(ED50) and log10(NF50) values obtained with 4, 5, or 6 ng/ml LF-A and 40 ng/ml LF-HMAL was calculated as Lin's concordance coefficient (rc values).

Article Snippet: The anti-rLF sera were pooled from female CD-1 mice that received two 0.5-ml intraperitoneal (i.p.) injections of 100 μg rLF-A with 750 μg Alhydrogel (Alhydrogel 2%; Brenntag Biosector, Frederikssund, Denmark) and 100 μg CPG 7909 on days 0 and 14.

Techniques: Neutralization, Vaccines, Negative Control

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

doi: 10.1128/CVI.00046-13

Figure Lengend Snippet: Evaluation of immune sera from multiple species that received different anthrax vaccines showed that TNA potency values obtained with 5 ng/ml rLF-A were equivalent to those obtained with 40 ng/ml rLF-HMAL. Anthrax LT neutralization potency (ED50 and NF50) of immune sera (n = 104) from humans, NHPs, rabbits, and mice immunized with different anthrax vaccines (AVA, AVA plus CPG 7909, or rPA plus Alhydrogel; Table 2 describes the serum samples) was evaluated in the TNA using 50 ng/ml rPA in the presence of 40 ng/ml rLF-HMAL or 5 ng/ml rLF-A, and log10(ED50) (A) and log10(NF50) (B) were reported. NF50 values are the quotient of test sample ED50 values divided by the reference serum ED50 values obtained from the respective TNA plate (lot MS011211; ED50 mean and SD of 2,130 ± 214). The dotted line represents the Deming linear regression (orthogonal least-squares estimates) of log10(ED50) and log10(NF50) values obtained with rLF-A and rLF-HMAL, and the solid line represents the line of equivalence (slope, 1; intercept, 0). The degree of agreement between the log10(ED50) and log10(NF50) values obtained with 5 ng/ml rLF-A and 40 ng/ml rLF-HMAL was calculated as Lin's concordance coefficient (rc values).

Article Snippet: The anti-rLF sera were pooled from female CD-1 mice that received two 0.5-ml intraperitoneal (i.p.) injections of 100 μg rLF-A with 750 μg Alhydrogel (Alhydrogel 2%; Brenntag Biosector, Frederikssund, Denmark) and 100 μg CPG 7909 on days 0 and 14.

Techniques: Vaccines, Neutralization

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

doi: 10.1128/CVI.00046-13

Figure Lengend Snippet: Additive neutralization activity of anti-rPA and anti-rLF mouse serum mixtures in the TNA. The anthrax LT neutralization capacity of anti-rPA (lot rPA-QC-H1) or anti-rLF-A (lot rLF-QC-2) mouse sera alone (A) or in combination (at ratios of 1:1, 1:2, 1:5, 1:10, or 1:20 anti-rLF to anti-rPA) (B to F) was evaluated using the TNA. (Anti-rPA sera were derived from a single immunization with rPA plus Alhydrogel, and that of anti-rLF was from two immunizations with rLF-A plus Alhydrogel and CPG 7909.) Serum mixture ratios were based on the neutralization capacity of each sample, in which the final 1:1 mixture contained equal ED50 values for the anti-rPA and anti-rLF serum samples. Accordingly, each serum sample evaluated alone was diluted in an equivalent manner with naive mouse serum. LT was composed of 20, 50, or 100 ng/ml rPA with 16, 4, and 3 ng/ml rLF-A, respectively. Data are the ED50s (means ± SD) of either 2 (1:2 and 1:5 mixtures) or 4 (1:1, 1:10, and 1:20 mixtures) replicate evaluations. (A) A number sign indicates significantly different ED50 values obtained with 20/16 and 100/3 ng/ml rPA/rLF combinations (anti-rPA sera; P < 0.05, two-tailed Student's t test). A dagger indicates a significantly different value from the ED50 value obtained with the 20/16 ng/ml (rPA/rLF) combination (anti-rLF sera; P < 0.05 by two-tailed Student's t test). (B to D) An asterisk indicates significant difference in ED50 value of the anti-rPA serum alone (control; white bar) from that of the respective anti-rLF/anti-rPA serum mixture (gray bars) (P < 0.05 by two-tailed Student's t test).

Article Snippet: The anti-rLF sera were pooled from female CD-1 mice that received two 0.5-ml intraperitoneal (i.p.) injections of 100 μg rLF-A with 750 μg Alhydrogel (Alhydrogel 2%; Brenntag Biosector, Frederikssund, Denmark) and 100 μg CPG 7909 on days 0 and 14.

Techniques: Neutralization, Activity Assay, Derivative Assay, Two Tailed Test, Control