Antibody production was investigated before and after infection, during the same period of the splenocyte cultures. For this, rSMP-3 and L. infantum SLA (0.5 and 1.0 µg per well, respectively) were added to the plates for 16 h at 4 °C. After blocking with phosphate buffer saline (PBS 1×) plus 0.05% Tween 20 (PBS-T), individual serum samples were diluted 1:100 in PBS-T, and incubation was processed for 1 h at 37 °C. After washing, the anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich) were diluted 1:5000 and 1:10,000, respectively, in PBS-T and added to the wells. A new incubation was processed for 1 h at 37 °C, at which time the plates were washed and reactions were developed using H2O2, ortho-phenylenediamine, and citrate-phosphate buffer pH 5.0, for 30 min, in the dark. Reaction interruption was performed using 2 N H2SO4, and optical density (O.D.) values were read in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Sunnyvale, CA, USA), at 492 nanometers. The ratios between IgG2a and IgG1 levels were calculated as described elsewhere [87 (link)].
Anti mouse igg1 and igg2a horseradish peroxidase conjugated antibodies
Manufactured by Merck Group
Sourced in United States
Anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies are laboratory reagents used for the detection and quantification of mouse IgG1 and IgG2a antibodies in various immunoassays. The antibodies are conjugated with horseradish peroxidase, an enzyme that can be used as a detection label.
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7 protocols using anti mouse igg1 and igg2a horseradish peroxidase conjugated antibodies
1
Antibody Response to Leishmania Antigens
2
Quantifying Anti-Parasite Antibody Levels
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Anti-parasite IgG1 and IgG2a isotype antibody levels were evaluated by ELISA assay in sera samples from infected and treated mice collected one and 15 days post-treatment. Briefly, L. infantum Soluble Leishmania Antigen (SLA) was used as the coating antigen (1.0 µg/well) and sera samples were diluted 1:100 in PBS-T (PBS plus 0.05% (v/v) Tween 20). Anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich, USA) were used both at a 1:10,000 dilution in PBS-T. Reactions were developed in the presence of H2O2, ortho-phenylenediamine and citrate–phosphate buffer pH 5.0 for 30 min and in the dark. Next, reactions were stopped by addition of 2 N H2SO4, after which OD values were measured at 492 nm in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Canada).
3
Humoral Response Evaluation after Vaccination
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For the analysis of the humoral response induced after vaccination, the rLiHyp1, rLiHyp6, rHRF, and SLA-specific IgG1 and IgG2a isotypes levels were evaluated using the sera samples collected from the vaccinated animals, four weeks after the third and last immunization and before infection; as well as in the 10th week after challenge, by an ELISA technique, as previously described [13 (link)]. The used concentration of the recombinant proteins were: 1.0, 1.0, and 0.5 μg per well for rLiHyp1, rLiHyp6, and rHRF proteins, respectively; 0.5 μg per well of each protein to their mixture, and 2.0 μg per well of SLA L. infantum. The sera samples were diluted at 1:200, and the anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich, USA) were employed both in a 1:5,000 dilution. The nitrite production in the cultures supernatant was assessed by the Griess reaction [37 (link)]. Data were expressed as μM per 106 cells.
4
Anti-SLA Antibody Isotype Quantification
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The anti-SLA IgG1 and IgG2a isotype levels were evaluated in sera samples of the treated and infected animals, one and 15 days after treatment. L. infantum SLA was used as an antigen (1.0 μg per well), and sera samples were 1:100 diluted in PBS-T (PBS 1× plus 0.05% Tween 20). Both anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich, USA) were used in a 1:10,000 dilution, which was performed using PBS-T. Reactions were developed by adding H2O2, ortho-phenylenediamine and citrate-phosphate buffer at pH 5.0, for 30 min and in the dark. Reactions were then stopped by adding 2N H2SO4, and the optical density (OD) values were read in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Canada) at 492 nm.
5
Evaluating Humoral Immune Response
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The humoral response was evaluated by collecting sera samples of the animals (n = 4, per group) after the last immunization and before infection (4 weeks after last doses), as well as at the 10th week after challenge. For this, The LiHyD-and L. infantum SLA-specific IgG1 and IgG2a isotypes levels were measured by an ELISA technique, as described (12) . Recombinant protein was employed at 5 lg/mL, and SLA was assayed at 10 lg/mL (100 lL per well). The sera samples were diluted at 1 : 100, and the anti-mouse IgG1 and IgG2a horseradish peroxidase-conjugated antibodies (Sigma-Aldrich) were used in a 1 : 5000 dilution.
6
Quantification of Nitrite and Antileishmanial Antibodies
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The nitrite production was evaluated in the culture supernatants that were used to quantify the cytokines, by Griess method [40] . The humoral response was evaluated by determining the antileishmanial IgG1 and IgG2a isotype antibodies levels, as described [41] . Briefly, L. infantum SLA was used as an antigen (1.0 μg per well) and serum samples were diluted at 1:100 in PBS-T (PBS 1× plus Tween-20 0.05%). The anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich) were used both 1:10,000 diluted in PBS-T, and reactions were developed through incubation with 2 mg ortophenylenediamine, 2 μL H 2 O 2 30 vol. and 10 mL citrate-phosphate buffer pH 5.0, for 30 min in the dark; when they were stopped by adding 25 μL H 2 SO 4 2 N. The optical density was read in an ELISA microplate spectrophotometer at 492 nm.
7
Quantifying Parasite-Specific Antibodies
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The antibody production was evaluated one-day after the end of the treatments. For this, the parasite-specific IgG1 or IgG2a isotype antibodies levels were measured by an ELISA technique, as described (Coelho et al., 2003) . Briefly, L. amazonensis SLA was used as an antigen (at a concentration of 1.0 mg per well), whereas serum samples were diluted at 1:100 in a PBS-T (PBS 1Â plus 0.05% Tween 20) buffer and anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich) were both used at a 1:10,000 dilution (also diluted in PBS-T). Reactions were developed by incubation with a solution composed by 2 mL H 2 O 2 , 2 mg ortophenylenediamine and 10 mL citrate-phosphate buffer at pH 5.0, for 30 min in the dark and stopped by adding 20 mL H 2 SO 4 2 N. The optical density was determined in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Canada), at 492 nm.
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