Hrp conjugated anti goat igg

Plasma samples were harvested from mice using sterile technique. ELISA
plates (cat# 3855, ThermoFisher Scientific, Waltham, MA) were coated
with LPS (2 µg/well, catalog# L2762 Sigma-Aldrich, St. Louis,
MO) at 4°C overnight. After washing 3× in buffer
(PBS/0.05% Tween 20) and blocking with BSA (250 µl/well,
1% in PBS buffer), diluted mouse plasma (1:5 or 1:10 in
Mg²⁺-EGTA buffer) was incubated on an ELISA plate at 37°C for
1 h (50 µl/well). The plates were washed 3× in washing buffer
and then goat anti-mouse C3 Ab (100 µl/well, 1:4000 dilution in
1% BSA in PBS buffer; MP Cappel, CA) was added for 1 h. Wells were
washed 3× as above and incubated with HRP-conjugated anti-goat IgG (100
µl/well, 1:2000 dilution in 1% BSA in PBS buffer; Jackson
ImmunoResearch, PA) for 1 h. After 3 washes, Substrate Reagent (R&D
Systems, MN) was added for 10 min (100 µl/well). The reaction was
stopped with 50 µl 1 M H₂SO₄, and the OD of
samples was measured at 450 nm. Similar experiments were performed with normal
human serum and FD-depleted human serum supplemented with purified human FD
(CompTech, Tyler, Texas). The primary and second antibodies used in the LPS
assay with human serum are: Mouse anti-human C3d (ThermoScientific, #HYB
005-01-02) and HRP Donkey anti-Mouse IgG (Jackson Immunoresearch)
respectively.

C57BL/6J mice (The Jackson Laboartory) were administered i.p. with various concentrations of Salp20. Plasma samples were collected at various time points (2 to 24 h) for an LPS-dependent AP activation assay [32 (link)–34 (link)]. Briefly, plasma was diluted (1:10 in Mg²⁺-EGTA GVB²⁺ buffer), applied to LPS-coated ELISA plates (2µg /well), and incubated at 37°C for 1 h. After washing, goat anti-mouse C3 Ab (1:4000 dilution, MP Cappel, cat#55463) was added followed by HRP-conjugated anti-goat IgG (1:2000 dilution; Jackson ImmunoResearch), after which substrate reagent (cat#DY999, R&D Systems) was added for 10 min. The reaction was stopped with 1M H2SO4 and the OD of samples measured at 450 nm.
To assess the AP activity in the OVA-induced asthma model, mice were sacrificed on day 17 (see below) and their lungs lavaged 3 times with 1 mL of sterile PBS. Samples were cleared by centrifugation at 14,000 rpm for 20 min at 4°C. Cleared samples were diluted at 1:2 in Mg²⁺-EGTA GVB²⁺ buffer prior to being applied to LPS-coated plates. Post lavage, lungs were harvested and homogenized in 2 mL of cold PBS. Homogenates were cleared twice with centrifugation at 14,000 rpm × 20 min at 4°C. Samples were normalized by protein concentration based on absorbance at 280 nm and diluted 1:2 in Mg²⁺-EGTA GVB²⁺ buffer prior to being applied to LPS-coated plates. AP activity was measured as above.