Goat anti human igg

To qualitatively discern the reactivity of subjects’ sera with Sm-TSP-2, Western blot screening was performed with a selected set of blinded samples. Recombinant Sm-TSP-2 [13 (link),14 (link),18 (link)] was loaded (2 μg) under non-reducing and reducing (with addition of 2-mercaptoethanol, βME) conditions on a 4–20% SDS-PAGE gel (Invitrogen) at 135 V for 80 min. An irrelevant control recombinant protein (Na-GST-1, also expressed in a Pichia Pink system) was loaded as a negative control. Gels were then either stained with SimplyBlue™ (Thermo) or transferred on to 0.2 μm nitrocellulose membranes for 60 min at 30 V. Following transfer, the blots were stained with Ponceau S (Sigma) to confirm protein transfer and protein loads, followed by blocking with 2% milk diluted in 1X-PBST-20 (0.05%). Serum samples were then diluted at 1:100 in blocking buffer and incubated for 60 min at room temperature. Blots were washed 3 times with 1X-PBST-20 (0.05%) and then incubated with alkaline phosphatase secondary antibody, goat antihuman IgG (Southern Biotech) at a dilution of 1:1000 in blocking buffer. Following subsequent 3 washes with 1X-PBST-20 (0.05%), the blots were developed for 15 min with BCIP/NBT substrate (Invitrogen), then stopped with multiple washes with ultra-pure water. Blots were scanned together on a flatbed scanner (Epson) for visual documentation.

To assess antibody binding, soluble protein was plated on Immulon 2HB plates (Thermo Fisher Scientific) at 2 μg/mL overnight at 4°C. In cases where capture ELISA was used, plates were pre-incubated for 2 h at room temperature (RT) with 5 μg/mL GNA lectin (Sigma) or 2 μg/mL anti-AviTag (Genscript) and washed 3× with PBS+ 0.05% Tween 20 (PBS-T) before antigen plating overnight. Between each of the subsequent incubation steps, plates were washed 3× with PBS-T. Non-specific binding was blocked by incubation with 5% fetal bovine serum (FBS) (Gibco) diluted in PBS-T for 1 h at RT. Primary monoclonal antibodies were diluted in 5% FBS-PBST starting at 20 μg/mL with a serial 1:5 dilution (unless otherwise specified) and then added to the plate for 1 h at RT. Secondary antibody, either goat anti-human IgG (Southern Biotech) or goat anti-human IgA (Invitrogen), was diluted 1:10,000 in 5% FBS diluted in PBS-T and added for 1 h at RT. Reaction was developed by 10 min incubation with One Step Ultra-TMB (Thermo Fisher Scientific) and stopped with 1N sulfuric acid. Plate absorbances were read at 450 nm (Biotek). Data are represented as mean ± SEM for one ELISA experiment performed in duplicate. ELISA experiments were repeated with at least 2 different antibody preparation aliquots. The area under the curve (AUC) was calculated using GraphPad Prism 8.0.0.

Total IgGs were quantified using 96-well MaxiSorp plates (Nunc) coated with goat
anti-human IgG (SouthernBiotech, 2040-01) using Certified Reference Material 470
(ERMs-DA470, Sigma-Aldrich) as a standard. To test specific binding of antibody
constructs, ELISA plates were coated with 2 μg ml⁻¹ of type I
recombinant human collagen (Millipore, CC050), 2 μg ml⁻¹ of an
anti-human LAIR1 antibody (clone DX26, BD Biosciences 550810), 1 μg
ml⁻¹ of recombinant human GM-CSF (Gentaur), 2 μg
ml⁻¹ of an anti-PD1 or an anti-SLAM antibody (R&D Systems,
AF1086 and AF164). Plates were blocked with 1% bovine serum albumin (BSA) and incubated
with titrated antibodies, followed by AP-conjugated goat anti-human IgG, Fcγ
fragment specific (Jackson Immuno Research, 109-056-098). Plates were then washed,
substrate (p-NPP, Sigma) was added and plates were read at 405 nm.

Nasal washes, tears and stool samples were collected as described
previously.(17 (link)) Immulon 2 plates were
coated with 4 μg/ml of lipoarabinomannan H37Ra LAM
(provided by Dr. Larry Schlesinger, Ohio State University) diluted in
100% ethanol. The plates were dried for 3–4 hours in a fume hood
then washed with PBS plus 0.05% tween 20. Plates were blocked overnight
at 4°C with 1% BSA in PBS with 0.05% tween 20. Following
blocking, the plates were washed and serum, tear, nasal wash and stool samples
were added to duplicate wells at optimal dilutions predetermined for each type
of sample. The plates were incubated overnight at 4°C. After washing and
the application of goat anti-human IgG or goat anti-human IgA horseradish
peroxidase (Southern Biotechnology Associates, Inc.), plates were incubated for
1 hour at room temperature in the dark. Then plates were washed and the ABTS
substrate (Kirkegaard & Perry, Gaithersburg, MD, USA) was added. After the
plates were incubated 20 minutes at room temperature, absorbance was read at 405
nm.