SGVB⁺⁺ (with Ca⁺⁺ and Mg⁺⁺)

S501131-1L

1L

170 mM Sucrose, 0.1 % gelatin, 5 mM Veronal, 57 mM NaCl, 0.025 % NaN3 , 0.15 mM calcium chloride, and 0.5 mM magnesium chloride, pH 7.3.

General Description SGVB⁺⁺ is a specialized buffer which is low in ionic strength (due to the low NaCl concentration), but isotonic (due to the sucrose). This combination of properties allows complement assays or binding assays to be performed with cells at low ionic strength, which improves binding, while the sucrose keeps the cells from lysing due to the high concentration of sucrose. This buffer is practically identical to DGVB buffer except that they use D-glucose instead of sucrose to maintain their isotonic characteristic. These buffers are isotonic with VBS, GVB, PBS, etc. meaning that the osmotic pressure on cell membranes will be the same even though the salt concentration is low. SGVB++ buffer is especially useful in C1 assays (Dodds, A.W. and Sim, R.B. (1997)) and in binding assays such as those measuring the binding of factor B or factor H to EsC3b cells (sheep erythrocytes bearing surface-bound C3b) (Pangburn, M.K.and Muller-Eberhard, H.J. (1978); Pangburn, M.K., et al. (1980)). These interactions are sensitive to thesalt concentration and lowering the ionic strength can enhance binding 5-to10-fold. Buffer Components Veronal is used as the buffer because in the mid-1900s this was the only buffer for pH range 7.2-7.4 that did not chelate metal ions and did not to inhibit complement reactions as did other buffers. Sodium chloride and sucrose are present to provide an isotonic environment so that cells do not lyse due to osmotic pressure. Gelatin is present to prevent loss of protein components due to adsorption onto tips or tubes during dilutions and in the assays themselves. Azide is present to prevent bacterial growth. Calcium is present because the classical and lectin pathways require it to hold the subunits of the C1, MBL and ficolin complexes together. Magnesium is required for formation of the C3 and C5 convertases of all three pathways of complement. Although total calcium in plasma and serum is about 2.3 mM, the free available concentration is about 1.07 mM. The remainder is complexed with proteins or small molecules. Complement buffers contain 0.15 mM calcium because it was found that under CH50 assay conditions the classical pathway activity is greater at 0.15 mM than at 1.0 mM calcium. Plasma and serum concentrations of total magnesium are about 0.87 mM and the free available concentration is 0.5 mM. Thus, SGVB⁺⁺contains 0.5 mM magnesium chloride. Physical Characteristics The concentrationof gelatin in this buffer is below the concentration that forms solid gels at room temperature. However, at 4℃ some strings of gelatin can form during standing. They can be redissolved easily by heating to 37℃ or by brief heating in a microwave oven. Applications SGVB⁺⁺ can be used to titer both purified C1 complex and C1 in normal human serum samples (Dodds, A.W. and Sim, R.B. (1997)). In these assays all components are diluted in SGVB⁺⁺ and the EA cells are washed into this buffer. Binding assays such as those measuring the binding of C5, factor B or factor H to EsC3b cells (sheep erythrocytes bearing surface-bound C3b) or ZymosanC3b cells also benefit from being performed in SGVB++ (Pangburn, M.K. and Muller-Eberhard, H.J. (1978); Pangburn, M.K., et al. (1980)). The enhancement of binding can be as much as 10-fold compared to using a buffer with physiological ionic strength such as GVB or PBS.