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

Abstract

A method for measuring the amount of sialic acid in immunoglobulin G and immunoglobulin G anti-ds DNA antibodies is disclosed. The method for measuring the amount of sialic acid in immunoglobulin G in the present invention uses culture fluid, blood, plasma, or serum to directly measure the amount of sialic acid in immunoglobulin G. Also, using a mouse monoclonal antibody immunoglobulin G as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml in phosphate buffered saline (PBS), produces good results. The method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies has never been done and the present invention produces good results as well.

1 . A method for measuring the amount of sialic acid in immunoglobulin G comprising steps of:
 Step 1: in an ELISA plate, adding 200 ng of  Sambucus nigra  agglutinin lectin dissolved in 100 μl PBS (pH=7.2) in each well, and placing said enzyme-linked immunosorbent assay (ELISA) plate in a refrigerator at 4° C. overnight;   Step 2: washing the plate four times with PBS-0.5% Tween 20, adding 300 μl of 1% bovine serum albumin (BSA) dissolved in phosphate buffered saline (PBS) in each well, and placing said ELISA plate at room temperature for 2 hours in order to block non-specific binding;   Step 3: washing (four times) the ELISA plate with PBS-0.5% Tween 20, adding plasma, serum, blood, or the separated immunoglobulin, and placing said ELISA plate at room temperature for 2 hours;   Step 4: washing (four times) the ELISA plate as in Step 2 with the PBS-0.5% Tween 20, adding 300 μl of 1% bovine serum albumin dissolved in PBS in each well, and placing said ELISA plate at room temperature for 1 hour;   Step 5: using PBS-0.5% Tween 20 to wash (four times) the ELISA plate, adding 100 μl of horseradish peroxidase-linked goat anti-mouse immunoglobulin G (at 1:8000 dilution) dissolved in phosphate buffered saline, and placing said ELISA plate in a room temperature for 1 hour;   Step 6: using PBS-0.5% Tween 20 as in Step 2 to wash (four times) the ELISA plate, adding mixture of tetramethyl benzidine (TMB) solution 50 μl and hydrogen peroxide solution 50 μl in each well, and incubating said ELISA plate at room temperature for 10 minutes, and adding 100 μl of 0.5N H 2 SO 4  to stop the reaction; and   Step 7: measuring the absorption value at 450 nm in each well by an ELISA reader.   
     
     
         2 . The method for measuring the amount of sialic acid in immunoglobulin G of  claim 1 , wherein a mouse monoclonal antibody immunoglobulin G is used as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml and incubated at room temperature for two hours. 
     
     
         3 . The method for measuring the amount of sialic acid in immunoglobulin G of  claim 1 , wherein said ELISA plate has ninety-six (96) wells. 
     
     
         4 . A method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies comprises steps of:
 Step 1: in an enzyme-linked immunosorbent assay (ELISA) plate, adding 150 μl of 0.5 mg/ml of protamine chloride in each well and placing said ELISA plate at room temperature for 2 hours;   Step 2: washing (three times) with PBS (pH=7.2) and adding 100 μl/well 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) 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 (Tween 20) solution to make 1% oxidized bovine serum albumin;   Step 4: using PBS-0.5% Tween 20 to wash the ELISA plate (four times), adding 300 μl of 1% oxidized bovine serum albumin into each well, and incubating said ELISA plate at room temperature for 2 hours in order to block non-specific binding;   Step 5: washing (four times) the ELISA plate as in Step 4 with PBS-0.5% Tween 20, adding 100 μl of immunoglobulin G isolated from a protein G column, and incubating said ELISA plate at room temperature for two hours;   Step 6: washing (four times) the ELISA plate as in Step 4 with PBS-0.5% Tween 20, and adding diluted (at 1:500 dilution) horseradish peroxidase-linked  Sambucus nigra  agglutinin lectin (SNA) 100 μl to each well;   Step 7: washing (four times) the ELISA plate as in Step 4 with PBS-0.5% Tween 20, adding 100 μl of tetramethyl benzidine (TMB) solution and peroxide hydrogen solution mixture (at equal volume) at room temperature for 5 minutes, and adding 100 μl of 0.5N H 2 SO 4  into each well to stop the reaction; and   Step 8: measuring the absorption value at 450 nm in each well by an ELISA reader.   
     
     
         5 . The method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies of  claim 4 , wherein a mouse monoclonal antibody immunoglobulin G anti-ds DNA antibodies is used as a standard, which is diluted from 1000 ng/ml to 15.625 ng/ml and incubated at room temperature for two hours. 
     
     
         6 . The method for measuring the amount of sialic acid in immunoglobulin G anti-ds DNA antibodies of  claim 4 , wherein said ELISA plate has ninety-six (96) wells.