Recombinant Mouse Anti-C. burnetii PI-LPS Antibody (1E4) (CAT#: FAMAB-0041CQ)

Figure 1 Characterization of 1E4 by ELISA

Isotyping of 1E4 by ELISA with PI Ag. A 96-well microtiter plate was coated with inactivated PI or PII Ag and incubated with the hybridoma supernatant from cloned hybridoma 1E4. The reactivity of 1E4 with anti-IgM, anti-IgG, anti-IgG1, anti-IgG2a, anti-IgG2b, and anti-IgG3 was measured by OD 490. The values represent the average absorbance at 490 nm of duplicate wells

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 2 Characterization of 1E4 by Western blotting.

The reactivity of 1E4 with PI and PII Ags, and proteinase K-digested PI and PII Ags in Western blotting. I, PI Ag; I/PK, proteinase K-treated PI Ag; II, PII Ag; II/PK, proteinase K-treated PII Ag

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 3 Evaluation of the ability of 1E4 to inhibit C. burnetii infection in vivo.

The inhibition of C. burnetii was performed by incubation of 1 × 10⁷ virulent C. burnetii NMI with 1, 10, 100, and 300 μg purified 1E4, or mouse IgG2a isotype control at 4˚C overnight. Six-week-old BALB/c mice were infected by i.p. injection with 1 × 10⁷ of 1E4 or control IgG2a-treated C. burnetii NMI. Mice infected with 1 × 10⁷ of PBS-treated C. burnetii NMI were used as negative controls. Splenomegaly and bacterial burden in the spleen were measured at 14 d postinfection and used as indicators to evaluate the ability of 1E4 to inhibit C. burnetii infection in BALB/c mice. (A) Splenomegaly was measured by spleen weight as a percentage of body weight. (B) Bacterial burden in the spleen was determined by realtime PCR and reported as log₁₀ of C. burnetii com1 gene copy numbers. (C) Pathological changes in the spleen at 14 d postchallenge.The data presented in each group are the average with SD of four mice. Original magnification ×410. *p < 0.05, **p < 0.01.

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 4 Identification of 1E4-specific phage clones.

The reactivity of recombinant phage clones with 1E4 was analyzed by ELISA. A 96-well microtiter plate was coated with individual 10¹⁰ of purified phage particles and incubated with 5 μg/ml 1E4 with or without 2 μg/ml PI Ag. The inhibition of 1E4 binding by PI Ag was measured by the inhibition index ([A490 without inhibitor - A490 with inhibitor]/A490 without inhibitor).

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 5 Evaluation of the binding ability of the synthetic mimetic peptide m1E41920 and m1E41920-KLH conjugate with 1E4 by ELISA and competitive ELISA.

(A) Binding activity of m1E41920 and m1E41920-KLH conjugate with 1E4 was measured by ELISA. A 96-well microtiter plate was coated with m1E41920, m1E41920-KLH, control peptide, control-KLH, or KLH and incubated with 5 μg/ml 1E4. The values represent the average absorbance at 490 nm duplicate wells. (B) Competitive inhibition ELISA analysis of the ability of synthetic peptide m1E41920 to inhibit 1E4 binding with PI Ag. A 96-well microtiter plate was coated with inactivated PI Ag and incubated with 5 μg/ml 1E4 mixed with different concentrations of m1E41920 or control peptide. Points plotted represent the average absorbance at 490 nm duplicate wells.

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 6 Analysis of m1E41920-KLH elicited Ab response to PI Ag.

Western blotting analysis of the reactivity of 1E4 and immune sera from KLH- or m1E41920-KLH–immunized mice with proteinase K-digested PI Ags.

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 7 Evaluation of the ability of immune sera from m1E41920-KLH-immunized mice to inhibit C. burnetii infection in BALB/c mice.

The inhibition of C. burnetii was performed by incubation of 1×10⁷ virulent C. burnetii with 30 μl of normal mouse sera or immune sera from m1E41920-KLH-immunized mice at 4 °C overnight. In addition, 1×10⁷ virulent C. burnetii NMI was treated with 30 μl of immune sera from PIV-vaccinated BALB/c mice or 300 μg of purified 1E4 in the same manner and used as positive controls. Six week-old BALB/c mice were infected by i.p. injection with 1×10⁷ of normal mouse sera, immune sera and 1E4-treated C. burnetii, respectively. Splenomegaly and bacterial burden in the spleen were measured at 14 days post infection and used as indicators to evaluate the ability of immune sera from m1E41920-KLH-immunized mice to inhibit C. burnetii infection in BALB/c mice with negative and positive controls. Panel A, splenomegaly was measured by spleen weight as percentage of body weight. Panel B, bacterial burden in the spleen was determined by real time-PCR and reported as log10 of C. burnetii com1 gene copy numbers. Panel C, Pathological changes in the spleen at 14 days post challenge. The data presented in each group is the average with standard deviation of four mice. *, P<0.05; **, P<0.01.

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 8 Evaluation of the protective efficacy of m1E41920-KLH against C. burnetii infection in BALB/c mice.

Panel A, m1E41920-KLH induced specific IgG response to PI antigen as measured by ELISA. A 96-well microtiter plate was coated with inactivated PI antigen and incubated with immune sera from KLH-, PIV- or m1E41920-KLH-immunized mice at 1:400 dilutions. The reactivity of immune sera with anti-IgG was measured by OD 490. The values represent the average absorbance at 490 nm of duplicate wells. Panel B, splenomegaly was measured by spleen weight as percentage of body weight. Panel C, bacterial burden in the spleen was determined by real time-PCR and reported as log10 of C. burnetii com1 gene copy numbers. Panel D, Pathological changes in the spleen at 14 days post challenge. The data presented in each group is the average with standard deviation of four mice. *, P<0.05; **, P<0.01; ***, P<0.001.

Peng, Y., Zhang, Y., Mitchell, W. J., & Zhang, G. (2012). Development of a lipopolysaccharide-targeted peptide mimic vaccine against Q fever. The Journal of Immunology, 189(10), 4909-4920.

Figure 9 The ability of 1E4 to inhibit C. burnetii infection in vivo.

Pathological changes in the spleen of mice infected with IgG2a isotype control, 10, 100, or 300 μg 1E4-treated C. burnetii.

Figure 10 The ability of 1E4 to confer protection in mice against C. burnetii aerosol infection.