anti-cfp10 antibody  (Advisains)

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: The primary antibodies used for CFP-10 and ESAT-6.

Article Snippet: Ab8 , Rabbit Polyclonal Anti-ESAT-6 antibody (AB45074) , Abcam PTY (LTD).

Techniques:

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: Stability of AuNPs with ESAT-6 and CFP-10 antibodies at different pH values. AuNPs of 14 nm at pH 7, 8, 9 and 10 were dispensed into wells of a 96-well plate. Anti-ESAT-6 and anti-CFP-10 antibodies were added into wells and incubated at room temperature for 5 min prior to addition of NaCl. Color changes, if any, were observed and with reference from AuNPs that had no antibody and NaCl, and optimum conditions for conjugation were chosen. The arrows indicate the selected conditions for conjugation.

Article Snippet: Ab8 , Rabbit Polyclonal Anti-ESAT-6 antibody (AB45074) , Abcam PTY (LTD).

Techniques: Incubation, Conjugation Assay

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: Compatibility of CFP-10 capture and detection antibody pairs. The capture and control antibodies were dotted on the nitrocellulose membrane using 1 µL of 1 mg/mL antibody. The membranes were left to dry for 15 min prior to testing. To test the samples, the negative control contained 30 µL of running buffer and 10 µL of AuNP–antibody conjugate, whilst the test sample also contained recombinant CFP-10 protein (4 µg/mL) or ESAT-6 protein (2.7 µg/mL). Tests were allowed to run for 15 min before analysis. ‘Ghost lines’ are indicated by blue circles.

Article Snippet: Ab8 , Rabbit Polyclonal Anti-ESAT-6 antibody (AB45074) , Abcam PTY (LTD).

Techniques: Negative Control, Recombinant

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: LOD of ESAT-6 ( A ) Ab6/Ab7 and ( B ) Ab7/Ab7 pair. ( A ) The Ab6 and ( B ) Ab7 capture antibodies were dispensed at 1 mg/mL on the nitrocellulose membrane, whilst the Ab7 conjugate was sprayed on the conjugate pad thereafter assembled on the backing cards. Recombinant ESAT-6 was serially diluted in the range indicated in order to compute the LOD. The arrows indicate the strip selected as the LOD. The results are representative of the same test performed in triplicate.

Article Snippet: Ab8 , Rabbit Polyclonal Anti-ESAT-6 antibody (AB45074) , Abcam PTY (LTD).

Techniques: Recombinant, Stripping Membranes

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: The primary antibodies used for CFP-10 and ESAT-6.

Article Snippet: Ab5 , Rabbit Polyclonal Anti-CFP-10 antibody (AB45073) , Abcam PTY (LTD) (Discovery Drive, Cambridge, UK).

Techniques:

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: Stability of AuNPs with ESAT-6 and CFP-10 antibodies at different pH values. AuNPs of 14 nm at pH 7, 8, 9 and 10 were dispensed into wells of a 96-well plate. Anti-ESAT-6 and anti-CFP-10 antibodies were added into wells and incubated at room temperature for 5 min prior to addition of NaCl. Color changes, if any, were observed and with reference from AuNPs that had no antibody and NaCl, and optimum conditions for conjugation were chosen. The arrows indicate the selected conditions for conjugation.

Article Snippet: Ab5 , Rabbit Polyclonal Anti-CFP-10 antibody (AB45073) , Abcam PTY (LTD) (Discovery Drive, Cambridge, UK).

Techniques: Incubation, Conjugation Assay

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: Compatibility of CFP-10 capture and detection antibody pairs. The capture and control antibodies were dotted on the nitrocellulose membrane using 1 µL of 1 mg/mL antibody. The membranes were left to dry for 15 min prior to testing. To test the samples, the negative control contained 30 µL of running buffer and 10 µL of AuNP–antibody conjugate, whilst the test sample also contained recombinant CFP-10 protein (4 µg/mL) or ESAT-6 protein (2.7 µg/mL). Tests were allowed to run for 15 min before analysis. ‘Ghost lines’ are indicated by blue circles.

Article Snippet: Ab5 , Rabbit Polyclonal Anti-CFP-10 antibody (AB45073) , Abcam PTY (LTD) (Discovery Drive, Cambridge, UK).

Techniques: Negative Control, Recombinant

Journal: Biosensors

Article Title: Development of Gold-Nanoparticle-Based Lateral Flow Immunoassays for Rapid Detection of TB ESAT-6 and CFP-10

doi: 10.3390/bios13030354

Figure Lengend Snippet: Determination of the LOD of CFP-10 ( A ) Ab1/Ab1 and ( B ) Ab1/Ab2 pairs. Ab1 was dispensed on the test line at a concentration of 0.5 mg/mL, and the anti-Mouse IgM antibody was dispensed at 0.3 mg/mL on the control line. The ( A ) Ab1 and ( B ) Ab2 were conjugated to AuNPs, then sprayed on the conjugate pad which was blocked using 3% BSA in sodium borate buffer. Following assembly of the nitrocellulose membrane, conjugate pad and absorbent pad on the backing cards, 3 mm strips were cut using the Kinbio cutter. Test samples were prepared at a range of 0.0769 × 10 6 and 0.0769 ng/mL recombinant CFP-10 in running buffer. Samples were then applied at a volume of 65 µL and analyzed after 15 min of incubation. These results are representative of the same test performed in triplicate.

Article Snippet: Ab5 , Rabbit Polyclonal Anti-CFP-10 antibody (AB45073) , Abcam PTY (LTD) (Discovery Drive, Cambridge, UK).

Techniques: Concentration Assay, Recombinant, Incubation

Journal: bioRxiv

Article Title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis

doi: 10.1101/2020.11.14.382572

Figure Lengend Snippet: A&B .CD63 and ESAT-6 antigens were detected in aerosol-EVs by ELISA. For all the samples, equal amounts of EVs (8μg/ml protein) were loaded in each well. AE-COPD: Aerosol-EVs of COPD subjects; AESN-PTB: Aerosol-EVs of smear negative PTB subjects. AESN-PTB+D: Aerosol-EVs of smear negative PTB subjects completing anti-TB medications. Number 1, 2 and 7 denotes patient samples used for the western blot. C. Western blot of EVs obtained from smear negative PTB (SN-PTB) subjects 1-3, showing the mild, highest and lowest level of ESAT-6 protein as depicted in . EVs were obtained by the direct immunomagnetic sorting of CD63+ EVs in the exhaled breath aerosols (EBA), and equal amount of EVs (4 μg/ml) was loaded in each well. D . In Cell Western assay image (merging of green and red channels), showing the induction of MHC II and CD11c expression in the mouse bone marrow derived mononuclear cells by AESN-PTB subject#2 versus #7. Equal amount of EVs (0.5 μg/ml) was added in each well, and CD11b expression (800 CW, green channel) was used for normalization of MHC II or CD11c expression (700 CW, red signal).Quantified data is given in the text. E. INF-γ assay in C57BL/6 mice (6-8 weeks, female). Mice were twice (a week apart) challenged with intranasal injection of 0.5 μg/ml (20 ul in 0.1 BSA) aerosol-EVs obtained from the study subjects. After 6 weeks, splenic cells were obtained and challenged with either ESAT-6 (10μg/ml) or Ag85 (10μg/ml), and then subjected to IFN-gamma measurement by ELISA. Red and chocolate color arrows represent patient#2, and 7respectively. Dormant infection was achieved by Mtb -m18b infection in mice (10^6 CFUs i.v. and 2 mg streptomycin/daily/two weeks) followed by two-months of streptomycin withdrawal (16960113). Data in A, B and E are presented as mean +/− SEM and each dot represents the mean value obtained from two mice injected with EVs of one study subject. **p<0.001, *** p<0.0001 (ANOVA and Dunnett post hoc test).

Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 1 µg/well of polyclonal rabbit anti-ESAT-6 Ab (#ab45073, Abcam Inc, USA) followed by blocking with 2% BSA for 1 h at 37° C or overnight at 4° C. EVs suspended in RIPA was centrifuged, and 50 μl of supernatant was added to the wells of the ELISA plate, covered and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, In-Cell ELISA, Expressing, Derivative Assay, Injection, Infection

Journal: bioRxiv

Article Title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis

doi: 10.1101/2020.11.14.382572

Figure Lengend Snippet: A. Schematic of the in vivo experimental plan in mice to test the hypothesis of nigudah-immunity depicted in . Mice were immunized or vaccinated with intranasal injection of 40 μl of 0.5 μg/ml/weekly/two weeks of aerosol-EVs obtained from COPD (n=15; 3 mice each for three subjects), or SN-PTB subjects(n=20; 5 for each of the four PTB subjects showing >1.5 ng/ml IFN-gamma response in ). Control groups of mice received intransal injection of either 40 μl of 0.1% BSA (n=15 mice) or BCG (5×10^5 CFU of ATCC # 35734; n=15 mice). Red-ink numbers 1-4 represent successive data collection plan to test for vaccine-induced protective immunity against Mtb -m18b infection (1×10^6 CFU by intravenous injection, and daily supplemented with 2 mg of stremptomycin sulphate s.c. for 4 weeks ( , ). B. Left histogram: IFN-gamma response of splenic cells obtained from healthy mice (n=4 in each group) subjected to aerosol inoculation (cumulative 50 minutes in two weeks; ∼15 μg EVs inhaled from 5 mice); four weeks after animals were sacrificed for the assay. Right histogram shows that the lungs or spleens of these mice were devoid of Mtb -DNA. The lungs obtained from a Mtb -m18b (1×10 5 CFUs, intranasal injection and 2 mg streptomycin/two weeks) infected mice (n=3)were used as positive control. D. EVs isolated from the aerosols and BAL fluid supernatant of AESN-PTB vaccinated and post-challenge Mtb -m18b infected mice (n=5 for each of the four subjects) shows the presence of ESAT-6 protein (ELISA). E. CD45+ and CD45-cells of BAL fluid were in vitro cultured for 24 hours in serum free media , and EVs were isolated from the supernatant by ultracentrifugation to perform ELISA for ESAT-6 (total ∼2×10^5 CD45-cells were pooled from ten mice). D. Mtb -m18b CFUs of lung and BAL fluid supernatant and CD45+/CD45-cell fractions (BAL fluid of 5 mice of each subject were pooled as one, and then immunomagnetic sorting of CD45+ cells were performed as described . Data in B, C-E are presented as mean +/− SEM. **p<0.001, *** p<0.0001 (ANOVA and Dunnett post hoc test).

Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 1 µg/well of polyclonal rabbit anti-ESAT-6 Ab (#ab45073, Abcam Inc, USA) followed by blocking with 2% BSA for 1 h at 37° C or overnight at 4° C. EVs suspended in RIPA was centrifuged, and 50 μl of supernatant was added to the wells of the ELISA plate, covered and incubated overnight at 4°C.

Techniques: In Vivo, Injection, Infection, Positive Control, Isolation, Enzyme-linked Immunosorbent Assay, In Vitro, Cell Culture

Journal: bioRxiv

Article Title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis

doi: 10.1101/2020.11.14.382572

Figure Lengend Snippet: Sorted CD45-cells of BAL fluid (AESN-PTB subject #2; on day-90 after aerosol-EV immunization, and infected with Mtb -m18b-GFP) were either cultured on a glass chamber coated with polylysine and fibronectin for IF imaging, or cultured in 48 well plates (1×10^3 cells/well in serum free media for naïve MSCs) for functional assays. A. Representative phase contrast and immunofluorescence images of immunomagnetically sorted CD45-cells showing intracellular Mtb -m18b-GFP (red arrow). 1. Phase contrast microscopy. 2-4: IF images, merged, DAPI staining for nucleus, and Mtb -m18b-GFP. Scalebar 20 μm. B. The CD45-cell intracellular Mtb -m18b-GFP bacteria were manually counted per microscopic field, and the data was used to find number of bacteria per 1×10^4 cells. The data was compared with the Mtb -DNA copy number, and Mtb -CFUs with and without early stationary phase supernatant (ESPSN) of Mtb -H37Ra, a conditioned media that can resuscitate viable but non culturable Mtb . C &D Sorted CD45-cells exhibit self-sufficiency and ASC phenotype on day-8 and differentiate/activate p53 on day-14, confirming the transient state of their enhanced stemness state (53;12;31). The gene expression data was compared with the MSCs of dormant Mtb -m18b infected mice . ND-not detected. E. Schematic of experimental design. A nimals (n=15; 5 mice for each subject #2-4) were injected with FM19G11 (5 mg/kg/ dissolved in 1.5% DMSO, i.p. thrice weekly for two weeks) for two weeks ,6 weeks after the infection with Mtb -m18b. The control groups were injected with 200 μl 1.5% DMSO in saline, i.p. thrice weekly for two weeks. Results showed that FM19G11 treatment lead to marp>ked reduction of Nigudah-immunity, including the reduction in ASCs (CD45-cells) in the BAL fluid, and corresponding reduction in the ESAT-6+ EVs in the aerosols (key component of the Nigudah immunity). EVs (average 0.5 ug/10^5 cells) were isolated by ultracentrifugation method; Data in B,C-E are presented as mean +/− SEM. **p<0.001, *** p<0.0001 (ANOVA and Dunnett post hoc test).

Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 1 µg/well of polyclonal rabbit anti-ESAT-6 Ab (#ab45073, Abcam Inc, USA) followed by blocking with 2% BSA for 1 h at 37° C or overnight at 4° C. EVs suspended in RIPA was centrifuged, and 50 μl of supernatant was added to the wells of the ELISA plate, covered and incubated overnight at 4°C.

Techniques: Infection, Cell Culture, Imaging, Functional Assay, Immunofluorescence, Microscopy, Staining, Expressing, Injection, Isolation

Journal: bioRxiv

Article Title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis

doi: 10.1101/2020.11.14.382572

Figure Lengend Snippet: A&B: Naïve CD271+ BM- MSCs treated with Mtb -m18b derived VBNC (1×10^4 /2×10^6 BM-MSCs in 6 well plates) led to enhanced stemness reprogramming, which can be inhibited by FM19G11 (0.1 μM/72 hours starting on day-9), a HIFs inhibitor known to inhibit enhanced stemness or altruistic stemness reprogramming . “ A ” shows an altered state of p53/MDM2 oscillation as measured by In-Cell ELISA, whereas “ B ” shows the expression of enhanced stemness genes, two key features of ASC phenotype (53; 58, 31). RNA data was compared with freshly obtained CD271+/CD45-BM-MSCs of one healthy donor. Noted that “-2” in “A” denotes cell seeding two days before VBNC addition. C-E. BM-MSC culture and the conditioned media of day-0-12 as shown in “A” were subjected Mtb -DNA, Mtb -CFU as well as EV isolation (by density gradient centrifugation) assays to study relation between VBNC resuscitation and the secretion of ESAT-6 rich EVs. “C” shows resuscitation of Mtb -m18b derived VBNC by CD271+ BM-MSCs on day-12 versus day-4 culture (blue arrows). “D” shows that the process of VBNC resuscitation did not cause extracellular secretion of Mtb , but (E) triggered the secretion of ESAT+ and CD63+ EVs. FM19G11 (0.1μM/72 hours starting on day-5) treated cells failed to resuscitate VBNC as well as secretion of ESAT-6 rich EVs, which was obtained by density gradient centrifugation of conditioned media of cumulative 5×10^6BM-MSCs; experiment was repeated four times. CD271+ BM-MSCs were obtained by direct flow cytometry sorting of CD271+/CD45-cells from the BM mononuclear cells of healthy donors following proper consent . **P< 0.001, ***P<0.0001, Student’s t-test; Error bar represents SEM.

Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 1 µg/well of polyclonal rabbit anti-ESAT-6 Ab (#ab45073, Abcam Inc, USA) followed by blocking with 2% BSA for 1 h at 37° C or overnight at 4° C. EVs suspended in RIPA was centrifuged, and 50 μl of supernatant was added to the wells of the ELISA plate, covered and incubated overnight at 4°C.

Techniques: Derivative Assay, In-Cell ELISA, Expressing, Isolation, Gradient Centrifugation, Flow Cytometry

Journal: bioRxiv

Article Title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis

doi: 10.1101/2020.11.14.382572

Figure Lengend Snippet: A. Identification of VBNC + smear negative PTB subjects by subjecting processed sputum to early stationary phase supernatant (ESPSN). The ESPSN is rich in resuscitation factors, and was obtained from Mtb -H37Ra as previously described . Smear negative sputum that exhibited Mtb -CFUs (as confirmed by AFB staining) following ESPSN treatment was identified as sputum containing VBNC. B. Western blot images of CD63 and ESAT-6 proteins present in the EVs (10 µg loaded in each well) obtained from the exhaled breath condensate (EBC) of VBNC versus non-VBNC subjects. EBC products were pooled together from the 4/6 VBNC and 4/4 non-VBNC subjects, and EVs were isolated by density gradient centrifugation method. EVs (10 μg loading in each well) obtained from CD271+ ASCs infected with VBNC served as the positive control. C . Correlation between the number of CD45-ASCs cells in the BAL fluid, and the optical density reading of the ELISA assay for ESAT-6 in the aerosol-EVs (5 μg/ml) of the corresponding mice. Data is obtained from the experimental results of . D . A schematic model of Nigudah-immunity: VBNC present in the granuloma of PTB subjects are recruited by BM-MSCs migrating to lung . VBNC harboring BM-MSCs reprogram to altruistic stem cells (ASCs) and migrate to the BAL fluid, where these ASCs releases EVs pack with Mtb -antigen including ESAT-6. Following coughing or Kirtan-chanting, these EVs spread in the community by aerosol based transmission, and come in contact with the mucosal immune cells (M cells and DC of nasal, laryngeal and bronchial mucosa) of healthy subjects, or subjects affected by active PTB. EVs efficiently deliver the Mtb antigen to DC and other antigen-presenting cells leading to T-cell priming. In the subjects with active PTB, EV-induced immunity may also activate the innate ASC defense mechanism including the extraction of Mtb -antigen from the VBNC, and export antigen rich EVs into the BAL fluid.

Article Snippet: Briefly, 96-well ELISA plates were coated overnight with 1 µg/well of polyclonal rabbit anti-ESAT-6 Ab (#ab45073, Abcam Inc, USA) followed by blocking with 2% BSA for 1 h at 37° C or overnight at 4° C. EVs suspended in RIPA was centrifuged, and 50 μl of supernatant was added to the wells of the ELISA plate, covered and incubated overnight at 4°C.

Techniques: Staining, Western Blot, Isolation, Gradient Centrifugation, Infection, Positive Control, Enzyme-linked Immunosorbent Assay, Transmission Assay