Method for measuring sialic acid in immunoglobulin g and immunoglobulin g anti-double-stranded dna antibodies

A method for measuring the absolute amount of sialic acid in immunoglobulin G anti-double-stranded DNA antibodies comprises steps of:

• Step 1:
• in an enzyme immunoassay petridish, adding 150 μl of 0.5 mg/ml of protamine chloride in each grid and placing said petridish at room temperature for 2 hours;
• Step 2:
• washing (three times) with PBS (pH=7.2) and adding 100 μl of 50 μg/ml calf thymus double-stranded DNA overnight at 4° C.;
• Step 3:
• preparing oxidized bovine serum albumin, wherein the bovine serum albumin is dissolved in 20 mM potassium periodate (PBS, pH=7.2) and 50 mM sodium acetate (final pH=4.0 of the mixture of sodium periodate and sodium acetate) at 4° C. (for 30 minutes) to obtain a mixture, and the mixture is dialyzed with tris-buffered saline (pH=7.4), and adding (0.1% by volume) of polyoxyethylene (20) sorbitan monolaurate solution to make 1% oxidized bovine serum albumin;
• Step 4:
• using PBS-0.5% Tween 20 as a flushing fluid to flush the enzyme immunoassay petridish (four times), adding 300 μl of 1% of oxidation bovine serum albumin into each grid, and placing said petridish at room temperature for 2 hours in order to block non-specific adhesion;
• Step 5:
• washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding 100 μl of immunoglobulin G isolated from protein G, and placing said petridish at room temperature for two hours;
• Step 6:
• washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, and adding diluted (500 times) horseradish peroxidase-linked sambucus nigra agglutinin lectin (SNA) 100 μl to each grid;
• Step 7:
• washing (four times) the enzyme immunoassay petridish as in Step 4 with PBS-0.5% Tween 20, adding mixture of tetramethyl benzidine solution 50 μl and peroxide hydrogen solution 50 μl at room temperature for 5 minutes, and adding 100 μl of 0.5N H2SO4 into each grid to prevent the reaction;
• and
• Step 8:
• measuring the absorption value at 450 nm in each grid in the enzyme immunoassay analyzer.