Antibodies presented in post-treatment sera were screened using ProtoArray Human Protein Microarray V5 (Invitrogen, Grand Island, NY) according to manufacturer instructions. Antibody targets were identified by a Z factor ≥ 0.4. The presence of galectin antibodies in the sera were confirmed by immunoblot analysis using recombinant human galectins-1, -3 and -9 (R&D Systems (Minneapolis, MN)). To compare antibody levels as a function of treatment, galectin-1, -3 and -9 immunoblots (blocking 5% BSA) were incubated with pre-treatment and post-treatment sera (1:2000 dilution in PBS with 2% BSA) overnight, followed by HRP-conjugated goat anti-human IgG antibody (Invitrogen), and visualized with ECL. Densities of protein bands and backgrounds were quantified using NIH ImmageJ software. After background subtraction, galectin antibody responses to treatment were determined by the formula: Fold-change = (DensityPost – DensityPre)/DensityPre. Fold-change ≥ 0.5 was considered a significant increase.
Hrp conjugated goat anti human igg antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HRP-conjugated goat anti-human IgG antibody is a secondary antibody used in immunoassays. It is designed to bind to human immunoglobulin G (IgG) and is conjugated with horseradish peroxidase (HRP) enzyme, which can be used for colorimetric or chemiluminescent detection.
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5 protocols using hrp conjugated goat anti human igg antibody
1
Profiling Galectin Autoantibodies in Treatment Response
2
Quantifying Ebola Virus Protein Interactions
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For detection of direct interactions between Ebola virus GPs and ADI-15946, His-tagged GPs 2 μg/ml were first coated with coating buffer (0.1 M Na2CO3 and NaHCO3 [pH 9.6]) on 96-well plates overnight at 4°C. Wells were washed with PBST (PBS plus 0.2% Tween 20) and saturated for 1 h at 37°C with PBS–4% nonfat milk. Three-fold dilutions of ADI-15946 were added, and the mixture was incubated for 1 h at 37°C. Bound particles were detected with HRP-conjugated goat anti-human IgG antibody (Invitrogen; A18817). PS-associated ELISAs were carried out as previously described (57 (link)). PS (Sigma; P7769) and phosphatidylcholine (PC) (Sigma; P3556) were first dissolved in chloroform as a stock solution. Then, the solution was diluted to 5 μg/mL with methanol and added to the ELISA plate. Upon being dried, the plates were blocked with Tris-buffered saline (TBS)–4% bovine serum albumin (BSA) fraction V. TIM-1 ECD, TIM-1 IgV, TIM-1 ΔIgV, and control protein were diluted in TBS–10 mM CaCl2, and the mixture was added to the wells and incubated at 37°C for 1 h. The plates were washed with TBST (TBS plus 0.05% Tween 20) before incubation with HRP-conjugated mouse anti-human His antibody (Biolegend; 652503). The absorbance at 450 nm was measured by microplate reader (Thermo Fisher Scientific).
3
Quantifying Ebola Virus Protein Interactions
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For detection of direct interactions between Ebola virus GPs and ADI-15946, His-tagged GPs 2 μg/ml were first coated with coating buffer (0.1 M Na2CO3 and NaHCO3 [pH 9.6]) on 96-well plates overnight at 4°C. Wells were washed with PBST (PBS plus 0.2% Tween 20) and saturated for 1 h at 37°C with PBS–4% nonfat milk. Three-fold dilutions of ADI-15946 were added, and the mixture was incubated for 1 h at 37°C. Bound particles were detected with HRP-conjugated goat anti-human IgG antibody (Invitrogen; A18817). PS-associated ELISAs were carried out as previously described (57 (link)). PS (Sigma; P7769) and phosphatidylcholine (PC) (Sigma; P3556) were first dissolved in chloroform as a stock solution. Then, the solution was diluted to 5 μg/mL with methanol and added to the ELISA plate. Upon being dried, the plates were blocked with Tris-buffered saline (TBS)–4% bovine serum albumin (BSA) fraction V. TIM-1 ECD, TIM-1 IgV, TIM-1 ΔIgV, and control protein were diluted in TBS–10 mM CaCl2, and the mixture was added to the wells and incubated at 37°C for 1 h. The plates were washed with TBST (TBS plus 0.05% Tween 20) before incubation with HRP-conjugated mouse anti-human His antibody (Biolegend; 652503). The absorbance at 450 nm was measured by microplate reader (Thermo Fisher Scientific).
4
SARS-CoV-2 Spike Trimer Protein Binding Assay
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The capacity of the antibodies to bind Spike trimer protein of SARS-CoV-2 was determined by ELISA. The wells of Nunc MaxiSorp flat-bottom 96-well plates were coated with purified SARS-CoV-2 Spike trimer protein (Icosagen Cell Factory, catalog number: P-309-100) at 1 μg/mL in PBS overnight at 4°C and blocked with a PBS solution containing 2% BSA, 0.05% Tween 20 (Carl Roth, catalog number: 9127.1) for 1 h at RT. The samples (purified mAbs) were diluted in assay buffer (1% BSA, 0.05% Tween 20 in PBS) and added to the blocked plate in a final volume of 100 μL and incubated for 1 h at RT at 450 rpm. Secondary HRP-conjugated goat anti-human IgG antibody (Invitrogen, catalog number: A18823) was diluted in assay buffer at 1:10 000, added to the washed plate, and the plate incubated for 30 min at RT at 450 rpm. Colorimetric development was performed using 3,3’,5,5’-tetramethylbenzidine VII substrate. The reaction was stopped using 0.5 mol/L H2SO4 and absorbance was measured at 450 nm. Half-maximal (EC50) values were calculated using the Fourth Party Logistic Model (4PL).
5
Phage-Based ELISA for M. pneumoniae Antibodies
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ELISA was performed as previously described (Wang et al. 2016) (link). For the binding of phages to M. pneumoniaepositive serum, the ELISA plate was coated with 200 µL purified positive serum (100 µg/mL) at 4 °C overnight, while a negative serum control group and a BSA control group were set up. Followed by blocking with 5% skim milk for 3 h, the plates were rinsed for five times with 0.5% PBST. Then 50 µL of the phages were added into the plates and incubated at 37 °C for 2 h. After rinsing with 0.5% PBST for five times, HRP-labeled anti-M13 pIII monoclonal antibody (NEB, Beverly, MA, USA) was added and incubated at 37 °C for 15 min.
For the binding of M. pneumoniae-positive serum to the purified heptapeptides, the ELISA plate was coated with 200 µL heptapeptides (20 µg/mL) at 4 °C overnight. The plate was then blocked with 5% skim milk for 3 h at room temperature. Then, 50 µL of diluted serum (1:10) was added and incubated at 37 °C for 45 min. After rinsing with 0.5% PBST for 5 times, the HRP-conjugated goat anti-human IgG antibody (1: 5000) (Cat # 62-8420, Invitrogen, USA) was added and incubated at 37 °C for 30 min. After rinsing, the reaction was terminated with coloration for 10 min. Then the OD value at a wavelength of 450 nm was measured by a microplate reader (Tecan, Infinite F50, Swiss).
For the binding of M. pneumoniae-positive serum to the purified heptapeptides, the ELISA plate was coated with 200 µL heptapeptides (20 µg/mL) at 4 °C overnight. The plate was then blocked with 5% skim milk for 3 h at room temperature. Then, 50 µL of diluted serum (1:10) was added and incubated at 37 °C for 45 min. After rinsing with 0.5% PBST for 5 times, the HRP-conjugated goat anti-human IgG antibody (1: 5000) (Cat # 62-8420, Invitrogen, USA) was added and incubated at 37 °C for 30 min. After rinsing, the reaction was terminated with coloration for 10 min. Then the OD value at a wavelength of 450 nm was measured by a microplate reader (Tecan, Infinite F50, Swiss).
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