Development of a fluorescence assay for the detection of L-ficolin-MASP in serum or purified samples.
A fluorescence assay for the detection of L-ficolin-MASP in human serum or purified sample was developed by measuring the cleavage of fluorescent amide substrate by L-ficolin associated MASPs bound to the lipoteichoic acid (LTA). LTA (Staphylococcus aureus DSM 20233) was coated on NuncMaxisorp microtiter plates and serum or purified sample incubated overnight at 4 degrees C to allow the L-ficolin-MASP to bind LTA. Assay conditions for binding and complete cleavage of fluorescent amide substrate were standardized. The optimum temperature, incubation time and molarity of NaCl for LTA-ficolin binding were found to be 4 degrees C for 6 h at 1 M NaCl concentration. The optimum incubation time and pH for complete cleavage of fluorescent amide substrate by LTA bound L-ficolin associated MASP were found to be 2 h at pH 8.5. LTA-ficolin binding was found to be highly specific and was inhibited completely by LTA but not with mannose. A calibration curve was prepared by using the purified ficolin-MASP complex (1 to 12 mug/ml) and could be used to find concentration of ficolin-MASP complex in normal human serum.
Gulla
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KC
School of Life Sciences, Vigyan Bhawan, Khandwa Road Campus, Devi Ahilya University, Indore, 452017, India.
Gupta
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Gupta
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RK
Vyas
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Hajela
K
K
D002623:Chemistry Techniques, Analytical / Q000379:methods*; D005453:Fluorescence; D006801:Humans; D037102:Lectins / Q000032:analysis* / Q000097:blood* / Q000302:isolation & purification; D008070:Lipopolysaccharides / Q000737:chemistry; D050606:Mannose-Binding Protein-Associated Serine Proteases / Q000032:analysis*; D013237:Stereoisomerism; D013682:Teichoic Acids / Q000737:chemistry; D000097604:Ficolins
D037102:Lectins; D008070:Lipopolysaccharides; D050606:Mannose-Binding Protein-Associated Serine Proteases; D013682:Teichoic Acids; D000097604:Ficolins; C009900:lipoteichoic acid