Goat anti human igm

To assess GM2 specificity, we utilized a modified version of the previously published anti-GM2 ELISA [3 (link), 26 (link)]. Wells of a MaxiSorp plate (NUNC) were coated with 70µL 0.1 µg/mL GM2 (Sigma), diluted in methanol, and left to evaporate O/N in a laminar flow. Wells were blocked with 200 µL 1% BSA-PBS for 1 h at RT. Patient sera were diluted 1:200 in 1% BSA-PBS, either or not pre-incubated with GM1 or GM2 (50 µg/µL for 30 min at RT), and incubated in GM2 coated wells 1 h at RT. Next, wells were washed 3 times with PBS, and IgM binding was detected using goat anti-human IgM (Sigma, 70µL 1:10000 in 1% BSA-PBS, 1 h at RT), followed by a 3-time wash in PBS, and incubation with streptavidin-POD (Sigma, 70µL 1:1000 in 1% BSA-PBS, 30 min at RT), and a final 3-time wash in PBS. For detection 100µL TMB (Invitrogen) was added to each well. Finally, the reaction was stopped using 1 M HCL (Fisher Chemical). All measurements were conducted in triplicate and OD_{450 nm} (read-out at a wave length of 450 nm) was analyzed using a SpectraMax M3 (Molecular devices). The d-OD_450nm was obtained by subtracting the OD_450nm from an uncoated well of the respective OD_450nm of a GM2 coated well. The % inhibition of IgM binding was calculated by setting the OD_450nm of the serum sample without GM1/GM2 preincubation at 0% inhibition.

Microplates (96-well) coated with nickel (Thermo Scientific Nunc Immobilizer Nickel—Chelate, Waltham, MA) were pre-washed 3X (300 μl/well) with PBST (PBS + 0.05% Tween 20 and covered with 50 μl of 10 μg/ml purified recombinant protein HspX-His previously dissolved in KCl 0.01 M. Plates were incubated at 4°C with shaking (450 rpm) overnight. Plates then were washed 3X with 300 μl PBST and blocked with 300 μl of 5% skim milk for 2 hr, followed by 3 washes with PBST (300 μl ea.). Prepared plates were loaded with 100 μl per well of patient serum (1:50 dilution in1% skim milk) and incubated at 37°C for 90 min. After incubation, the plates were washed with PBST (3 x 300 μl) and 100 μl of secondary antibody-HRP (goat anti-human IgM at 1:10,000 or anti-human IgG at 1:30,000) (Sigma Aldrich) were added and incubated for 1 hr at 37°C. Plates were washed again with PBST (3 x 300 μl) and 100 μl of Sigma Fast OPD solution (Peroxidase substrate, Sigma Aldrich, St Louis, MO) was added to each well. After 30 min incubation, the reaction was stopped with 50 μl of 3N sulfuric acid. ELISA absorbance readings at 492 nm were taken using a microplate reader Synergy 2 (BioTek, Inc., Winooski, VT).

LFAs were performed with luminescent up-converting reporter particles (UCP) as a sensitive background-free label for quantitative detection of targeted analytes (Corstjens et al., 2003 (link), 2005 (link); Zuiderwijk et al., 2003 (link)). UCP nanomaterials (200 nm NaYF₄:Yb³⁺, Er³⁺ particles, functionalized with carboxyl groups) were obtained from Intelligent Material Solutions Inc. (Princeton, New Jersey, United States). UCP conjugates were prepared with goat anti-human IgM (I0759, Sigma-Aldrich, St. Louis, Missouri, United States) at a concentration of 50 μg antibody per mg UCP according to the method described previously (Bobosha et al., 2014 (link); Corstjens et al., 2019 (link)).

Serum was obtained from all patients and stored at −80 °C until analysis. Sera were screened for antibodies against the LPS serotype O157 with an ELISA as previously described by Chart [15 ]. In brief, the ELISA plates were coated with LPS from E. coli O157:H7 [List Biological Laboratories Inc., Campbell, CA; product code 206, diluted in carbonate buffer (pH 9.6) to a concentration of 20 ug/ml]. After incubation overnight at 4 °C, the plates were blocked, and diluted serum was added to the plate together with predetermined positive and negative control sera. After addition of the antibody goat anti-human IgM (Sigma-Aldrich, St. Louis, MO; product code A0420, diluted 1/500 with phosphate buffered saline with bovine serum albumin) and substrate (p-nitrophenyl phosphate tablets in diethanolaminebuffer), the absorbance was measured at 405 nm. A positive IgM reaction was defined by a mean absorbance of >0.800; values of <0.400 were to be considered negative and values between 0.400 and 0.800 were considered to be dubious and as such taken to be negative for the purpose of this study.