Immunology & Immunological Disorders Poster Session
Materials & Methods
Discussion & Conclusion
A Novel Anti-inflammatory Activity of Human Lysozyme: Modulation of Serum Complement Activation
Contact Person: Michael O. Ogundele (firstname.lastname@example.org)
Materials and Methods
Gelatin Veronal buffered saline (GVB++) and Sucrose gelatin veronal-bufered saline (SGVB++) with Ca++ and Mg++ were prepared as described (19). GVB-- buffer containing 10mM ethylene diacetyl ethylamine (EDTA) was used as the stopping solution. Sucrose-GVB++ was utilized for the serum complement assay in place of GVB++ buffer, with ionic strength of 0.065 and 0.149 respectively
Purified human lysozome
Purified lysozome crystals was obtained from Serva Feinbiochemica-Heidelberg, Germany. It was dissolved in SGVB++ and added to serum complement assays in final concentrations ranging from 50 to 1500µg/ml.
Sensitized sheep red blood cells
Sheep red blood cells (SRBC) (ORAW 30/31) obtained from Behringwerke, Marburg, Germany were washed twice in GVB++ buffer and resuspended in the same buffer. The SRBC concentration was standardized sphectrometrically by adding 100µl of the SRBC suspension to 1.9ml water and reading the absorbance at 541nm (A541)with a spectrophotometer (Stasar III Gilford instrument laboratories, Inc. Oberlin, Ohio). GVB++ was added to the SRBC until the A541 was 0.468, corresponding to an approximate cell suspension of 1 x 109/ml.
A sensitized cell suspension was prepared by a dropwise addition of an equal volume of anti-Forsman hemolysin (diluted 1:300 in GVB++ containing 20mM EDTA) to the previously prepared SRBC suspension. The cells were incubated at 37°C for 30 min and at 0-2°C for another 30minutes. The cells were then washed once in SGVB++ and resuspended in this buffer. A final suspension of 5 x 108 cells/ml, obtained by an equal addition of an equal volume of buffer, was employed in the test assays.
Normal human serum haemolytic assay
Serum samples were obtained from 10 healthy blood donors. A micro-modification of the standard hemolytic assay was used for the assessment of serum complement activity. A 1:20 dilution of serum was prepared, different volumes (12.5-100µl) of which is pipetted into the assay tubes, as well as 40µl of sensitized SRBC, to obtain a total reaction volume of 375µl. SGVB++ buffer was used for the serum assay, incubating the mixture for 60 minutes at 37°C, with occasional mixing. A further modification of the standard assay procedure was introduced by stopping the reaction with 1 ml of GVB-- containing 10mM EDTA. After pelleting the remaining intact cells, 200µl of each supernatant was pipetted into a 96 well flat-bottomed microtitre plates (Greiner labortechnik, Frickenhausen, Germany) and the amount of the released heamoglobin recorded by a plate sphectrophotometer (MR 700 microplate reader, Dynatech Instruments, Inc. Torrance, California) at a test wavelength of 410 against a reference wavelength of 630. The degree of lysis produced was obtained from the equation:
Y = Test OD - Spontaneous lysis OD - HBM/Serum blank OD X 100
H2O lysis OD - Spontaneous lysis OD
Assays of serum complement inhibition by lysozyme
Using the modified CH50 assay as described above, a constant volume of serum, containing approxmately one CH50 unit, and SRBC, were pipetted into assay tubes. Lysozyme was added before incubation of the reaction of the reaction mixtures, at final concentrations ranging from 50 to 1500 microgrammes per millilitre.
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