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ELISA of IgG antibody to oxidized low-density lipoprotein: effects of blocking buffer and method of data expression
W Y Craig1, S E Poulin, C P Nelson
1Foundation for Blood Research, Scarborough, ME 04070-0190.
We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.
We describe an ELISA for serum IgG antibodies against malondialdehyde-modified low-density lipoprotein (mLDL). Optimal antigen concentration, serum dilution, and dilution of enzyme-conjugated second antibody were 25 mg/L, 1:250, and 1:5000, respectively, when 5 g/L human serum albumin was used for blocking. When data were expressed as mLDL/LDL (the ratio of IgG binding to mLDL vs LDL), within-run and between-run CVs were 7.0% and 8.9%, respectively. Antibody concentrations expressed as mLDL/LDL or as mLDL-LDL (the difference between IgG binding to mLDL and to LDL) were higher in women with systemic lupus erythematosus (n = 20) than in controls (n = 20) (P < 0.001). With bovine serum albumin or Superblock blocking buffers, only the mLDL-LDL data were significant. Thus, the choice of blocking agent and the method of data expression should be carefully considered when assaying IgG antibodies against mLDL.