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Development of a fluorescence assay for the detection of L-ficolin–MASP in serum or purified samples

2006; Elsevier BV; Volume: 66; Issue: 1-3 Linguagem: Inglês

10.1016/j.jbbm.2005.12.001

1872-857X

Krishana Gulla, Kavita Gupta, Rajesh Kumar Gupta, Vinay Vyas, Krishnan Hajela,

Coagulation, Bradykinin, Polyphosphates, and Angioedema

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.