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The slides were viewed on a Zeiss Axioplan 2 imaging system with Plan Apochromat 100 /1.4 oil immersion objective. Images were captured using Axiovision 4.6.3 software and edited using Adobe Photoshop. For immunogold labeling, merozoites were fixed in 4 paraformaldehyde in 0.1 M phosphate buffer at pH 7.4 for 1 h at room temperature, rinsed three times in buffer, and infiltrated with 1 and then 10 gelatin before immersing in 2.3 M sucrose in phosphate buffer overnight at 4 for cryoprotection. Frozen samples were prepared by mounting onto aluminum pins and rapidly immersing in liquid nitrogen in preparation for ultrathin 80 nm sectioning on a Leica EM FC6 ultramicrotome. Ultra thin sections were labeled as per Tokuyasu (62), with a rabbit anti-human IgM antiserumVOLUME 291 ?NUMBER 27 ?JULY 1,14296 JOURNAL OF BIOLOGICAL CHEMISTRYHuman IgM Binds Broadly Diverged DBLMSP Protein Variants(Abcam) diluted 1:25, and detected with 10-nm protein A gold. Imaging was performed on an FEI 120kV Spirit Biotwin with a Tietz F4.15 CCD camera. Cell Culture of Transfectomas Secreting Anti-dinitrophenyl Human IgM--Transfectomas expressing anti-dinitrophenyl IgM antibodies containing the human constant heavy chains either with (Xp) or without (Gp) the mouse J-chain (22) were a kind gift of Prof. Marc Shulman. Transfectomas were grown in DMEM supplemented with 10 fetal bovine serum, 0.6 mg/ml G418, and 0.02 -mercaptoethanol, and supernatants were harvested and filtered before use in ELISA and AVEXIS experiments. ELISA and Primary Antibodies--Biotinylated recombinant P. falciparum proteins were normalized and immobilized on streptavidin-coated plates, preblocked with HBS and 0.1 Tween 20 (HBST), 2 BSA for 30 min. After washing with HBST, serial dilutions of either pooled human sera from 10 malaria-exposed or malaria-na e individuals, transfectoma cell culture supernatants, or purified primary antibodies were incubated for 90 min. The plates were again washed with HBST, and appropriate alkaline phosphatase-conjugated secondary antibodies were incubated for 1 h. The plates were washed with HBST and once with HBS before adding p-nitrophenyl at 1 mg/ml, and absorbance at 405 nm was quantified on either a PHERAstar Plus or FLUOstar Optima plate reader (BMG Labtech). For domain mapping experiments involving chimeric human antibodies, an HRP-conjugated anti-human secondary antibody was used. Following PBS washes, tetramethylbenzidine substrate was added, and absorbance was quantified at 450 nm. All procedures were performed at room temperature. Primary antibodies used were: anti-Cd4 OX68 (1:1000; AbD Serotec); purified human IgA, IgE, IgM (3 g/ml; Sigma), or IgG (3 g/ml; Bethyl Laboratories); human, goat, rabbit, guinea pig, or bovine serum (1:1000; Sigma); purified mouse IgM (10 g/ml MEM-150; Abcam); or domain-swapped human antibodies at 25 nM (25, 26). All alkaline-phosphatase-conjugated secondary antibodies were from Sigma, except anti-rabbit immunoglobulins (Jackson ImmunoResearch). Mapping DBLMSP and DBLMSP2 Binding Site on Human IgM--Domain-specific monoclonal antibodies that bind the human constant chain (23) were incubated for 90 min with either biotinylated human IgM or biotinylated human IgG (used as a control) before washing and adding pentamerized, -lactamase-tagged DBLMSP or DBLMSP2 and incubating for a https://britishrestaurantawards.org/members/neck40layer/activity/360513/ further hour. After washes, the -lactamase substrate nitrocefin was added at 125 g/ml, and colorimetric turnover was measured by absorbance.