An Effective Tip For RHOBTB1
05%. Binding inhibition assay A binding inhibition assay was carried out to study the ability of antibodies raised in mice against PvDBP_RII to inhibit the binding of this protein to its receptor DARC, as previously described (42, 47). Sera diluted as indicated were pre-incubated with 0.025?��g/mL of recombinant PvDBP_RII (SalI strain sequence) for 1?h at RT before adding to ELISA plates pre-coated with recombinant DARC (nDARC-Fc; N-terminal extracellular 60 amino acids of DARC fused to Fc region of human IgG) protein at 1?��g/mL. Bound PvDBP_RII was detected with rabbit polyclonal anti-PvDBP_RII sera (1:4000 dilution), followed by goat anti-rabbit IgG HRP-conjugated antibodies (1:6000 dilution). A standard curve generated using binding of PvDBP_RII (0.002�C0.025?��g/mL) RHOBTB1 to DARC was used to convert OD values into amount of protein bound in each well. Percent inhibition was calculated as 100% minus %?protein bound. A positive control serum (raised in mice against PvDBP_RII using Freund��s adjuvant) and selleckchem a negative control serum (from na?ve mice) were included in each assay. The negative control sample had a median of 4% binding inhibition across all replicates tested across the different dilutions. Indirect immunofluorescence assay Plasmodium vivax-infected blood was collected from malaria patients attending the clinics of the Shoklo Malaria Research Unit (SMRU), Mae Sot region northwest of Thailand, after written informed consent (OXTREC 027-025; University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK). The P. vivax-infected erythrocytes were cultured to the late schizont stage prior to being concentrated and washed as previously described (48), before being smeared onto glass slides, air dried, and fixed with cold acetone for 20?min. For the immunofluorescence assays (IFAs), the thin-smear preparations were blocked with 1% Casein, 10% goat serum in PBS for 1?h at RT. Mouse sera (diluted 1:50) or rabbit sera (diluted 1:100) were applied to the slides and incubated for 1.5?h at 37.5��C or 1?h Selleck Gefitinib at RT in a humidified incubator, respectively. The slides were then washed three times with PBS before the addition of anti-mouse or anti-rabbit IgG conjugated to Alexa Fluor 488 (Molecular Probes) diluted 1:100 (mouse) or 1:1000 (rabbit) with 1% Casein, 10% goat serum in PBS for 1?h at RT, and mounted with Vectashield (Vectorlabs) with DAPI (4��,6-diamidino-2-phenylindole) (Invitrogen). Binding was visualized using a Nikon TS 100 epifluorescence microscope. Statistical analysis All statistical analysis was carried out using Prism version 5.04 (Graphpad, USA). For non-parametric data, a Kruskal�CWallis test with Dunn��s multiple comparison post-test was used to compare more than two groups, and a Mann�CWhitney U-test was used to compare two groups. A two-way ANOVA with Bonferroni��s multiple comparison post-test was used to explore the effect of two variables. P?
