Journal:

Article Title: Detection by Enzyme Immunoassay of Serum Immunoglobulin G Antibodies That Recognize Specific Cryptosporidium parvum Antigens

doi:

Figure Lengend Snippet: Western blot of serum samples from symptomatic and asymptomatic Carrollton, Ga., residents. A crude antigen supernatant prepared from sonicated oocysts was resolved on an SDS–10 to 22.5% polyacrylamide gel under nonreducing conditions (600 ng/mm of gel width). Following electrotransfer to Immobilon P, 2-mm-wide strips of membrane were incubated overnight at 4°C with serum from individual patients with cryptosporidiosis at a dilution of 1:100 in 0.3% Tween 20-PBS. The blots were developed with a biotinylated anti-human IgG monoclonal antibody and alkaline phosphatase-labeled streptavidin as described in Materials and Methods. (A) Antigens recognized by serum collected from patients with cryptosporidiosis 28 to 66 days after symptom onset (late outbreak). (B) Antigens recognized by sera collected from patients less than 10 days after symptom onset (early outbreak). The locations of the 27- and 17-kDa-antigen families are indicated.

Article Snippet: Bound antibodies were detected with a biotin-labeled mouse monoclonal antibody against human IgG (clone HP6017; Zymed Laboratories, South San Francisco, Calif.) and alkaline phosphatase-labeled streptavidin (Life Technologies).

Techniques: Western Blot, Sonication, Electrotransfer, Incubation, Labeling

Journal:

Article Title: Detection by Enzyme Immunoassay of Serum Immunoglobulin G Antibodies That Recognize Specific Cryptosporidium parvum Antigens

doi:

Figure Lengend Snippet: Extraction of 27- and 17-kDa antigens by Triton X-114. A crude antigen supernatant prepared from sonicated oocysts was fractionated by Triton X-114 phase partition extraction as described in Materials and Methods. Total unfractionated proteins (Ttl), proteins found in the detergent-depleted aqueous phase (AQ), and proteins extracted into the Triton X-114 detergent-rich phase (TX) were resolved by SDS-polyacrylamide gel electrophoresis as described in the legend to Fig. ​Fig.1.1. The proteins were blotted onto Immobilon P and incubated overnight at 4°C with either a 1:100 dilution of human serum from a patient with cryptosporidiosis in 0.3% Tween 20-PBS (Human serum), a 1:1 dilution of tissue culture supernatant in 0.3% Tween 20-PBS containing a monoclonal antibody (C6C1) against the 17-kDa antigen (anti-17-kDa MAb), a 1:1 dilution of tissue culture supernatant in 0.3% Tween 20-PBS containing a monoclonal antibody (C6B6) against the 27-kDa antigen (anti-27-kDa MAb), or AuroDye-forte to stain all proteins (AuroDye). The blots were developed with either a biotinylated anti-human IgG monoclonal antibody and streptavidin-labeled alkaline phosphatase (human serum) or a horseradish peroxidase-labeled goat anti-mouse polyclonal antibody (anti-17-kDa MAb and anti-27-kDa MAb) as described in Materials and Methods. The positions of the molecular mass markers and the 27- and 17-kDa-antigen families are indicated.

Article Snippet: Bound antibodies were detected with a biotin-labeled mouse monoclonal antibody against human IgG (clone HP6017; Zymed Laboratories, South San Francisco, Calif.) and alkaline phosphatase-labeled streptavidin (Life Technologies).

Techniques: Sonication, Polyacrylamide Gel Electrophoresis, Incubation, Staining, Labeling

Journal:

Article Title: Detection by Enzyme Immunoassay of Serum Immunoglobulin G Antibodies That Recognize Specific Cryptosporidium parvum Antigens

doi:

Figure Lengend Snippet: ELISA responses for paired serum samples. Paired serum samples from patients with cryptosporidiosis were assayed for antigen (Ag)-specific IgG antibodies using the Triton antigen ELISA (A) or the recombinant-27-kDa-antigen ELISA (B) as described in Materials and Methods. The 76-arbitrary unit (A) and 206-unit (B) threshholds for positivity in the two ELISAs are indicated by dotted lines across the graphs. Samples with ELISA responses near or below this cutoff were assayed on three separate occasions, and all others were assayed twice. The mean values and 1 standard deviation are indicated. Each kind of line represents an individual patient.

Article Snippet: Bound antibodies were detected with a biotin-labeled mouse monoclonal antibody against human IgG (clone HP6017; Zymed Laboratories, South San Francisco, Calif.) and alkaline phosphatase-labeled streptavidin (Life Technologies).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: rsFcγRIIa dimer binding to IgG-opsonized envelope is dependent on the clonal diversity (polyclonality) of opsonizing Abs. HIV Env gp140-AD8 was coated at concentrations of 1.25, 2.5, 5, or 10 μg/mL and opsonized with HIV+ patient serum at 1/2,000 dilution (∼5–10 μg/mL total Abs) or three mAb IgG antibodies, separately (at 20 μg/mL) and in combination (mAb mix, 60 μg/mL total), (A) anti-IgG binding and (B) dimeric rsFcγRIIa-H131 binding.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Binding Assay

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: The initial anti-HIV IgG response of patient SC49 had high dimeric rsFcγRIIIa activity and low FcγRII binding activity. (A) Left axis ( green symbols) AD8-gp140 opsonizing IgG1 titer, mean DF50 ± range ( n = 3); right axis ( red symbols) viral load (Roche) is shown with dotted lines for the assay detection limits at 400 and subsequently 50 viral copies per milliliter. (B) Left axis ( orange closed symbols), opsonizing IgG3 levels at 1/5 dilution, mean AU ± range, n = 3, open symbols show HIV−human serum samples ( n = 3), background anti-IgG1 ( green ), and anti-IgG3 ( orange ) binding to gp140; right axis, dimeric rsFcγRIIa-H131, AU binding to AD8-gp140 opsonized at 1/50 sample dilution, mean ± SEM, n = 4. (C) Left axis, dimeric rsFcγRIIb binding ( blue symbols), normalized AU, and right axis filled triangle ( magenta ) dimeric rsFcγRIIIa-V158 binding, normalized AU. The arrow marks the time of ART initiation, and the period of poor ART compliance is shown by a hatched gray background pattern and complete ART removal is indicated by the plain gray background. Means for the early and late peak activities were compared using an unpaired Mann–Whitney t -test, * p < .05 and ** p ≤ .01.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Activity Assay, Binding Assay, MANN-WHITNEY

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: The anti-HIV IgG response and FcγR activities were detected in SC samples by ELISA using immobilized AD8-gp140; (A–D) opsonizing IgG1 titer, mean DF50 ± range ( n = 3); (E–H) opsonizing IgG3, AU ± range (at 1/5 dilution, n = 3); (I–P) dimeric rsFcγR binding to Env opsonized with serum samples or plasma at 1/50 dilution was measured and (I–L) dimeric rsFcγRIIa activity presented as mean AU ± SEM, n = 4; (M–P) dimeric rsFcγRIIIa activity (mean normalized AU ± SEM, n = 4); (Q–T) the ratio of IgG3, AU/IgG1 titer, mean DF50; and (U–X) the ratio of dimeric rsFcγRIIIa activity/dimeric rsFcγRIIa activity. The open bars indicate an off-ART sample for SC49. Change from baseline was assessed by one-way ANOVA, Tukey's multiple comparison test, * p < .05, ** p < .01, *** p < .001, and **** p < .0001. AU, absorbance units; ELISA, enzyme-linked immunosorbent assay; FcγR, Fcγ receptor; SC, seroconverter.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

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

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: Anti-Env IgG1 titer and dimeric rsFcγR binding activity decline during ART. Mean half-lives (t 1/2 ) ± SEM ( n = 5–8 as indicated), one-way ANOVA, Tukey's multiple comparison test, * p < .05. ART, antiretroviral therapy; SEM, standard error of the mean.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Binding Assay, Activity Assay

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: STI in subject SC21 allowed viral rebound and stimulated stepwise boosting of anti-Env IgG1 and dimeric rsFcγR activity. (A) Left axis, dimeric rsFcγRIIa-H131 binding to Env opsonized with a 1/150 dilution of samples (normalized AU, mean ± range, n = 3, blue symbols) and anti-IgG1 binding to a 1/400 dilution of samples with and without gp140 ( green closed and open symbols, respectively). Right axis, viral load ( red symbols) is shown with dotted lines indicating the assay detection limits at 400 and subsequently 50 viral copies per milliliter. Assay upper limit is 750,000 copies/mL. At recruitment, SC21 had been on ART for 1 month and periods of STIs are shown with a gray background. (B) Left axis, dimeric rsFcγRIIIa binding (normalized AU, magenta symbols, n = 3). Right axis, p24-stimulated T cell proliferation ( red symbols) and (C) IFNγ ELISPOTs/10 6 PBMCs stimulated with gag, pol, env, and nef peptides. Dimeric rsFcγR activities at the indicated times were compared using an unpaired Mann–Whitney t -test, ** p ≤ .01 and *** p < .001. STI, structured treatment interruption.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Activity Assay, Binding Assay, MANN-WHITNEY

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: Boosted Ab, dimeric rsFcγR activity, and T cell immunity in STI in subject SC24 are contemporaneous with partial viral control. At recruitment, SC24 had been on ART for 6 months. STI periods of no ART are shown with the gray background. (A) Anti-IgG and dimeric rsFcγRIIa binding to Env opsonized at 1/1,000 or 1/150 sample dilution, respectively, is shown for 1 representative experiment. (B, C) Left axis, dimeric rsFcγR binding (filled triangles ) to Env opsonized at 1/10 sample dilution, mean ± SEM, n = 6, and nonspecific binding in the absence of Env ( open triangles , n = 1). Open circles show, placed arbitrarily on the x-axis, background binding to Env treated with 1/10 HIV− serum samples ( n = 3). (B) Right axis, viral load with dotted lines indicating the assay detection limits at 400 and subsequently 50 viral copies per milliliter. (C) Right axis, p24-stimulated T cell proliferation and (D) IFNγ ELISPOTs/10 6 PBMCs stimulated with gag, pol, env, and nef peptides. Dimeric rsFcγR activities at indicated times were compared using an unpaired Mann–Whitney t -test, ** p ≤ .01.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Activity Assay, Binding Assay, MANN-WHITNEY

Journal: AIDS Research and Human Retroviruses

Article Title: Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption

doi: 10.1089/aid.2019.0047

Figure Lengend Snippet: STI in patient SC84 stimulates anti-Env IgG with dimeric rsFcγR binding activity, and sustained ART interruption is marked by episodic control of viremia. (A, B) Dimeric rsFcγR binding to Env opsonized at 1/150 sample dilution, mean ± range, n = 3. (A) Left axis, dimeric rsFcγRIIa-H131 activity, normalized AU, and anti-IgG1 binding to a 1/400 dilution of samples with and without gp140 ( green closed and open symbols, respectively); right axis, viral load with dotted lines indicating the assay detection limits at 400 and subsequently 50 viral copies per milliliter. The arrow marks the initiation of ART and periods of ART removal are shown with a gray background. (B) Left axis, dimeric rsFcγRIIIa activity, normalized AU, right axis p24-stimulated T cell proliferation and (C) IFNγ ELISPOTs/10 6 PBMCs stimulated with gag, pol, env, and nef peptides. Activities at indicated times were compared using an unpaired Mann–Whitney t -test, * p < .05 and *** p < .001.

Article Snippet: Detection was performed with biotinylated mouse anti-huIgG1 (A10650, clone HP6069; Thermo Fisher) or mouse anti-IgG3 (05-3640, clone HP6047; Thermo Fisher) at 1 μg/mL in PBS/1% BSA for 1 h at 37°C, followed by 1:10,000 high-sensitivity streptavidin-HRP conjugate (Pierce, Thermo Fisher) in PBS/1% BSA for 1 h, then developed with TMB.

Techniques: Binding Assay, Activity Assay, MANN-WHITNEY