Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) Isotype-specific competition WB. LVS lysate nitrocellulose strips were pretreated with either 800 µg/ml of Ab12 (competitor mAb, when indicated), or were mock-treated, and then 5 µg/ml of Ab12, Ab53 or Ab64 (primary mAb) was incubated with the membrane strips. Anti (a)-m (mouse)-IgG (H+L, binds IgG1, IgG2b and IgG3) or a-m-IgG2a-specific alkaline phosphatase (AP) conjugates (second Ab) were used for development, as indicated. ( B ) Immunoprecipitation. Twelve µg of the indicated mAb were incubated with 100 µl (0.25 OD 600 ) of LVS lysate (+) or just buffer, followed by capture of the antibody: antigen complex with 7.5 µl of Protein G agarose resin. After washing the resin to remove unbound components of the sample, Ab and Ab-bound antigen were visualized by SDS-PAGE on 4–15% polyacrylamide gradient gels under reducing (and boiling) or non-reducing conditions. The positions of prestained molecular weight standards (in kDa) are indicated. The positions of Ab, Ab H and L chains, and GroEL are indicated by arrows. Ft mAbs Ab58 (IgG1, anti-histone-like protein HU form B) and FB11 (IgG2a, anti-LPS) were used as specificity controls.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Incubation, Membrane, Immunoprecipitation, SDS Page, Molecular Weight

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A–D ) Western blot. Lysates of E. coli transformed with FtGroEL-encoding vector, empty vector, or vectors encoding the K344E and Y476E FtGroEL mutants were electrophoresed in preparative 7.5% polyacrylamide gels under denaturing conditions, allowing the dye in the sample buffer to run out of the gel in an attempt to obtain maximal separation between the Ft and Ec GroEL monomers (hence the stretched-out appearance of the GroEL bands compared with the GroEL bands in the Western blots of and ). After transfer to nitrocellulose, strips were probed with secondary supernatants of 24 IgG-producing hybridomas or of the Ab53 or Ab64 hybridoma cell lines. ( E ) Classification of FtGroEL mAbs into five epitope types based on Western blot results from A to D. Binding (+) or lack of binding (–) are indicated in black and red font, respectively, and mAbs selected for further analysis are bolded.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Western Blot, Transformation Assay, Plasmid Preparation, Binding Assay

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) ELISA. Human or mouse recombinant Hsp60 was coated on ELISA plates, and the plates were probed with serial dilutions of the indicated mAbs (made from the same stocks as the ones used in the experiments in , except for the ab13532 anti-Hsp60 mAb, which was used only for the experiments in this figure). ( B ) Western blot. Recombinant human and mouse Hsp60 were separately electrophoresed in preparative 4–15% polyacrylamide gels under denaturing conditions and, after transfer to nitrocellulose, strips were probed with 10 µg/ml of the indicated mAbs followed by AP-conjugated-anti-mouse IgG (H+L) (Promega). The positions of prestained molecular weight standards (in kDa) are indicated.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Western Blot, Molecular Weight

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) Competition ELISA. HRP-conjugated streptavidin was used to detect the binding of biotinylated reporter antibodies (indicated by *) to native GroEL (except for Ab12, which was detected by HRP-conjugated anti-mouse IgG3), in the presence of the indicated competitor mAbs. ( B ) Data summary. Competition, defined as a ≥80% increase in percent inhibition is indicated by +; a 40–79% increase in percent inhibition is indicated by ±; and ≤39% increase in percent inhibition is indicated by –. Four groups of mAbs based on similarity in crossblocking profiles are colored red, orange, green or blue. NA, not applicable; ND, not done.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Inhibition

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) BALB/cJ female mice (n = 15) were inoculated i.n. with 91–164 CFU of SchuS4, injected i.p. with 50 µg of the indicated mAbs 2 hours post inoculation, and bled then euthanized 3 days post inoculation for blood CFU determination. Data were pooled from 3 experiments with 5 mice per group for each mAb and compared for statistical significance with PBS only for groups that were tested at the same time (same panel). ( B ) BALB/cJ female mice (n = 4) were inoculated i.n. with 93 (n = 2) or 114 (n = 2) CFU of SchuS4, injected i.p. with 50 µg of the indicated mAbs 2 hours post inoculation, and bled then euthanized 3 days post inoculation for blood, lung and spleen CFU determination. Percent CFU reduction compared with PBS was calculated from the median CFU numbers and the P value (indicated below the percent CFU reduction by *p≤0.05, **p≤0.01, or ***p≤0.001) was determined using the two-tailed Mann-Whitney test. All mAbs are IgG2a except for Ab12 and TIB-114, which are IgG3 (indicated as G3). The Ab52 mAb (anti-Ft O-antigen) was used as standard. The TIB-114 (anti-sheep red blood cells) and CO17-1A (anti-human tumor-associated antigen EpCam) mAbs were used as isotype controls. All other mAbs are anti-Ft GroEL.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Injection, Two Tailed Test, MANN-WHITNEY

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) Difference heat-maps for FtGroEL complexed with Fab (left) or IgG (right) of the indicated mAbs in the indicated molar ratio of mAb to FtGroEL monomer. The times of FtGroEL-Ab interaction (10 seconds, 100 seconds, and 1000 seconds) are specified. As indicated in the color bar, blue shades suggest more buried regions (which exchange slower upon antibody-binding), and red shades suggest more exposed regions (which exchange faster upon antibody-binding). The darkest blue region(s) in each difference heat-map were taken as the DXMS-epitopes and their spans are indicated. Note that the heat map for FtGroEL in complex with Ab53 Fab (top left, the first one to be performed) is in a slightly different format than the other heat-maps. ( B ) Linear representation of the DXMS-epitopes on the sequence/secondary structure template of FtGroEL (see legend to for description, except partial view). When both Fab and IgG data were available, the results were combined. Epitopes for the indicated mAbs are represented as labeled colored boxes below the FtGroEL sequence.

Article Snippet: The Ab52 and FB11 IgG2a hybridoma mAbs, specific for Ft LPS, were generated in our laboratory or purchased from GeneTex Inc. (Irvine, CA), respectively.

Techniques: Binding Assay, Sequencing, Labeling