Alexa fluor 546 goat anti human igg

Amine coated slides and protein arrays were prepared as described in previous studies [14 (link)–16 (link)]. Briefly, serum samples from 96 people with P. vivax malaria and 96 unexposed individuals were used for humoral immune response analysis. Purified rPv32 was spotted into duplicate wells for arrays at 25 ng/μl in PBS and incubated for 1 h at 37 °C. After blocking with 1.0 μl of blocking buffer (5% BSA in PBS with 0.1% Tween 20, PBS/T) for 1 h at 37 °C, the chips were probed with human sera from malaria patients or healthy individuals (1:50 dilution) that were first pre-absorbed against wheat germ lysate (1:100 dilution) to block anti-wheat germ antibodies. Samples were detected by Alexa Fluor 546 goat anti-human IgG (10 ng/μl, Invitrogen Corp.) in PBS/T, quantified as described previously and scanned by a fluorescence scanner (ScanArray Express, PerkinElmer, Boston, MA, USA) [14 (link)]. The cut-off value is equal to the mean ± three standard deviations (SD) of the mean fluorescence intensity (MFI) of the 96 negative samples.

Protein probing onto a microarray-specific slide and serum screening constitute the main components of this innovative technology. A well-type amine array as described by Chen et al. [21 (link)] will help identify serological biomarkers of human parasites as the project progresses. First, crude WGCF recombinant proteins, as well as positive and negative controls will be spotted as probes in duplicate on a microarray-specific slide and incubated. Then, serum samples from parasite-specific exposed versus unexposed individuals will be used to screen the probes. Bound antibodies will be visualized using Alexa Fluor 546 goat anti-human IgG (Invitrogen), scanned in a fluorescent microarray scanner (CapitalBio), and fixed circle approach will be used to quantify the arrays.
Furthermore, screening of the highly immuno-reactive antigens/epitopes will be performed to profile their immune responses using also sera from infected individuals and healthy subjects.

Protein microarray was performed to evaluate total IgG reactivity. 3 aminopropyl-coated slides were prepared as described previously [31 (link)]. The slides were printed to each spot with recombinant protein (RII, 200 ng/μl and RIII-V, 12.5 ng/μl) as a saturated concentration and incubated for 2 hours at 37°C. The recombinant protein coated slide was blocked with blocking buffer (5% BSA in PBS-T) for 1 hour at 37°C. Vivax patient and healthy individual sera were diluted in PBS-T to 1:25 and probed on the chip for 1 hour at 37°C. The arrays were visualized with 10 ng/μl of Alexa Fluor 546 goat anti-human IgG (Invitrogen, Carlsbad, CA, USA) in PBS-T for 1 h at 37°C and scanned with ScanArray Gx laser confocal scanner (PerkinElmer, Norwalk, CT, USA). The positive cut-off values calculated by negative control mean fluorescence intensity (MFI) plus two standard deviations.

An array of 22 peptides, 18-mer each (Additional file 1: Table S1) overlapping by nine amino acids with >90% purity and spanning the conserved C terminus of PvMSP8 Sal-1 sequences was custom synthesized, purified, and used for epitope mapping (Peptron Co., Ltd., Daejeon, Korea). In the process of coating step, 1 μL of peptides (10–40 μg/mL) in 200 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Thermo Fisher Scientific Inc., Rockford, IL, USA) and 50 mM N-hydroxysuccinimide (NHS, Thermo Fisher Scientific Inc.) with coupling buffer were coated on amine-coated slide, respectively. After blocking, rPvMSP8-immunized mouse sera, pre-immunized mouse sera at 1:200 dilution, pooled sera from 10 high IgG titers of vivax-infected patients at 1:50 dilution, or 10 vivax-unexposed human sera at 1:50 dilution was added, respectively. Alexa Fluor 546 goat anti-mouse IgG (50 μg/mL, Invitrogen) or Alexa Fluor 546 goat anti-human IgG (10 μg/mL, Invitrogen) antibodies were used for detection of binding activity.

Amine-coated slides were prepared as described previously [15 (link)]. To develop protein arrays, sera samples from 56 cases of vivax malaria and 40 unexposed individuals were used for humoral immune response analyses using well-type amine arrays. A series of double dilutions was developed to optimize the coating concentration (0.1–200 μg/ml) of PvETRAMP11.2, and PvEXP1. The purified recombinant PvETRAMP11.2 and PvEXP1 proteins were spotted onto the duplicate wells of the arrays in PBS at 50 and 100 μg/ml, respectively, and incubated for 1 h at 37°C. Each well was blocked with 1.0 μl of blocking buffer (5% BSA in PBS with 0.1% Tween 20, PBS-T) and incubated for 1 h at 37°C. The chips were pre-absorbed against wheat germ lysate (1:100 dilution) to block anti-wheat germ antibodies and then probed with human malaria patients or healthy individuals (1:200 dilution). Alexa Fluor 546 goat anti-human IgG (10 μg/ml, Invitrogen) in PBS-T was used to detect antibodies, which was quantified as described previously, and the antibodies were scanned in a fluorescence scanner (ScanArray Express; PerkinElmer, Boston, MA, USA) [15 (link)]. The cutoff value was equal to the mean fluorescence intensity (MFI) plus two standard deviations (SDs) of the negative samples.

Three aminopropyl-coated slides were prepared as described previously48 (link). Briefly, the slides were spotted and then each recombinant protein was applied to the spot at concentrations of 50 ng/μl for PvRBP1a-34 and 100 ng/μl for PvRBP1b-32. The slides were incubated for 2 h at 37 °C. After blocking, each spot was probed with 1 μl of patient or healthy serum (1:25 dilution in PBS-T) followed by incubation for 1 h at 37 °C. The arrays were visualized with 10 ng/μl of Alexa Fluor 546 goat anti-human IgG (Invitrogen, Carlsbad, CA, USA) in PBS-T for 1 h at 37 °C and scanned with a ScanArray Gx laser confocal scanner (PerkinElmer, Norwalk, CT, USA). The positive cut-off values of the negative control plus two standard deviations were used.